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Dondi, D., R. Maggi, E. Scaccianoce, L. Martini, M. Motta, and A. Poletti. "Expression and role of functional glucocorticoid receptors in the human androgen-independent prostate cancer cell line, DU145." Journal of Molecular Endocrinology 26, no. 3 (June 1, 2001): 185–91. http://dx.doi.org/10.1677/jme.0.0260185.
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We investigated the presence of glucocorticoid receptors (GR) as well as the role of glucocorticoids (Gc) in the control of proliferation of the androgen-independent prostate cancer cell line, DU145. We detected the presence of a specific high affinity binding site (K(d) 2.3 nM) for [(3)H]dexamethasone ([(3)H]Dex) in the cytosolic preparations of DU145 cells; the density of these binding sites is significantly higher than that detected in HA22T/VGH and in HepG2, two hepatoma cell lines classically considered models for the study of GR. Immunocytochemistry studies confirmed the presence of GR in the cytosolic compartment of DU145 cells; GR undergo translocation to the nucleus following exposure to dexamethasone (Dex). The functional activity of GR present in DU145 cells was also studied by analyzing the potency of Dex in inducing chloramphenicol acyltransferase (CAT) activity in DU145 cells transfected with a glucocorticoid/progesterone response element (GRE/PRE) tkCAT plasmid (GRE/PREtkCAT plasmid). The results have shown that Dex stimulates the transcriptional activity of GR in transfected DU145 cells with an EC(50) of 9.65 nM and a maximal induction of sevenfold above basal levels. Finally, a dose-dependent (IC(50) 3.14 nM) decrease of DU145 cell numbers was observed after their exposure to Dex for 4 days; this effect was counteracted by the presence of the steroid antagonist, RU486. In conclusion, the present data suggest a possible role of corticoids in the control of the growth of androgen-independent prostate cancer.
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Wanyan, Yangke, Xixi Xu, Kehang Liu, Huidan Zhang, Junai Zhen, Rong Zhang, Jumei Wen, Ping Liu, and Yuqing Chen. "2-Deoxy-d-glucose Promotes Buforin IIb-Induced Cytotoxicity in Prostate Cancer DU145 Cells and Xenograft Tumors." Molecules 25, no. 23 (December 7, 2020): 5778. http://dx.doi.org/10.3390/molecules25235778.
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Inhibition of the glycolytic pathway is a critical strategy in anticancer therapy because of the role of aerobic glycolysis in cancer cells. The glycolytic inhibitor 2-Deoxy-d-glucose (2-DG) has shown potential in combination with other anticancer agents. Buforin IIb is an effective antimicrobial peptide (AMP) with broad-spectrum anticancer activity and selectivity. The efficacy of combination treatment with 2-DG and buforin IIb in prostate cancer remains unknown. Here, we tested the efficacy of buforin IIb as a mitochondria-targeting AMP in the androgen-independent human prostate cancer cell line DU145. Combining 2-DG with buforin IIb had a synergistic toxic effect on DU145 cells and mouse xenograft tumors. Combination treatment with 2-DG and buforin IIb caused stronger proliferation inhibition, greater G1 cell cycle arrest, and higher apoptosis than either treatment alone. Combination treatment dramatically decreased L-lactate production and intracellular ATP levels, indicating severe inhibition of glycolysis and ATP production. Flow cytometry and confocal laser scanning microscopy results indicate that 2-DG may increase buforin IIb uptake by DU145 cells, thereby increasing the mitochondria-targeting capacity of buforin IIb. This may partly explain the effect of combination treatment on enhancing buforin IIb-induced apoptosis. Consistently, 2-DG increased mitochondrial dysfunction and upregulated Bax/Bcl-2, promoting cytochrome c release to initiate procaspase 3 cleavage induced by buforin IIb. These results suggest that 2-DG sensitizes prostate cancer DU145 cells to buforin IIb. Moreover, combination treatment caused minimal hemolysis and cytotoxicity to normal WPMY-1 cells. Collectively, the current study demonstrates that dual targeting of glycolysis and mitochondria by 2-DG and buforin IIb may be an effective anticancer strategy for the treatment of some advanced prostate cancer.
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Melzer, Catharina, Juliane von der Ohe, and Ralf Hass. "Involvement of Actin Cytoskeletal Components in Breast Cancer Cell Fusion with Human Mesenchymal Stroma/Stem-Like Cells." International Journal of Molecular Sciences 20, no. 4 (February 18, 2019): 876. http://dx.doi.org/10.3390/ijms20040876.
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Cell fusion as a rare event was observed following the co-culture of human MDA-MB-231cherry breast cancer cells or benign neoplastic MCF10Acherry breast epithelial cells together with different mesenchymal stroma/stem-like cells (MSCGFP) cultures, respectively, resulting in the generation of double-fluorescing hybrid cells. Analysis of potential molecular mechanisms for the formation of cancer hybrid cells revealed cytoskeletal components, including F-actin. Thus, a sub-lethal concentration of cytochalasin D, which blocks elongation of actin filaments, was able to significantly reduce cancer hybrid cell formation. Simultaneously, cell cycle progression of the different co-cultures remained unaffected following treatment with cytochalasin D, indicating continued proliferation. Moreover, exposure to 50 nM cytochalasin D revealed little if any effect on the expression of various integrins and cell adhesion molecules in the different co-cultures. However, LC-MS proteome analysis of the different control co-cultures compared to corresponding cytochalasin-treated co-cultures demonstrated predominant differences in the expression of actin-associated cytoskeletal proteins. In addition, the requirement of structured actin to provide an appropriate cytoskeletal network for enabling subsequent fusion processes was also substantiated by the actin filament disrupting latrunculin B, which inhibits the fusion process between the breast cancer populations and mesenchymal stroma/stem-like cells (MSC). Together, these findings suggest an important role of distinct actin structures and associated cytoskeletal components during cell fusion and the formation of breast cancer hybrid cells.
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Tanaudommongkon, Irin, Asama Tanaudommongkon, Priyanka Prathipati, Joey Trieu Nguyen, Evan T. Keller, and Xiaowei Dong. "Curcumin Nanoparticles and Their Cytotoxicity in Docetaxel-Resistant Castration-Resistant Prostate Cancer Cells." Biomedicines 8, no. 8 (July 30, 2020): 253. http://dx.doi.org/10.3390/biomedicines8080253.
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Most prostate cancer patients develop resistance to anti-androgen therapy. This is referred to as castration-resistant prostate cancer (CRPC). Docetaxel (DTX) is the mainstay treatment against CRPC. However, over time patients eventually develop DTX resistance, which is the cause of the cancer-related mortality. Curcumin (CUR) as a natural compound has been shown to have very broad pharmacological activities, e.g., anti-inflammatory and antioxidant properties. However, CUR is very hydrophobic. The objective of this study was to develop CUR nanoparticles (NPs) and evaluate their cytotoxicity in DTX-resistant CRPC cells for the treatment of DTX-resistant CRPC. We tested solubility of CUR in different lipids and surfactants. Finally, Miglyol 812 and D-alpha-tocopheryl poly (ethylene glycol) succinate 1000 (TPGS) were chosen to prepare lipid-based NPs for CUR. We fully characterized CUR NPs that had particle size < 150 nm, high drug loading (7.5%), and entrapment efficiency (90%). Moreover, the CUR NPs were successfully lyophilized without using cryoprotectants. We tested the cytotoxicity of blank NPs, free CUR, and CUR NPs in sensitive DU145 and PC3 cells as well as their matching docetaxel-resistant cells. Cytotoxicity studies showed that blank NPs were very safe for all tested prostate cancer cell lines. Free CUR overcame the resistance in PC3 cells, but not in DU145 cells. In contrast, CUR NPs significantly increased CUR potency in all tested cell lines. Importantly, CUR NPs completely restored CUR potency in both resistant DU145 and PC3 cells. These results demonstrate that the CUR NPs have potential to overcome DTX resistance in CRPC.
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Morrison, T. G., and L. J. McGmnes. "Cytochalasin D accelerates the release of Newcastle disease virus from infected cells." Virus Research 4, no. 1 (December 1985): 93–106. http://dx.doi.org/10.1016/0168-1702(85)90023-1.
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Park, Jee-Young, Jong-Wook Park, Seong-Il Suh, and Won-Ki Baek. "d-Glucosamine down-regulates HIF-1α through inhibition of protein translation in DU145 prostate cancer cells." Biochemical and Biophysical Research Communications 382, no. 1 (April 2009): 96–101. http://dx.doi.org/10.1016/j.bbrc.2009.02.129.
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Marchiani, Sara, Lorella Bonaccorsi, Pietro Ferruzzi, Clara Crescioli, Monica Muratori, Luciano Adorini, Gianni Forti, Mario Maggi, and Elisabetta Baldi. "The vitamin D analogue BXL-628 inhibits growth factor-stimulated proliferation and invasion of DU145 prostate cancer cells." Journal of Cancer Research and Clinical Oncology 132, no. 6 (February 17, 2006): 408–16. http://dx.doi.org/10.1007/s00432-006-0086-8.
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Gallegos-Martínez, Salvador, Itzel Montserrat Lara-Mayorga, Mohamadmahdi Samandari, Christian Mendoza-Buenrostro, Brenda Giselle Flores-Garza, Luisa María Reyes-Cortés, Juan Carlos Segoviano-Ramírez, Yu Shrike Zhang, Grissel Trujillo-de Santiago, and Mario Moisés Álvarez. "Culture of cancer spheroids and evaluation of anti-cancer drugs in 3D-printed miniaturized continuous stirred tank reactors (mCSTRs)." Biofabrication 14, no. 3 (April 21, 2022): 035007. http://dx.doi.org/10.1088/1758-5090/ac61a4.
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Abstract Cancer continues to be a leading cause of mortality in modern societies; therefore, improved and more reliable in vitro cancer models are needed to expedite fundamental research and anti-cancer drug development. Here, we describe the use of a miniaturized continuous stirred tank reactor (mCSTR) to first fabricate and mature cancer spheroids (i.e. derived from MCF7 cells, DU145 cells, and a mix of MCF7 cells and fibroblasts), and then to conduct anti-cancer drug assays under continuous perfusion. This 3 ml mCSTR features an off-center agitation system that enables homogeneous chaotic laminar mixing at low speeds to support cell aggregation. We incubated cell suspensions for 3 d in ultra-low-attachment plates to allow formation of discoid cell aggregates (∼600 µm in diameter). These cell aggregates were then transferred into mCSTRs and continuously fed with culture medium. We characterized the spheroid morphology and the expression of relevant tumor biomarkers at different maturation times for up to 4 weeks. The spheroids progressively increased in size during the first 5–6 d of culture to reach a steady diameter between 600 and 800 µm. In proof-of-principle experiments, we demonstrated the use of this mCSTR in anti-cancer drug testing. Three drugs commonly used in breast cancer treatment (doxorubicin, docetaxel, and paclitaxel) were probed at different concentrations in MCF7-derived spheroids. In these experiments, we evaluated cell viability, glucose consumption, spheroid morphology, lactate dehydrogenase activity, and the expression of genes associated with drug resistance (ABCB1 and ABCC1) and anti-apoptosis (Bcl2). We envision the use of this agitated system as a tumor-on-a-chip platform to expedite efficacy and safety testing of novel anti-cancer drugs and possibly in personalized medicine applications.
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Diaz Cruz, Maria Araceli, Sandra Karlsson, Ferenc Szekeres, Maria Faresjö, Dan Lund, and Dennis Larsson. "Differential expression of protein disulfide-isomerase A3 isoforms, PDIA3 and PDIA3N, in human prostate cancer cell lines representing different stages of prostate cancer." Molecular Biology Reports 48, no. 3 (March 2021): 2429–36. http://dx.doi.org/10.1007/s11033-021-06277-1.
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AbstractProstate cancer (PCa) is a highly heterogeneous and unpredictable progressive disease. Sensitivity of PCa cells to androgens play a central role in tumor aggressiveness but biomarkers with high sensitivity and specificity that follow the progression of the disease has not yet been verified. The vitamin D endocrine system and its receptors, the Vitamin D Receptor (VDR) and the Protein Disulfide-Isomerase A3 (PDIA3), are related to anti-tumoral effects as well as carcinogenesis and have therefore been suggested as potential candidates for the prevention and therapy of several cancer forms, including PCa. In this study, we evaluated the mRNA expression of VDR and PDIA3 involved in vitamin D signaling in cell lines representing different stages of PCa (PNT2, P4E6, LNCaP, DU145 and PC3). This study further aimed to evaluate vitamin D receptors and their isoforms as potential markers for clinical diagnosis of PCa. A novel transcript isoform of PDIA3 (PDIA3N) was identified and found to be expressed in all PCa cell lines analyzed. Androgen-independent cell lines showed a higher mRNA expression ratio between PDIA3N/PDIA3 contrary to androgen-dependent cell lines that showed a lower mRNA expression ratio between PDIA3N/PDIA3. The structure of PDIA3N differed from PDIA3. PDIA3N was found to be a N-truncated isoform of PDIA3 and differences in protein structure suggests an altered protein function i.e. cell location, thioredoxin activity and affinity for 1,25(OH)2D3. Collectively, PDIA3 transcript isoforms, the ratio between PDIA3N/PDIA3 and especially PDIA3N, are proposed as candidate markers for future studies with different stages of PCa progression.
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Karr, Joan F., Robert P. Apkarian, and John A. Petros. "Cationic Peptide Mediated Oligonucleotide Delivery to DU145 Prostate Cancer Cells - Cell Surface Binding Detected by High Resolution SEM." Microscopy and Microanalysis 5, S2 (August 1999): 392–93. http://dx.doi.org/10.1017/s1431927600015282.
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Transfection of mammalian cells can be enhanced by forming complexes between exogenous DNA and cationic peptides which facilitate DNA transport through cell membranes. Although complexes of plasmid DNA and cationic peptides have been characterized by microscopy, little is known about the complexes formed between oligonucleotides (ODN) and cationic peptides or how the ultrastructure of these complexes affects the intracellular delivery via the cell membrane. Because ODN have potential as therapeutic reagents in the treatment of many diseases, there is a need to develop improved methods for effective delivery of these molecules. By analyzing molecular interactions between cationic peptides and ODN and their effects on cell membranes, cell viability, and DNA delivery, improved delivery compounds can be designed. In this paper, we compare the interactions of a 28 base ODN with three cationic peptides: a novel compound, MNLEK (H-Met-(Nle-Lys4)7-Nle-NH2), an established transfection reagent, poly(L-lysine) (∼ 54 kDa), and a recently described cationic peptide which delivers both plasmid DNA and antisense ODN to cells, KALA (WEAKLAKALAKALAKHLAKALAKALKACEA).For high resolution scanning electron microscopy (HRSEM), DU145 human prostate cancer cells were grown overnight on poly-(D-lysine) coated, UV irradiated silicon chips.
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Małecki, Jędrzej Mieczyslaw, Anna Bentke, Barbara Ostrowska, and Piotr Laidler. "Cytochalasin D, LY294002 and olomoucine synergize in promoting death of melanoma cells through activation of caspase-3 and apoptosis." Melanoma Research 20, no. 1 (February 2010): 52–58. http://dx.doi.org/10.1097/cmr.0b013e328332f1e6.
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Jonnala, Sandhya, Bhaskar Nameta, Murthy Chavali, Rajashaker Bantu, Pallavi Choudante, Sunil Misra, B. Sridhar, S. Dilip, and B. V. Subba Reddy. "Design, Synthesis, Molecular Docking and Biological Evaluation of 1-(benzo[d]thiazol-2-ylamino)(phenyl)methyl)naphthalen-2-ol Derivatives as Antiproliferative Agents." Letters in Organic Chemistry 16, no. 10 (August 23, 2019): 837–45. http://dx.doi.org/10.2174/1570178616666190408101233.
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A class of 1-((benzo[d]thiazol-2-ylamino)(phenyl)methyl)naphthalen-2-ol derivatives (4a-t) has been synthesized in good yields through a three component coupling reaction. The newly synthesized compounds were evaluated for their in vitro antiproliferative activity against five cell lines such as DU145 (human prostate cancer), MDA-MB-B231 (human breast cancer), SKOV3 (human ovarian cancer), B16-F10 (mouse skin melanoma) and CHO-K1 (Chinese hamster ovary cells), a noncancerous cell line. In vitro inhibitory activity indicates that compounds 4a, 4b, 4c, 4d, 4g, 4j, and 4o exhibited potent anti-proliferative behavior. Among them, compounds 4g, 4j and 4o found to be the most active members exhibiting remarkable growth inhibitory activity. Molecular docking facilitates to investigate the probable binding mode and key active site interactions in tubulins α and β proteins. The docking results are complementary to experimental results.
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Kari, Suresh Chandra, Aravind Kancharla, Harshita B. Gupta, April Risinger, and Tyler J. Curiel. "Tumor-intrinsic PD-L1 reduces actin cytoskeleton polymerization to promote mTORC1 signals driving tumor stemness." Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 137.8. http://dx.doi.org/10.4049/jimmunol.202.supp.137.8.
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Abstract Tumor-intrinsic PD-L1 signals contribute to tumor virulence and pathology, but mechanisms are poorly understood. We previously showed that tumor-intrinsic PD-L1 increases mTORC1 and tumor initiating cell (TIC) generation and function in ovarian cancer and melanoma. We used CRISPR/Cas9 (KO) and shRNA (lo) to reduce PD-L1 in mouse melanoma (B16) and ovarian cancer (ID8agg), and human ovarian cancer (ES2). PD-L1lo reduced mTORC1 and TIC as expected, but unexpectedly increased actin polymerization and filopodia, and altered cell morphology. The actin depolymerizing agents Latrunculin-A or Cytochalasin-D reversed actin polymerization in PD-L1lo ID8agg cells and increased mTORC1 (p-rpS6) without affecting mTORC2 (pAktS473). Latrunculin-A and Cytochalasin-D increased TIC numbers and stemness gene expression (Oct4) in PD-L1lo ID8agg cells with no effect on control ID8agg cells. Similar actin effects were seen in PD-L1KO B16 and PD-L1KO ES2. To confirm TIC function, Latrunculin-A increased tumorospheres (TIC self-renewal function) in PD-L1lo, but not control ID8agg cells. The mTORC1 inhibitor rapamycin reduced TIC numbers and functions with no effect on actin polymerization, suggesting mTORC1 is downstream of actin polymerization. Rptorlo ID8agg cells (with low mTORC1 signaling) did not exhibit the PD-L1lo actin polymerization, nor did Latrunculin-A increase Rptorlo ID8agg TIC numbers, tumorospheres, or stemness genes, confirming the flow of signals is PD-L1 to actin to mTORC1 to TIC (stemness). Our data define a highly novel tumor virulence control mechanism of cell-intrinsic PD-L1 through inhibiting actin polymerization suggesting new drug discovery targets. Preliminary data show some similar effects of tumor PD-1.
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El-Zawahry, Ahmed M., Xiang Liu, Saeed ElOjeimy, David Holma, Ayman Elmahdy, Nabil K. Bissada, Hashim M. Rashwan, et al. "924. D-MAPP Analogue, LCL-204 Sensitizes DU145 Prostate Cancer Cells (PCa) to FasL Gene Therapy through Down-Regulation of Survivin." Molecular Therapy 13 (2006): S357. http://dx.doi.org/10.1016/j.ymthe.2006.08.1015.
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Ogawa, Tetsuya, Ayako Sasaki, Koki Ono, Shusa Ohshika, Yasuyuki Ishibashi, and Katsuya Yamada. "Uptake of fluorescent d- and l-glucose analogues, 2-NBDG and 2-NBDLG, into human osteosarcoma U2OS cells in a phloretin-inhibitable manner." Human Cell 34, no. 2 (January 17, 2021): 634–43. http://dx.doi.org/10.1007/s13577-020-00483-y.
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AbstractMammalian cells take in d-glucose as an essential fuel as well as a carbon source. In contrast, l-glucose, the mirror image isomer of d-glucose, has been considered merely as a non-transportable/non-metabolizable control for d-glucose. We have shown that 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), a d-glucose analogue combining a fluorophore NBD at the C-2 position, is useful as a tracer for monitoring d-glucose uptake through glucose transporters (GLUTs) into mammalian cells. To more precisely evaluate the stereoselectivity of 2-NBDG uptake, we developed an l-glucose analogue 2-NBDLG, the mirror-image isomer of 2-NBDG. Interestingly, 2-NBDLG was taken up into mouse insulinoma MIN6 cells showing nuclear heterogeneity, a cytological feature of malignancy, while remaining MIN6 cells only exhibited a trace amount of 2-NBDLG uptake. The 2-NBDLG uptake into MIN6 cells was abolished by phloretin, but persisted under blockade of major mammalian glucose transporters. Unfortunately, however, no such uptake could be detected in other tumor cell lines. Here we demonstrate that human osteosarcoma U2OS cells take in 2-NBDLG in a phloretin-inhibitable manner. The uptake of 2-NBDG, and not that of 2-NBDLG, into U2OS cells was significantly inhibited by cytochalasin B, a potent GLUT inhibitor. Phloretin, but neither phlorizin, an inhibitor of sodium-glucose cotransporter (SGLT), nor a large amount of d/l-glucose, blocked the 2-NBDLG uptake. These results suggest that a phloretin-inhibitable, non-GLUT/non-SGLT, possibly non-transporter-mediated yet unidentified mechanism participates in the uptake of the fluorescent l-glucose analogue in two very different tumor cells, the mouse insulinoma and the human osteosarcoma cells.
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Wu, Wei, Su-Ni Tang, Yong Zhang, Manohar Puppala, Timothy K. Cooper, Chengguo Xing, Cheng Jiang, and Junxuan Lü. "Prostate Cancer Xenograft Inhibitory Activity and Pharmacokinetics of Decursinol, a Metabolite of Angelica gigas Pyranocoumarins, in Mouse Models." American Journal of Chinese Medicine 45, no. 08 (January 2017): 1773–92. http://dx.doi.org/10.1142/s0192415x17500963.
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We have previously shown that the ethanol extract of dried Angelica gigas Nakai (AGN) root exerts anticancer activity against androgen receptor (AR)-negative human DU145 and PC-3 prostate cancer xenografts and primary carcinogenesis in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. The major pyranocoumarin isomers decursin (D) and decursinol angelate (DA), when provided at equi-molar intake to that provided by AGN extract, accounted for the inhibitory efficacy against precancerous epithelial lesions in TRAMP mice. Since we and others have shown in rodents and humans that D and DA rapidly and extensively convert to decursinol, here we tested whether decursinol might be an in vivo active compound for suppressing xenograft growth of human prostate cancer cells expressing AR. In SCID-NSG mice carrying subcutaneously inoculated human LNCaP/AR-Luc cells overexpressing the wild type AR, we compared the efficacy of 4.5[Formula: see text]mg decursinol per mouse with equi-molar dose of 6[Formula: see text]mg D/DA per mouse. The result showed that decursinol decreased xenograft tumor growth by 75% and the lung metastasis, whereas D/DA exerted a much less effect. Measurement of plasma decursinol concentration, at 3[Formula: see text]h after the last dose of respective dosing regimen, showed higher circulating level in the decursinol-treated NSG mice than in the D/DA-treated mice. In a subsequent single-dose pharmacokinetic experiment, decursinol dosing led to 3.7-fold area under curve (AUC) of plasma decursinol over that achieved by equi-molar D/DA dosing. PK advantage notwithstanding, decursinol represents an active compound to exert in vivo prostate cancer growth and metastasis inhibitory activity in the preclinical model.
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YAO, MIN, JINGBO YANG, LANQING CAO, LIAN ZHANG, SHANSHAN QU, and HONGWEN GAO. "Saikosaponin-d inhibits proliferation of DU145 human prostate cancer cells by inducing apoptosis and arresting the cell cycle at G0/G1 phase." Molecular Medicine Reports 10, no. 1 (April 15, 2014): 365–72. http://dx.doi.org/10.3892/mmr.2014.2153.
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Amawi, Haneen, Noor Hussein, Sai H. S. Boddu, Chandrabose Karthikeyan, Frederick E. Williams, Charles R. Ashby, Dayanidhi Raman, Piyush Trivedi, and Amit K. Tiwari. "Novel Thienopyrimidine Derivative, RP-010, Induces β-Catenin Fragmentation and Is Efficacious against Prostate Cancer Cells." Cancers 11, no. 5 (May 23, 2019): 711. http://dx.doi.org/10.3390/cancers11050711.
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Thienopyrimidines containing a thiophene ring fused to pyrimidine are reported to have a wide-spectrum of anticancer efficacy in vitro. Here, we report for the first time that thieno[3,2-d]pyrimidine-based compounds, also known as the RP series, have efficacy in prostate cancer cells. The compound RP-010 was efficacious against both PC-3 and DU145 prostate cancer (PC) cells (IC50 < 1 µM). The cytotoxicity of RP-010 was significantly lower in non-PC, CHO, and CRL-1459 cell lines. RP-010 (0.5, 1, 2, and 4 µM) arrested prostate cancer cells in G2 phase of the cell cycle, and induced mitotic catastrophe and apoptosis in both PC cell lines. Mechanistic studies suggested that RP-010 (1 and 2 µM) affected the wingless-type MMTV (Wnt)/β-catenin signaling pathway, in association with β-catenin fragmentation, while also downregulating important proteins in the pathway, including LRP-6, DVL3, and c-Myc. Interestingly, RP-010 (1 and 2 µM) induced nuclear translocation of the negative feedback proteins, Naked 1 and Naked 2, in the Wnt pathway. In addition, RP-010 (0.5, 1, 2 and 4 µM) significantly decreased the migration of PC cells in vitro. Finally, RP-010 did not produce significant toxic effects in zebrafish at concentrations of up to 6 µM. In conclusion, RP-010 may be an efficacious and relatively nontoxic anticancer compound for prostate cancer. Future mechanistic and in vivo efficacy studies are needed to optimize the hit compound RP-010 for lead optimization and clinical use.
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Jirsch, J. D., D. W. Loe, S. P. Cole, R. G. Deeley, and D. Fedida. "ATP is not required for anion current activated by cell swelling in multidrug-resistant lung cancer cells." American Journal of Physiology-Cell Physiology 267, no. 3 (September 1, 1994): C688—C699. http://dx.doi.org/10.1152/ajpcell.1994.267.3.c688.
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During whole cell recording with 4 mM ATP and 0.1 mM GTP in the pipette, outwardly rectifying Cl- currents (155 +/- 20.5 pA/pF) were repetitively activated on reduction of bath solution osmolarity from 290 mosM (control) to 210 mosM. These currents were sensitive to 0.1-1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Omission of ATP from the pipette solution reduced the current magnitude to 42.7 +/- 9.5 pA/pF and prevented repetitive activation. More hyposmotic solutions (160 mosM) usually elicited current repetitively despite an ATP-free pipette solution. In cells depleted of ATP (to < 5% of control) by preincubation with 2-deoxyglucose (10 mM) and rotenone (100 nM), hyposmotic solutions failed to activate significant current. Cell volume increased to 230 +/- 18% of control (19.1 +/- 1.2 microns) in 210 mosM bath (normal cells) but only to 114 +/- 13% of control in ATP-depleted cells exposed to 160 mosM solution. This failure of ATP-depleted cells to swell in hypotonic external solutions was reversed by overnight pretreatment with cytochalasin D (2 micrograms/ml; n = 6) but not by colchicine (250 microM; n = 8). In outside-out patches of membrane dialyzed with zero ATP and excised from swollen cells, we observed sustained activation of a 53-pS outwardly rectifying channel (chord conductance, +100 mV; open probability approximately 1.0). In cell-attached patches from normal and ATP-depleted cells, we activated similar channels by suction. ATP does not appear to be an absolute requirement for the activation of this Cl- channel in H69AR cells but may be essential for the normal volume response and channel activation mediated through cytoskeletal elements within cells.
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Pu, Tainjie, Jing Wang, Chia-Hui Chen, Tzu-Ping Lin, and Boyang Wu. "Abstract 1579: Increased stromal-oriented MAOB expression associated with poor clinical outcomes promotes prostate cancer progression." Cancer Research 82, no. 12_Supplement (June 15, 2022): 1579. http://dx.doi.org/10.1158/1538-7445.am2022-1579.
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Abstract Background. Stromal fibroblasts, the predominant cell type within the tumor microenvironment, plays a critical regulatory role in prostate cancer (PC) by engaging in stromal-epithelial interactions to dictate cancer initiation, growth, and progression. Monoamine oxidase B (MAOB) catalyzes oxidative deamination of biogenic and dietary monoamines and generates hydrogen peroxide as a by-product, which can predispose stromal cells to a reactive, cancer-promoting state with elevated production of extracellular matrix molecules, growth factors, cytokines, and chemokines. This study investigated the clinical relevance and previously unrecognized function of stromal MAOB in PC. Methods. The stromal MAOB expression levels were determined in PC clinical specimens by immunohistochemistry and in publicly available multi-omics cancer datasets, which were associated with different clinical parameters. The human prostatic stromal fibroblast PrSC cells engineered for stable overexpression or knockdown of MAOB were co-cultured with a panel of human PC cells in 2-D or 3-D fashion, followed by examination of cancer cell behavior by cell proliferation, migration, and invasion assays. MAOB-knockdown PrSC cells were co-inoculated with luciferase-tagged PC cells as subrenal tissue recombinant xenografts into mice for assessment of MAOB’s impact on tumor growth by bioluminescence imaging. Results. MAOB expression was induced in the stroma associated with primary PC compared to normal prostatic tissues of clinical samples, which was corroborated by the same MAOB expression trend in three matched pairs of patient-derived fibroblasts. Following the analysis of three independent clinical cohorts, increased stromal MAOB levels were shown to be correlated with higher Gleason scores, castration resistance, survival, and appearance of neuroendocrine differentiation as evidenced by CHGA expression in the adjacent epithelia. Co-culturing MAOB-overexpressing PrSC cells with several human PC cell lines (C4-2, PC-3, DU145 and C4-2BENZR) accelerated cancer cell proliferation, migration, and invasion. Conversely, these cancer cell phenotypes were suppressed in the presence of MAOB-knockdown PrSC cells. Additionally, C4-2 tumor xenografts when co-inoculated with MAOB-knockdown PrSC grew to a substantially smaller size in mice, which was paralleled by reduced Ki-67 index and reactive stromal marker αSMA expression in the tumor and stromal compartments respectively, compared to controls. Conclusion. Our results suggest that the elevated MAOB expression in PC stromal cells is associated with poor clinical outcomes and further contributes to adjacent PC growth, which provides new insights into understanding the stromal support of PC pathogenesis and progression. Funding provided by the NIH/NCI grant R01CA258634 and WSU CPPS startup fund (to B.W). Citation Format: Tainjie Pu, Jing Wang, Chia-Hui Chen, Tzu-Ping Lin, Boyang Wu. Increased stromal-oriented MAOB expression associated with poor clinical outcomes promotes prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1579.
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Kwon, Sojung, Areum Kwak, Hyejin Shin, Soyoung Choi, Soohyun Kim, and Hyunjung Jade Lim. "Application of a novel cell-permeable peptide-driven protein delivery in mouse blastocysts." REPRODUCTION 146, no. 2 (August 2013): 145–53. http://dx.doi.org/10.1530/rep-13-0203.
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Cell-permeable peptides (CPPs) mediate the delivery of macromolecules into cells. However, whether CPPs are usable in mammalian oocytes and embryos for the modulation of protein expression has not been widely investigated. We have previously designed a novel 12-mer CPP from the conserved region of the human papillomavirus L1 capsid protein. In this study, we tested whether this peptide, LDP12, effectively delivers a protein cargo to mouse oocytes and preimplantation embryos. We prepared a LDP12–EGFP fusion protein having LDP12 as an N-terminal tag. This fusion protein readily enters HeLa cells, a cervical cancer cell line. The entry of LDP12–EGFP was partially blocked by amiloride, while cytochalasin D or methyl-β-cyclodextrin slightly increased the uptake. LDP12–EGFP shows efficient transduction in mouse blastocysts, but not in oocytes, two-cell-stage, or morula-stage-preimplantation embryos. LDP12-mediated delivery of EGFP–LC3, a widely used marker of autophagic activation, is successful in HeLa cells and mouse blastocysts, as it enters cells and exhibits a signature punctate pattern. The lipidation of EGFP–LC3 also normally occurs after transduction, suggesting that the transduced protein retains the functional characteristics. Collectively, we show that LDP12-driven protein delivery is a fast and convenient method applicable to mouse blastocysts and reproductive cancer cells.
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Tseng, Ahmed, Huang, Tsai, Tai, Orfali, Hwang, Wang, Dai, and Sheu. "Bioactive Capnosanes and Cembranes from the Soft Coral Klyxum flaccidum." Marine Drugs 17, no. 8 (August 7, 2019): 461. http://dx.doi.org/10.3390/md17080461.
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Two new capnosane-based diterpenoids, flaccidenol A (1) and 7-epi-pavidolide D (2), two new cembranoids, flaccidodioxide (3) and flaccidodiol (4), and three known compounds 5 to 7 were characterized from the marine soft coral Klyxum flaccidum, collected off the coast of the island of Pratas. The structures of the new compounds were determined by extensive spectroscopic analyses, including 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, and spectroscopic data comparison with related structures. The rare capnosane diterpenoids were isolated herein from the genus Klyxum for the first time. The cytotoxicity of compounds 1 to 7 against the proliferation of a limited panel of cancer cell lines was assayed. The isolated diterpenoids also exhibited anti-inflammatory activity through suppression of superoxide anion generation and elastase release in the N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLF/CB)-stimulated human neutrophils. Furthermore, 1 and 7 also exhibited cytotoxicity toward the tested cancer cells, and 7 could effectively inhibit elastase release. It is worth noting that the biological activities of 7 are reported for the first time in this paper.
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Stoicov, Calin, Xun Cai, Hanchen Li, Kristine Klucevsek, Jane Carlson, Reza Saffari, and JeanMarie Houghton. "Major Histocompatibility Complex Class II Inhibits Fas Antigen-Mediated Gastric Mucosal Cell Apoptosis through Actin-Dependent Inhibition of Receptor Aggregation." Infection and Immunity 73, no. 10 (October 2005): 6311–21. http://dx.doi.org/10.1128/iai.73.10.6311-6321.2005.
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ABSTRACT Escape from normal apoptotic controls is thought to be essential for the development of cancer. During Helicobacter pylori infection, the leading cause of gastric cancer, activation of the Fas antigen (Fas Ag) apoptotic pathway is responsible for early atrophy and tissue loss. As disease progresses, metaplastic and dysplastic glands arise which express Fas Ag but are resistant to apoptosis and are believed to be the precursor cells for adenocarcinoma. In this report, we show that one mechanism of acquired Fas resistance is inhibition of receptor aggregation via a major histocompatibility complex class II (MHCII)-mediated, actin-dependent mechanism. For these studies we used the well-described C57BL/6 mouse model of Helicobacter pylori and Helicobacter felis infection. Under normal conditions, Fas Ag is expressed at low levels, and MHCII expression on gastric mucosal cells is negligible. With infection and inflammation, both receptors are upregulated, and 6.1% of gastric mucosal cells express MHCII in combination with Fas Ag. Using the rat gastric mucosal cell line RGM-1 transfected with murine Fas Ag and MHCIIαβ chains, we demonstrate that MHCII prevents Fas receptor aggregation and inhibits Fas-mediated signaling through its effects on the actin cytoskeleton. Depolymerization of actin with cytochalasin D allows receptors to aggregate and restores Fas sensitivity. These findings offer one mechanism by which gastric mucosal cells acquire Fas resistance.
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Sipos, Arnold, Kwang-Jin Kim, Constantinos Sioutas, and Edward D. Crandall. "Evidence for Nanoparticle-Induced Lysosomal Dysfunction in Lung Adenocarcinoma (A549) Cells." International Journal of Molecular Sciences 20, no. 21 (October 23, 2019): 5253. http://dx.doi.org/10.3390/ijms20215253.
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Background: Polystyrene nanoparticles (PNP) are taken up by primary rat alveolar epithelial cell monolayers (RAECM) in a time-, dose-, and size-dependent manner without involving endocytosis. Internalized PNP in RAECM activate autophagy, are delivered to lysosomes, and undergo [Ca2+]-dependent exocytosis. In this study, we explored nanoparticle (NP) interactions with A549 cells. Methods: After exposure to PNP or ambient pollution particles (PM0.2), live single A549 cells were studied using confocal laser scanning microscopy. PNP uptake and egress were investigated and activation of autophagy was confirmed by immunolabeling with LC3-II and LC3-GFP transduction/colocalization with PNP. Mitochondrial membrane potential, mitophagy, and lysosomal membrane permeability (LMP) were assessed in the presence/absence of apical nanoparticle (NP) exposure. Results: PNP uptake into A549 cells decreased in the presence of cytochalasin D, an inhibitor of macropinocytosis. PNP egress was not affected by increased cytosolic [Ca2+]. Autophagy activation was indicated by increased LC3 expression and LC3-GFP colocalization with PNP. Increased LMP was observed following PNP or PM0.2 exposure. Mitochondrial membrane potential was unchanged and mitophagy was not detected after NP exposure. Conclusions: Interactions between NP and A549 cells involve complex cellular processes leading to lysosomal dysfunction, which may provide opportunities for improved nanoparticle-based therapeutic approaches to lung cancer management.
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Ikezoe, Takayuki, Yang Yang, Kentaro Bandobashi, Nobuo Sekiguchi, Shuichi Sakai, H. Phillip Koeffler, and Hirokuni Taguchi. "HIV-1 Protease Inhibitor Ritonavir Potentiates the Effect of 1,25-Dihydroxyvitamin D3 to Induce Growth Arrest and Differentiation of Human Myeloid Leukemia Cells Via Inhibition of CYP24." Blood 104, no. 11 (November 16, 2004): 2543. http://dx.doi.org/10.1182/blood.v104.11.2543.2543.
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Abstract Ritonavir (RTV) is a potent inhibitor of cytochrome p450 (CYPs) enzymes. This study explores the effects of RTV on CYP24 which converts 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] to its inactive form 1,24,25,(OH)3. Real-time RT-PCR showed that exposure of HL-60 cells to 1,25(OH)2D3 (10−7 M, 1h) induced expression of CYP24 by 80-fold, and pre-incubation of these cells with RTV (2X10−5 M, 3h) decreased 1,25(OH)2D3-stumulated expression of CYP24 transcripts by 30 %, resulting in increased levels of 1,25(OH)2D3 in culture media (50.7 vs 82.4 nmol/L). Importantly, pre-incubation of HL-60 cells with RTV (2X10−5 M, 3h) and then exposure to 1,25(OH)2D3 (10−7 M, 24 h) also increased intracellular levels of 1,25(OH)2D3 (1.20 vs 2.46 nmol/L) and potentiated the ability of 1,25(OH)2D3 to induce growth arrest of HL-60 cells. Clonogenic assay showed that either RTV (5X10−6 M) or 1,25(OH)2D3 (10−8 M) alone inhibited colonal growth of HL-60 cells by either a mean 30 ± 12 (±SD) % and 40 ± 12 %, respectively; when both were combined, colony formation was inhibited by a mean 80 ± 13 % (p<0.01). Measurement of CD14 expression, NBT reduction, and non-specific esterase staining showed that RTV augmented the prodifferentiative effects of 1,25(OH)2D3 in HL-60 cell. For example, 1,25(OH)2D3 (10−8 M, 2 days) induced approximately 48 % of HL-60 cells to become CD14 antigen positive. RTV (2X10−5 M, 48h) alone did not stimulate expression of CD14 antigen; when 1,25(OH)2D3 was combined with RTV, approximately 65 % of cells expressed CD14. Furthermore, androgen-independent DU145 human prostate cancer cells, that are resistant to growth inhibition by 1,25(OH)2D3, expressed 80-fold higher levels of CYP24 transcripts compared to HL-60 cells, and intracellular levels of 1,25(OH)2D3 were not detectable (<0.3 nmol/L) after exposure of these cells to 1,25(OH)2D3 (10−7 M, 24 h). Of note, pre-incubation of these cells with RTV (2X10−5 M, 3h) and then exposure to 1,25(OH)2D3 (10−7 M, 24 h) increased intracellular levels of 1,25(OH)2D3 to 0.94 nmol/L and sensitized DU145 cells to growth inhibition mediated by 1,25(OH)2D3 as measured by MTT assay. Taken together, inhibition of CYP24 might open a new paradigm for therapy using vitamin D compounds.
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Ojeda, Paola, Alejandra Pérez, Lorena Ojeda, Mauricio Vargas-Uribe, Coralia I. Rivas, Monica Salas, Juan Carlos Vera, and Alejandro M. Reyes. "Noncompetitive blocking of human GLUT1 hexose transporter by methylxanthines reveals an exofacial regulatory binding site." American Journal of Physiology-Cell Physiology 303, no. 5 (September 1, 2012): C530—C539. http://dx.doi.org/10.1152/ajpcell.00145.2012.
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Glucose transporter (GLUT)1 has become an attractive target to block glucose uptake in malignant cells since most cancer cells overexpress GLUT1 and are sensitive to glucose deprivation. Methylxanthines are natural compounds that inhibit glucose uptake; however, the mechanism of inhibition remains unknown. Here, we used a combination of binding and glucose transport kinetic assays to analyze in detail the effects of caffeine, pentoxifylline, and theophylline on hexose transport in human erythrocytes. The displacement of previously bound cytochalasin B revealed a direct interaction between the methylxanthines and GLUT1. Methylxanthines behave as noncompetitive blockers (inhibition constant values of 2–3 mM) in exchange and zero- trans efflux assays, whereas mixed inhibition with a notable uncompetitive component is observed in zero- trans influx assays (inhibition constant values of 5–12 mM). These results indicate that methylxanthines do not bind to either exofacial or endofacial d-glucose-binding sites but instead interact at a different site accessible by the external face of the transporter. Additionally, infinite- cis exit assays (Sen-Widdas assays) showed that only pentoxifylline disturbed d-glucose for binding to the exofacial substrate site. Interestingly, coinhibition assays showed that methylxanthines bind to a common site on the transporter. We concluded that there is a methylxanthine regulatory site on the external surface of the transporter, which is close but distinguishable from the d-glucose external site. Therefore, the methylxanthine moiety may become an attractive framework for the design of novel specific noncompetitive facilitative GLUT inhibitors.
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Ferguson, Peter J., Mark D. Vincent, Yousef Najajreh, Brian Shilton, Stephen Ritter, Rima Al-awar, Richard Marcellus, Mohammed Mohammed, Methvin Isaac, and James Koropatnick. "Abstract 346: Synergistic antiproliferative activity of novel RAD51 inhibitor JKYN-1 and its mesylate salt with standard-of-care cancer drugs." Cancer Research 82, no. 12_Supplement (June 15, 2022): 346. http://dx.doi.org/10.1158/1538-7445.am2022-346.
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Abstract The inherent genetic instability of cancer cells and the dependence of many tumor types on oncogenic drivers contribute selectivity of anticancer agents against tumor cells. That selectivity is limited, and toxicity to normal cells remains a major limitation to the success of chemotherapy. To increase selectivity by exploiting cancer cell genetic instability, we demonstrated that the small molecule IBR2 (an inhibitor of the DNA repair protein RAD51) enhanced cytotoxicity of numerous anticancer drugs including agents that do not directly target DNA (J Pharmacol Expt Ther, 364: 46-54, 2018. doi.org/10.1124/jpet.117.241661). We demonstrated the ability of IBR2 and a derivative [IBR120, (R)-3-(2-(benzylsulfonyl)isoindolin-1-yl)-1h-indole] to synergistically inhibit proliferation of a wider range of cancer cell lines in combination with a broad range of anticancer drugs (Proc. Amer. Assoc. Cancer Res., 60: Abst. 3057, 2019). To improve the activity and potentially increase selectivity for inhibiting RAD51, modifications were made to the structure of IBR120 using a virtual drug-protein docking program, yielding the compound JKYN-1. JKYN-1 inhibits proliferation of cancer cell lines approximately 5 times more strongly than IBR120. Given the potential importance of combining JKYN-1 with targeted anticancer drugs to increase therapeutic index, and the synergy previously observed between IBR120 and agents targeted against specific tumor types, JKYN-1 was tested in combination with targeted agents against a panel of tumor cell lines. Four- to five-day drug exposures were conducted in 96-well plates. Relative cell density determined using vital stains (alamarBlue©, neutral red) was reported as a percent of the fluorescence/absorbance of control cultures. Cell lines were representative of tumors from breast (MCF-7), prostate (DU145, LNCaP), stomach (N87), pancreas (PANC-1, Capan-1, Capan-2) and lung (A549b, H1650). The chemotherapy agents included inhibitors of epidermal growth factor receptor (osimertinib, afatinib), other tyrosine kinases (regorafenib, imatinib), sex steroid receptors (4-OH-tamoxifen, enzalutamide), and microtubule function (docetaxel). To improve solubility, a methylsulfonate salt of JKYN-1 was used for most experiments. JKYN-1-mesylate decreased the concentration of drugs that inhibited proliferation by 50% (IC50) by up to 90%, depending on the drug and cell line, indicating synergy between the agents. There were some combinations in which additivity but no synergy was observed, indicating selectivity for this interaction. Individual combinations will be presented. The ability of JKYN-1 to enhance antiproliferative activity of a wide variety of anticancer agents, and its potential selectivity for cancer cells, make possible the future use of RAD51 inhibitors as systemic therapy potentiators to improve clinical outcomes. Citation Format: Peter J. Ferguson, Mark D. Vincent, Yousef Najajreh, Brian Shilton, Stephen Ritter, Rima Al-awar, Richard Marcellus, Mohammed Mohammed, Methvin Isaac, James Koropatnick. Synergistic antiproliferative activity of novel RAD51 inhibitor JKYN-1 and its mesylate salt with standard-of-care cancer drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 346.
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ANNABI, Borhane, Marie-Paule LACHAMBRE, Nathalie BOUSQUET-GAGNON, Martine PAGÉ, Denis GINGRAS, and Richard BÉLIVEAU. "Localization of membrane-type 1 matrix metalloproteinase in caveolae membrane domains." Biochemical Journal 353, no. 3 (January 25, 2001): 547–53. http://dx.doi.org/10.1042/bj3530547.
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Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a membrane-associated MMP that has been recently reported to have a central role in tumour cell invasion. Here we report that both the native and overexpressed recombinant forms of MT1-MMP are highly enriched in low-density Triton X-100-insoluble membrane domains that contain the caveolar marker protein caveolin 1. Moreover, the MT1-MMP-dependent activation of proMMP-2 induced by concanavalin A and cytochalasin D was correlated with the processing of MT1-MMP to its proteolytically inactive 43kDa fragment in U-87 glioblastoma and HT-1080 fibrosarcoma tumour cell lines; this processing was also preferentially observed within the caveolar fraction. Interestingly, whereas the expression of caveolin 1had no effect on the MT1-MMP-dependent activation of proMMP-2, its co-expression with MT1-MMP antagonized the MT1-MMP-increased migratory potential of COS-7 cells. Taken together, our results provide evidence that MT1-MMP is preferentially compartmentalized and proteolytically processed in caveolae of cancer cells. The inhibition of MT1-MMP-dependent cell migration by caveolin 1 also suggests that the localization of MT1-MMP to caveolin-enriched domains might have an important function in the control of its enzymic activity.
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Saatci, Ozge, Ozge Akbulut, Metin Cetin, Vitali Sikirzhytski, and Ozgur Sahin. "Abstract P4-08-20: Inhibition of TACC3 blocks the growth of highly aggressive breast cancers with centrosome amplification." Cancer Research 83, no. 5_Supplement (March 1, 2023): P4–08–20—P4–08–20. http://dx.doi.org/10.1158/1538-7445.sabcs22-p4-08-20.
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Abstract Centrosome amplification (CA) is a hallmark of cancer that is strongly associated with highly aggressive disease and worse clinical outcome. Enhanced mitotic progression via clustering of extra centrosomes is a major coping mechanism utilized by cancer cells with CA that would otherwise undergo mitotic cell death due to formation of multipolar spindles. However, the underlying molecular mechanisms have largely been unexplored. Furthermore, mitosis-targeting inhibitors have mostly been unsuccessful in clinical settings with poor efficacy and severe side effects. Therefore, there is a dire need to uncover novel molecular mechanisms of CA-driven tumor growth and identify therapeutic targets playing key roles not only in mitosis, but also in interphase of cancer cells with CA to achieve durable anti-tumor effect with minimal toxicity. Here, we identified Transforming Acidic Coiled-Coil Containing Protein 3 (TACC3) as a novel CA-directed dependency, driving highly aggressive cell growth by forming distinct functional interactomes during cell cycle progression. We demonstrated, for the first time, that TACC3 interacts with the Kinesin Family Member C1 (KIFC1) via its TACC domain in mitotic cells with CA to promote centrosome clustering (CC) and facilitate mitotic progression. On the other hand, TACC3 interacts with the members of the nucleosome remodeling and deacetylase (NuRD) complex (HDAC2 and MBD2) in the nucleus of interphase cells with CA, thereby suppressing the transcription of key tumor suppressors to facilitate G1/S progression and cell survival. Inhibiting TACC3 in mitotic cells blocks the formation of TACC3/KIFC1 complex, leading to formation of multipolar spindles and activation of spindle assembly checkpoint (SAC)/CDK1/p-Bcl2 axis that ultimately results in mitotic cell death; whereas TACC3 inhibition in interphase cells blocks TACC3/HDAC2/MBD2 complex, leading to enhanced transcription of cyclin-dependent kinase inhibitors (e.g., p21 and p16) and apoptosis regulators (e.g., APAF1), ultimately causing p53-independent G1 arrest and strong apoptosis. Notably, inducing CA by chemical (cytochalasin D) or genomic (PLK4 overexpression or p53 loss) modulations renders cancer cells highly sensitive to TACC3 inhibition, showing the dependency of cells with CA to TACC3. Targeting TACC3 by small molecule inhibitors or CrispR-CAS9-mediated knock-out significantly reduces colony formation ability, inhibits the growth of organoids of patient-derived xenografts (PDXs) with CA, and strongly inhibits tumor growth in breast cancer cell line xenografts and PDXs with CA. Notably, we demonstrated that high CA tumors express much higher levels of TACC3, and high TACC3 expression, in association with its downstream effectors, KIFC1, HDAC2 and MBD2, leads to drastically worse clinical outcome in cancer patients with CA. Altogether, our results show, for the first time, that TACC3 is a multifunctional driver of the growth of the highly aggressive breast tumors with CA and that targeting TACC3 is a promising approach to tackle this aggressive disease. Citation Format: Ozge Saatci, Ozge Akbulut, Metin Cetin, Vitali Sikirzhytski, Ozgur Sahin. Inhibition of TACC3 blocks the growth of highly aggressive breast cancers with centrosome amplification [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-20.
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Kopecká, Marie. "Microtubules and Actin Cytoskeleton of Cryptococcus neoformans as Targets for Anticancer Agents to Potentiate a Novel Approach for New Antifungals." Chemotherapy 61, no. 3 (December 10, 2015): 117–21. http://dx.doi.org/10.1159/000437134.
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Background: We investigated the targeting of microtubules (MT) and F-actin cytoskeleton (AC) of the human pathogenic yeast Cryptococcus neoformans with agents for cancer therapy, in order to examine whether this yeast cytoskeleton could become a new antifungal target for the inhibition of cell division. Methods: Cells treated with 10 cytoskeleton inhibitors in yeast extract peptone dextrose medium were investigated by phase-contrast and fluorescence microscopy, and growth inhibition was estimated by cell counts using a Bürker chamber and measuring absorbance for 6 days. Results: Docetaxel, paclitaxel, vinblastine sulfate salt, cytochalasin D and chlorpropham [isopropyl N-(3-chlorophenyl) carbamate] did not inhibit proliferation. The MT inhibitors methyl benzimidazole-2-ylcarbamate (BCM), nocodazole, thiabendazole (TBZ) and vincristine (VINC) disrupted MT and inhibited mitoses, but anucleated buds emerged on cells that increased in size, vacuolated and seemed to die after 2 days. The response of the cells to the presence of the actin inhibitor latrunculin A (LA) included the disappearance of actin patches, actin cables and actin rings; this arrested budding and cell division. However, in 3-4 days, resistant budding cells appeared in all 5 inhibitors. Disruption of the MT and AC and inhibition of cell division and budding persisted only when the MT and AC inhibitors were combined, i.e. VINC + LA, BCM + LA or TBZ + LA. Conclusion: The MT and AC of C. neoformans are new antifungal targets for the persistent inhibition of cell division by combined F-actin and MT inhibitors.
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Nanni, Samuel Burke, Jonathan Pratt, David Beauchemin, Khadidja Haidara, and Borhane Annabi. "Impact of Concanavalin-A-Mediated Cytoskeleton Disruption on Low-Density Lipoprotein Receptor-Related Protein-1 Internalization and Cell Surface Expression in Glioblastomas." Biomarkers in Cancer 8 (January 2016): BIC.S38894. http://dx.doi.org/10.4137/bic.s38894.
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The low-density lipoprotein receptor-related protein 1 (LRP-1) is a multiligand endocytic receptor, which plays a pivotal role in controlling cytoskeleton dynamics during cancer cell migration. Its rapid endocytosis further allows efficient clearance of extracellular ligands. Concanavalin-A (ConA) is a lectin used to trigger in vitro physiological cellular processes, including cytokines secretion, nitric oxide production, and T-lymphocytes activation. Given that ConA exerts part of its effects through cytoskeleton remodeling, we questioned whether it affected LRP-1 expression, intracellular trafficking, and cell surface function in grade IV U87 glioblastoma cells. Using flow cytometry and confocal microscopy, we found that loss of the cell surface 600-kDa mature form of LRP-1 occurs upon ConA treatment. Consequently, internalization of the physiological α 2-macroglobulin and the synthetic angiopep-2 ligands of LRP-1 was also decreased. Silencing of known mediators of ConA, such as the membrane type-1 matrix metalloproteinase, and the Toll-like receptors (TLR)-2 and TLR-6 was unable to rescue ConA-mediated LRP-1 expression decrease, implying that the loss of LRP-1 was independent of cell surface relayed signaling. The ConA-mediated reduction in LRP-1 expression was emulated by the actin cytoskeleton-disrupting agent cytochalasin-D, but not by the microtubule inhibitor nocodazole, and required both lysosomal- and ubiquitin-proteasome system-mediated degradation. Our study implies that actin cytoskeleton integrity is required for proper LRP-1 cell surface functions and that impaired trafficking leads to specialized compartmentation and degradation. Our data also strengthen the biomarker role of cell surface LRP-1 functions in the vectorized transport of therapeutic angiopep bioconjugates into brain cancer cells.
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Haubold, Katja, Michael Rink, Brigitte Spath, Ali Amirkhosravi, John L. Francis, Barbara Eifrig, Carsten Bokemeyer, and Florian Langer. "Microparticle-Associated Tissue Factor: A Molecular Link Between Coagulation Activation, Inflammation and Disease Progression in Early-Stage Prostate Cancer?" Blood 112, no. 11 (November 16, 2008): 3813. http://dx.doi.org/10.1182/blood.v112.11.3813.3813.
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Abstract Activation of coagulation and inflammation is a characteristic finding in patients with advanced malignancies, including prostate cancer. Tissue factor (TF), a molecule involved in hemostasis, thrombosis and pro-inflammatory signaling pathways, is over-expressed on tumor cells and cells of the tumor microenvironment (i.e. endothelial cells, fibroblasts and tissue macrophages). Moreover, the enhanced release of TF into plasma in association with sub-cellular membrane vesicles, so-called plasma microparticles (MPs), has recently been associated with key events in molecular oncogenesis and cancer progression. In this study, we measured TF-specific procoagulant activity (PCA) of plasma MPs in 58 consecutive patients with clinically localized prostate cancer (mean age, 64±5 years) to explore its potential as a prognostic marker in this tumor entity. MPs were isolated from pre-operative plasma samples by sequential high-speed centrifugation for 1 h at 16,100 × g. TF-specific PCA of plasma MPs was quantified using a highly sensitive one-stage clotting assay in the presence and absence of inhibitory TF monoclonal antibody and calibration of clotting times against serial dilutions (1:10–1:105) of lipidated recombinant human full-length TF (rhTF1–263), showing a linear correlation in a log-log plot with R2>0.99. The lower detection limit of this assay for rhTF1–263 (33 kDa) was <5 pg/ml (<150 fM), and the intra- and inter-assay coefficients of variation were 7.3% and 5.4%, respectively. Total numbers of TF-positive MPs were measured by single-color flow cytometry using PE-conjugated TF monoclonal antibody (HTF-1) and microspheres for size calibration (1 μm) and sample flow standardization. TF antigen was quantified in plasma by ELISA. Calibrated automated thrombography (CAT) was used to monitor thrombin generation in platelet-free plasma samples over a 2-h period without the addition of exogenous TF or phospholipids. Intravascular coagulation activation was assessed by measuring plasma D-dimer. All assay systems were validated using MPs spontaneously shed from prostate cancer cell lines (PC-3, LNCaP and DU145) or from whole blood monocytes after challenge with endotoxin. Based on plasma fibrinogen and C-reactive protein levels, patients were stratified into those with (n=26) and those without (n=32) laboratory evidence of an acute-phase reaction (APR). Compared to healthy male controls (n=20), patients had significantly increased levels of both D-dimer (0.46±0.19 vs. 0.21±0.05 mg/l) and TF-specific PCA of plasma MPs (563±301 vs. 292±74 U/ml) (P<0.001). Among patients, laboratory evidence of an APR was associated with a significant increase in MP-associated TF PCA (699±351 vs. 452±196 U/ml) (P=0.001). Overall, we found a significant correlation between MP-associated TF PCA and plasma D-Dimer (P=0.015), suggesting that plasma MPs contributed to in-vivo coagulation activation in a TF-dependent manner. CAT also revealed significantly increased thrombin generation in patient compared to control plasmas, as indicated by a shortening in lag phase (25±4 vs. 29±5 min) and an increase in both peak thrombin generation (184±76 vs. 127±71 nM) and the endogenous thrombin potential, defined as the area under the thrombin generation curve (3576±509 vs. 2980±562 nM*min) (P<0.01). Importantly, TF-specific PCA of plasma MPs correlated neither with absolute numbers of TF-positive MPs nor with plasma TF antigen, suggesting that a substantial and variable fraction of the total plasma TF pool circulated as an inactive variant. Interestingly, systemic levels of IL-8, an inflammatory cytokine involved in TF/FVIIa-dependent, PAR-2-mediated pro-migratory signaling pathways in tumor cells and shown to be of biological relevance in advanced, hormone-refractory prostate cancer, were elevated in patients compared to controls (9±11 vs. 4±6 pg/ml) (P<0.01). In summary, our findings suggest that TF-specific PCA of plasma MPs contributes to intravascular coagulation activation in patients with early-stage prostate cancer and may represent an important molecular link between hypercoagulability, inflammation and disease progression. The above-described assay for the quantification of MP-associated TF PCA could thus be of prognostic value in the risk stratification of patients with localized prostate cancer with respect to thromboembolic complications and/or tumor recurrence.
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Robillard, Liliane, Kevin K. Lin, Andrea Loehr, Tanya Kwan, Rachel Dusek, Andrew D. Simmons, Thomas C. Harding, Brieuc Sautois, and Minh Nguyen. "Abstract 1260: Nonclinical evaluation of rucaparib in tumors with mutations in non-BRCA1/2 homologous recombination repair (HRR) genes." Cancer Research 82, no. 12_Supplement (June 15, 2022): 1260. http://dx.doi.org/10.1158/1538-7445.am2022-1260.
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Abstract Background: Rucaparib is a PARP inhibitor approved to treat ovarian cancer (OC) and metastatic castration-resistant prostate cancer (mCRPC). Deficiencies in HRR resulting from genetic or epigenetic alterations in BRCA1/2 sensitize tumors to rucaparib through synthetic lethality. Inactivation of other HRR genes may also confer sensitivity to PARP inhibition. Here, we evaluated rucaparib efficacy in preclinical models with genomic or epigenetic alterations in an HRR gene panel. Methods: BRCA1/2 and 14 additional genes were selected for their role in DNA repair and mutation frequency in OC and mCRPC. Small interfering RNA (siRNA) knockdowns were performed in OC (OVCAR-3, OAW28, SK-OV-3) and mCRPC (DU145, PC-3) cell lines to model their inactivation and assess impact on rucaparib sensitivity (IC50) in cell viability assays. Patient-derived xenografts (PDX) harboring deleterious alterations in BRCA1/2, BARD1, BRIP1, NBN, PALB2, RAD51B, RAD51C, or RAD51D, or BRCA1/RAD51C promoter hypermethylation were treated with up to 150 mg/kg rucaparib BID for 25-42 days and tumor growth was monitored. Mutational status was confirmed by next-generation sequencing. Results: In addition to BRCA1/2 knockdown, depletion of BARD1, PALB2, or RAD51 resulted in potent rucaparib activity in OC and mCRPC cells, where the rucaparib IC50 was reduced by >70% vs control in at least 4 of the 5 cell lines tested. Rucaparib also displayed greater potency in cells with siRNA-mediated decreased expression of FANCA, NBN, RAD51C, or RAD54L, where at least 1 cell line had >50% decrease in IC50. Rucaparib treatment in 19/33 HRR-deficient PDX models resulted in significant tumor growth inhibition (TGI). These included BRCA1/2, PALB2, NBN, RAD51B, RAD51C, or RAD51D-mutated and BRCA1/RAD51C-hypermethylated models. Efficacy was strongly associated with biallelic inactivation of BRCA1/2, PALB2, RAD51C, or RAD51D with 87% vs 29% mean TGI for monoallelic BRCA1/2 alterations (P<0.0001). In addition, a single PDX model each for the exploratory genes NBN and RAD51B had 101% and 80% TGI, respectively. No significant difference was observed between PDX models with BRCA1/2 vs non-BRCA1/2 (PALB2, NBN, RAD51B, RAD51C, or RAD51D) biallelic alterations (98% vs 80% mean TGI, respectively; P=0.21). In support of these nonclinical findings, we highlight a patient with RAD51B-mutant mCRPC treated with rucaparib in the TRITON2 trial (NCT02952534) who had confirmed radiographic and PSA responses. Conclusions: Cell lines and PDX models with biallelic alterations in HRR genes other than BRCA1/2, including RAD51B, RAD51C, RAD51D, PALB2, and NBN, are comparably sensitive to rucaparib. Rucaparib is being evaluated in the LODESTAR trial (NCT04171700) in patients with tumors associated with deleterious alterations in BRCA1/2, PALB2, RAD51C, and RAD51D (cohort A) and BARD1, BRIP1, FANCA, NBN, RAD51, and RAD51B (exploratory cohort B). Citation Format: Liliane Robillard, Kevin K. Lin, Andrea Loehr, Tanya Kwan, Rachel Dusek, Andrew D. Simmons, Thomas C. Harding, Brieuc Sautois, Minh Nguyen. Nonclinical evaluation of rucaparib in tumors with mutations in non-BRCA1/2 homologous recombination repair (HRR) genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1260.
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Ravera, Mauro, Elisabetta Gabano, Elena Perin, Beatrice Rangone, Diego Bonzani, and Domenico Osella. "Can the Self-Assembling of Dicarboxylate Pt(IV) Prodrugs Influence Their Cell Uptake?" Bioinorganic Chemistry and Applications 2021 (June 19, 2021): 1–8. http://dx.doi.org/10.1155/2021/9489926.
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The possibility of spontaneous self-assembly of dicarboxylato Pt(IV) prodrugs and the consequences on their uptake in cancer cells have been evaluated in different aqueous solutions. Four Pt(IV) complexes, namely, (OC-6-33)-diacetatodiamminedichloridoplatinum(IV), Ace, (OC-6-33)-diamminedibutanoatodichloridoplatinum(IV), But, (OC-6-33)-diamminedichloridodihexanoatoplatinum(IV), Hex, and (OC-6-33)-diamminedichloridodioctanoatoplatinum(IV), Oct, have been dispersed in i) milliQ water, ii) phosphate buffered saline, and iii) complete cell culture media (RPMI 1640 or DMEM) containing fetal bovine serum (FBS). The samples have been analyzed by dynamic light scattering (DLS) to measure the size and distribution of the nanoparticles possibly present. The zeta potential offered an indication of the stability of the resulting aggregates. In the case of the most lipophilic compounds of the series, namely, Oct and to a lesser extent Hex, the formation of nanosized aggregates has been observed, in particular at the highest concentration tested (10 μM). The cell culture media had the effect to disaggregate these nanoparticles, mainly by virtue of their albumin content, able to interact with the organic chains via noncovalent (hydrophobic) interactions. For Oct, at the highest concentration employed for the uptake tests (10 μM), the combination between passive diffusion and endocytosis of the self-assembled nanoparticles makes the cellular uptake higher than in the presence of passive diffusion only. During the study of cellular uptake on A2780 ovarian cancer cells pretreated with cytochalasin D, a statistically significant inhibition of endocytosis was observed for Oct. In these experimental conditions, the relationship between uptake and lipophilicity becomes almost linear instead of exponential. Since Oct anticancer prodrug is active at nanomolar concentrations, where the aggregation in culture media is almost abolished, this phenomenon should not significantly impact its antiproliferative activity.
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Cairo, M. S., C. Mallett, C. VandeVen, P. Kempert, G. A. Bennetts, and J. Katz. "Impaired in vitro polymorphonuclear function secondary to the chemotherapeutic effects of vincristine, adriamycin, cyclophosphamide, and actinomycin D." Journal of Clinical Oncology 4, no. 5 (May 1986): 798–804. http://dx.doi.org/10.1200/jco.1986.4.5.798.
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The present study investigated the in vitro effect of four different chemotherapeutic agents, namely, cyclophosphamide (CTX), vincristine (VCR), Adriamycin (Adria Laboratories, Columbus, Ohio) (ADR), and actinomycin D (ACT-D) on human polymorphonuclear leukocyte (PMN) function. Human PMNs suspended in phosphate-buffered saline (PBS) at 1 X 10(7) cells/mL were incubated with increasing concentrations of CTX (0, 10(-5), 10(-4), 10(-3) mol/L) or VCR (0, 10(-7), 10(-6), 10(-5), 10(-4) mol/L), ADR (0, 10(-6), 10(-5), 10(-4), 10(-3) mol/L), or ACT-D (0, 5 X 10(-8), 1 X 10(-7), 5 X 10(-7), and 10(-6) mol/L). The cells were then tested for bacterial killing against Staphylococcus aureus, chemotaxis activity stimulated by Escherichia coli endotoxin, N-formyl-methionyl-leucyl-phenylalanine (FMLP)-stimulated aggregation, and cytochalasin B (Cyto B)/FMLP-stimulated superoxide production and enzyme degranulation. High concentration of CTX, an alkylating agent, showed a significant depression of PMN superoxide production, (124 +/- 13 v 161 +/- 15 nmol/10(7) cells, 5 minutes, P less than or equal to .025). ADR, an intercalating agent and membrane inhibitor, showed a significant depression of PMN degranulation and lysozyme release at 10(-4) and 10(-3) mol/L (15.3% +/- 1.7% v 24% +/- 7%, P less than .01; and 15.0% +/- 2.5% v 24% +/- 7%, P less than or equal to .025). VCR, a microtubule inhibitor, showed a significant depression of PMN aggregation at 10(-6), 10(-5), and 10(-4) mol/L (P less than .05), lysozyme release at 10(-4) mol/L (P less than .004), and beta-glucuronidase release at 10(-4) mol/L (P less than .004). In addition, chemotaxis was inhibited by VCR in a dose-dependent manner at all concentrations (10(-7) mol/L, P less than .02; 10(-6) mol/L, P less than .007; 10(-5) mol/L, P less than .006, and 10(-4) mol/L, P less than .003). ACT-D showed no significant effect on the PMN functions tested. These studies conclude that chemotherapeutic agents have modulating in vitro effects on PMN function. Further in vivo studies are therefore needed to assess PMN abnormalities in patients receiving cancer chemotherapy to determine their role in infectious complications.
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Kim, Sujeong, Hye-Young Kim, Seungmin Lee, Sung Woo Kim, Seonghyang Sohn, Kyongmin Kim, and Hyeseong Cho. "Hepatitis B Virus X Protein Induces Perinuclear Mitochondrial Clustering in Microtubule- and Dynein-Dependent Manners." Journal of Virology 81, no. 4 (December 6, 2006): 1714–26. http://dx.doi.org/10.1128/jvi.01863-06.
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ABSTRACT The hepatitis B virus (HBV) X protein (HBx) is thought to play a key role in HBV replication and the development of liver cancer. It became apparent that HBx induces mitochondrial clustering at the nuclear periphery, but the molecular basis for mitochondrial clustering is not understood. Since mitochondria move along the cytoskeleton as a cargo of motor proteins, we hypothesized that mitochondrial clustering induced by HBx occurs by an altered intracellular motility. Here, we demonstrated that the treatment of HBx-expressing cells with a microtubule-disrupting drug (nocodazole) abrogated mitochondrial clustering, while the removal of nocodazole restored clustering within 30 to 60 min, indicating that mitochondrial transport is occurring in a microtubule-dependent manner. The addition of a cytochalasin D-disrupting actin filament, however, did not measurably affect mitochondrial clustering. Mitochondrial clustering was further studied by observations of HBV-related hepatoma cells and HBV-replicating cells. Importantly, the abrogation of the dynein activity in HBx-expressing cells by microinjection of a neutralizing anti-dynein intermediate-chain antibody, dynamitin overexpression, or the addition of a dynein ATPase inhibitor significantly suppressed the mitochondrial clustering. In addition, HBx induced the activation of the p38 mitogen-activated protein kinase (MAPK) and inhibition of the p38 kinase activity by SB203580-attenuated HBx-induced mitochondrial clustering. Taken together, HBx activation of the p38 MAPK contributed to the increase in the microtubule-dependent dynein activity. The data suggest that HBx plays a novel regulatory role in subcellular transport systems, perhaps facilitating the process of maturation and/or assembly of progeny particles during HBV replication. Furthermore, mitochondrion aggregation induced by HBx may represent a cellular process that underlies disease progression during chronic viral infection.
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Mordue, Kathryn E., Timothy J. Satchwell, and Ashley M. Toye. "CD47 Is Dependent on the Actin Cytoskeleton for Its Membrane Stability Prior to Protein 4.2 Expression during Early Erythroblast Differentiation." Blood 124, no. 21 (December 6, 2014): 2665. http://dx.doi.org/10.1182/blood.v124.21.2665.2665.
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Abstract CD47 is a ubiquitously expressed ‘Marker of Self’ that protects cells from phagocytosis, through recognition by SIRPα on macrophages (Oldenborg et al Science 2000). CD47 was originally isolated on ovarian tumour cells (Poels et al J Natl Cancer Inst 1986) and has subsequently been detected on leukemic stem cells, where increased CD47 levels ensure immune evasion (Jaiswal et al Cell 2009). CD47 is also a ‘Marker of Self’ on red cells, but is reduced at the cell surface in certain patients with Hereditary Spherocytosis. In red cells, ~60% of CD47 is connected to the cytoskeleton (Dahl et al Blood 2004). Cytoskeletal connectivity of CD47 in the red cell membrane is dependent on the band 3 complex associated protein 4.2, demonstrated by an ~80% reduction in CD47 levels in protein 4.2 null red cells (Mouro-Chanteloup et al Blood 2003). Previous work (van den Akker et al Haematologica 2009) established that CD47 becomes dependent on protein 4.2 at the basophilic erythroblast stage (48 hours post-differentiation), but it is unknown what interactions support CD47 membrane stability prior to protein 4.2 expression during expansion and early erythroid differentiation. CD47 mRNA is alternatively spliced giving rise to four potential isoforms. The most abundant isoforms are form 2, expressed in all bone-marrow derived cells, and form 4 (and form 3), found predominantly in neural tissues (Reinhold et al J Cell Sci 1995). CD47 isoform 2 is the only form expressed on mature red cells, but we hypothesized that expression of other CD47 isoforms with different trafficking or binding characteristics could explain the independence of CD47 prior to band 3 complex assembly. Using specific polyclonal antibodies to multiple CD47 isoforms, we demonstrate that isoform 2 is expressed prior to and throughout in vitroerythroid differentiation. CD47 isoforms 3 and 4 were detected by western blotting until the late polychromatic erythroblast stage (96 hours post-differentiation), but only CD47 isoform 2 was detected at the cell surface. Therefore, we next hypothesised that CD47 must interact with another protein or exhibit different trafficking characteristics to maintain its membrane stability early during terminal differentiation. To identify a candidate protein or associated protein complex, CD47 was immunoprecipitated from expanding erythroblasts (Exp), proerythroblasts (T0), and basophilic erythroblasts (T48), and analysed via Nano-LC mass spectroscopy. In Exp and T0 erythroblasts, CD47 pulled down actin and multiple actin-associated proteins. These interactions were not observed in T48 erythroblasts, corresponding to the time during terminal differentiation when CD47 is dependent on protein 4.2. To confirm a dependence on actin for CD47 membrane stability, well-characterised drugs that disrupt actin dynamics were employed. CD47 expression at the cell membrane, as judged by flow cytometry, was markedly reduced within 30 minutes using actin stabilising drugs (Cytochalasin D (5µM): Exp 13.7±5.4% versus T48 0.5±5.7%; Latrunculin A (1µM): Exp 18.9±3.5% versus T48 9.9±5.9%, of the DMSO control), and destabilising drug (Jasplakinolide (1µM): Exp 24.2±1.9% versus T48 -6±1.8%, of the DMSO control), until the basophilic erythroblast stage. In K562 cells, which predominantly express CD47 isoforms 3 and 4, a larger actin dependency is observed (37±14.9% reduction in CD47 with Cytochalasin D versus a DMSO control) suggesting that dependence on actin by CD47 is not isoform specific. In summary, we propose a role for actin in the maintenance of CD47 at the cell surface before and during early erythroid differentiation. We have shown that CD47 isoform 2 is the major isoform present at the cell surface and that this version is initially dependent on the actin cytoskeleton for its membrane stability by an as yet undetermined mechanism. Once band 3 complex assembly initiates at the surface of the basophilic erythroblast (48 hours post-differentiation), CD47 is selectively incorporated via an interaction with protein 4.2, and is preferentially retained whilst the actin cytoskeleton remodels. In addition to explaining how CD47 expression is maintained during the formation of the red cell membrane, this work raises the possibility that the dependence on actin by CD47 for its membrane stability in hematopoietic stem cells may be exploited for the development of therapeutics that render the leukemic cells susceptible to phagocytosis. Disclosures No relevant conflicts of interest to declare.
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Alduaij, Waleed, Sandeep Potluri, Andrei Ivanov, Jamie Honeychurch, Stephen A. Beers, Claude Chan, Kazayuki Shimada, Martin J. Glennie, Mark S. Cragg, and Tim Illidge. "New-Generation Anti-CD20 Monoclonal Antibody (GA101) Evokes Homotypic Adhesion and Actin-Dependent, Lysosome-Mediated Cell Death in B-Cell Lymphoma." Blood 114, no. 22 (November 20, 2009): 725. http://dx.doi.org/10.1182/blood.v114.22.725.725.
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Abstract Abstract 725 The addition of the anti-CD20 monoclonal antibody (mAb) rituximab to chemotherapy has substantially improved the clinical outcome of patients with a wide range of B-cell malignancies. Despite this success, many patients are not cured by standard approaches and there is intense investigation into the development of new-generation anti-CD20 mAbs with further improved therapeutic efficacy. Although Fc-FcgR interactions appear to underlie much of the therapeutic success with Rituximab, certain Type II anti-CD20 mAbs, can directly induce programmed cell death (PCD), whereas rituximab-like Type I anti-CD20 mAbs do not (Chan et al. Cancer Res 63: 5480-5489, 2003). We have demonstrated that Type II mAbs are more effective at B-cell depletion in syngeneic human CD20 transgenic mice (Beers et al. Blood 112: 4170-4177, 2008). Recently, we elucidated the mechanism underlying PCD induced by the Type II anti-CD20 mAb Tositumomab, demonstrating a novel non-apoptotic mode of cell death, defined by homotypic adhesion, peripheral relocalization of actin and lysosomal activity (Ivanov et al. J Clin Invest, doi: 10.1172/JCI37884, 2009). Here we confirm that the humanized anti-CD20 mAb GA101 and derivatives harboring non-glycomodified human IgG1 or mouse IgG2a Fc regions are bone fide Type II reagents, lacking the ability to translocate CD20 into lipid rafts or initiate calcium flux. Furthermore, GA101 initiates extensive non-apoptotic cell death in a range of B-lymphoma cell lines in contrast to rituximab (e.g. in Raji cells 48 ± 1.8% versus 13 ± 0.2%, p<0.001 by Student's t-test) quantified using the Annexin V/propidium iodide cell death assay. Inhibitors of actin polymerization (latrunculin B and cytochalasin D) inhibited cell death elicited by GA101 from 45 ± 1.5% to 15 ± 3.1% (p<0.01). The importance of cell to cell contact in this form of antibody induced cell death was confirmed by the addition of low-melting point agarose which physically blocked cell to cell contact and markedly attenuated cell death induced by GA101. The role of lysosomal activity in GA101-induced PCD was assessed using an inhibitor of the lysosomal cysteine protease cathepsin B, which significantly inhibited cell death induced by GA101 from 53 ± 4.3% to 18 ± 1.9%, (p<0.001). To confirm that this mode of death is non-apoptotic, we demonstrated that GA101-induced PCD occurred independently of BCL-2 over-expression and caspase activation. Complement-dependent cytotoxicity (CDC) assays using human serum as a source of complement reveal that GA101 has significantly weaker CDC activity than rituximab, consistent with our previous work on Type II anti-CD20 mAbs (Cragg et al Blood 101: 2738-2743, 2003). Taken together, these findings demonstrate that GA101 is the first humanized anti-CD20 mAb with Type II properties, potently eliciting a novel mode of cell death in B-cell malignancies, which potentially can lead to improved B-cell depletion over rituximab. Furthermore, we are currently investigating the relative ability of GA101 and rituximab to delete B cells in vivo using directly comparable versions of these mAb with human or mouse Fc regions in human CD20 transgenic mice and will present these data. Disclosures: No relevant conflicts of interest to declare.
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Korzuch, Julia, Monika Rak, Katarzyna Balin, Maciej Zubko, Olga Głowacka, Mateusz Dulski, Robert Musioł, Zbigniew Madeja, and Maciej Serda. "Towards water-soluble [60]fullerenes for the delivery of siRNA in a prostate cancer model." Scientific Reports 11, no. 1 (May 19, 2021). http://dx.doi.org/10.1038/s41598-021-89943-5.
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AbstractThis paper presents two water-soluble fullerene nanomaterials (HexakisaminoC60 and monoglucosamineC60, which is called here JK39) that were developed and synthesized as non-viral siRNA transfection nanosystems. The developed two-step Bingel–Hirsch reaction enables the chemical modification of the fullerene scaffold with the desired bioactive fragments such as d-glucosamine while keeping the crucial positive charged ethylenediamine based malonate. The ESI–MS and 13C-NMR analyses of JK39 confirmed its high Th symmetry, while X-ray photoelectron spectroscopy revealed the presence of nitrogen and oxygen-containing C–O or C–N bonds. The efficiency of both fullerenes as siRNA vehicles was tested in vitro using the prostate cancer cell line DU145 expressing the GFP protein. The HexakisaminoC60 fullerene was an efficient siRNA transfection agent, and decreased the GFP fluorescence signal significantly in the DU145 cells. Surprisingly, the glycofullerene JK39 was inactive in the transfection experiments, probably due to its high zeta potential and the formation of an extremely stable complex with siRNA.
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Beebe, Maureen, Rami Najjar, Darren Chan, Chappell Madhani, Manal Elfakhani, Sophie Yount, Xiangming Ji, Rafaela Feresin, Desiree Wanders, and Huanbiao Mo. "Synergistic Impact of Xanthorrhizol and d-δ-tocotrienol on the Proliferation of Murine B16 Melanoma Cells and Human DU145 Prostate Carcinoma Cells (P06-042-19)." Current Developments in Nutrition 3, Supplement_1 (June 1, 2019). http://dx.doi.org/10.1093/cdn/nzz031.p06-042-19.
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Abstract Objectives Xanthorrhizol, a sesquiterpene, and d-δ-tocotrienol, a vitamin E molecule, each suppresses the proliferation of a number of tumor cells. This study aims to examine the potentially synergistic effect of xanthorrhizol and d-δ-tocotrienol in tumor cells. Methods Murine B16 melanoma and human DU145 prostate carcinoma cells were incubated for 48 h (B16) or 72 h (DU145) with xanthorrhizol or d-δ-tocotrienol before cell populations were determined by CellTiter 96â Aqueous One Solution. Cells incubated with the agents for 24 hours were stained with propidium iodide and analyzed for cell cycle using flow cytometry and MultiCycle AV. Isobologram and combination index (CI) were used to demonstrate their synergistic anti-proliferative impacts. Results Xanthorrhizol (0–200 µmol/L) and d-δ-tocotrienol (0–40 µmol/L) each elicited a concentration-dependent suppression of the proliferation of B16 cells. A blend of 16.25 µmol/L xanthorrhizol and 10 µmol/L d-δ-tocotrienol achieved 69% (P < 0.05) growth suppression of B16 cells, exceeding the sum of individual effects. B16 cells incubated with 5 and 10 µmol/L d-δ-tocotrienol for 24-h had a concentration-dependent increase in the percentage of cells in the G1 phase with a concomitant decrease in the percentage of cells in the S phase. The G1/S ratio, an indicator of cell cycle arrest at the G1 phase, increased from 1.73 ± 0.05 (Control) to 2.01 ± 0.10 (5 µmol/L) and 2.73 ± 0.05 (10 µmol/L). A parallel pattern of concentration-dependent increase in the G1/S ratio was induced by xanthorrhizol at concentrations equivalent to 25% (16.25 µmol/L) and 50% (32.5 µmol/L) of its IC50 value. A blend of 5 µmol/L d-δ-tocotrienol and 16.25 µmol/L xanthorrhizol, each at no-effect concentrations, significantly increased the percentage of B16 cells in the G1 phase to 62.6 ± 0.6%. Isobologram and CI confirmed the synergistic effect of xanthorrhizol (50 and 100 μmol/L) and d-δ-tocotrienol (10 and 20 μmol/L) on the proliferation of DU145 cells. Conclusions Xanthorrhizol and d-δ-tocotrienol synergistically suppress tumor cell proliferation by inducing G1 arrest and may have potential in cancer prevention and therapy. Funding Sources American River Nutrition, Inc. and the University Assistantship Program and the Department of Nutrition of Georgia State University.
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Chan, Darren, Maureen L. Meister, Chappell R. Madhani, Manal Elfakhani, Sophie T. Yount, Xiangming Ji, Rafaela G. Feresin, Desiree Wanders, and Huanbiao Mo. "Synergistic Impact of Xanthorrhizol and d-δ-Tocotrienol on the Proliferation of Murine B16 Melanoma Cells and Human DU145 Prostate Carcinoma Cells." Nutrition and Cancer, August 18, 2020, 1–12. http://dx.doi.org/10.1080/01635581.2020.1807573.
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Chen, Wenbin, Shengren Cen, Xumin Zhou, Taowei Yang, Kaihui Wu, Libin Zou, Junqi Luo, Chuanyin Li, Daojun Lv, and Xiangming Mao. "Circular RNA CircNOLC1, Upregulated by NF-KappaB, Promotes the Progression of Prostate Cancer via miR-647/PAQR4 Axis." Frontiers in Cell and Developmental Biology 8 (January 8, 2021). http://dx.doi.org/10.3389/fcell.2020.624764.
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BackgroundCircRNAs recently have shown critical roles in tumor biology. However, their roles in prostate cancer (PCa) remains largely unclear.MethodsCircRNA microarrays were performed in immortal prostate cell line RWPE1 and PCa cell lines as DU145, PC3, LNCaP, C4-2, and 22RV1. Combined with upregulated circRNAs in PCa tissues, circNOLC1 expression was validated in PCa cells and tissues via qRT-PCR and FISH. Sanger sequencing, actinomycin D, gDNA, and cDNA, RNase R assays were used to assess the circular characteristics of circNOLC1. CCK-8, colony formation, transwell migration assays, and mice xenograft models were conducted to evaluate the functions of PCa cells after circNOLC1 knockdown and overexpression. RNA pulldown, luciferase reporter assay, FISH (fluorescence in situ hybridization), and CHIP were utilized to illustrate the further mechanisms of circNOLC1.ResultsOur research indicated that circNOLC1 was overexpressed in PCa cells and tissues, and circNOLC1 was more stable than linear NOLC1 mRNA. CircNOLC1 promoted PCa cells proliferation and migration in vitro and vivo. Additionally, we found that circNOLC1 could upregulate PAQR4 expression by sponging miR-647, leading to the activation of PI3K/Akt pathway. Moreover, NF-kappaB was identified to bind to the NOLC1 promoter sites and upregulated both NOLC1 and circNOLC1 expression.ConclusionCircNOLC1, elevated by transcription factor NF-kappaB, promotes PCa progression via a miR-647/PAQR4 axis, and circNOLC1 is a potential biomarker and target for PCa treatment.
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Chen, Wenbin, Shengren Cen, Xumin Zhou, Taowei Yang, Kaihui Wu, Libin Zou, Junqi Luo, Chuanyin Li, Daojun Lv, and Xiangming Mao. "Circular RNA CircNOLC1, Upregulated by NF-KappaB, Promotes the Progression of Prostate Cancer via miR-647/PAQR4 Axis." Frontiers in Cell and Developmental Biology 8 (January 8, 2021). http://dx.doi.org/10.3389/fcell.2020.624764.
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BackgroundCircRNAs recently have shown critical roles in tumor biology. However, their roles in prostate cancer (PCa) remains largely unclear.MethodsCircRNA microarrays were performed in immortal prostate cell line RWPE1 and PCa cell lines as DU145, PC3, LNCaP, C4-2, and 22RV1. Combined with upregulated circRNAs in PCa tissues, circNOLC1 expression was validated in PCa cells and tissues via qRT-PCR and FISH. Sanger sequencing, actinomycin D, gDNA, and cDNA, RNase R assays were used to assess the circular characteristics of circNOLC1. CCK-8, colony formation, transwell migration assays, and mice xenograft models were conducted to evaluate the functions of PCa cells after circNOLC1 knockdown and overexpression. RNA pulldown, luciferase reporter assay, FISH (fluorescence in situ hybridization), and CHIP were utilized to illustrate the further mechanisms of circNOLC1.ResultsOur research indicated that circNOLC1 was overexpressed in PCa cells and tissues, and circNOLC1 was more stable than linear NOLC1 mRNA. CircNOLC1 promoted PCa cells proliferation and migration in vitro and vivo. Additionally, we found that circNOLC1 could upregulate PAQR4 expression by sponging miR-647, leading to the activation of PI3K/Akt pathway. Moreover, NF-kappaB was identified to bind to the NOLC1 promoter sites and upregulated both NOLC1 and circNOLC1 expression.ConclusionCircNOLC1, elevated by transcription factor NF-kappaB, promotes PCa progression via a miR-647/PAQR4 axis, and circNOLC1 is a potential biomarker and target for PCa treatment.
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Ali, Roshia, Hilal Ahmad Mir, Rabia Hamid, Basharat Bhat, Riaz A. Shah, Firdous A. Khanday, and Sahar Saleem Bhat. "Actin Modulation Regulates the Alpha-1-Syntrophin/p66Shc Mediated Redox Signaling Contributing to the RhoA GTPase Protein Activation in Breast Cancer Cells." Frontiers in Oncology 12 (February 21, 2022). http://dx.doi.org/10.3389/fonc.2022.841303.
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SNTA1 signaling axis plays an essential role in cytoskeletal organization and is also implicated in breast cancers. In this study, we aimed to investigate the involvement of actin cytoskeleton in the propagation of SNTA1/p66shc mediated pro-metastatic cascade in breast cancer cells.The effect of actin filament depolymerization on SNTA1-p66Shc interaction and the trimeric complex formation was analyzed using co-immunoprecipitation assays. Immunofluorescence and RhoA activation assays were used to show the involvement of SNTA1-p66Shc interaction in RhoA activation and F-actin organization. Cellular proliferation and ROS levels were assessed using MTT assay and Amplex red catalase assay. The migratory potential was evaluated using transwell migration assay and wound healing assay.We found that cytochalasin D mediated actin depolymerization significantly declines endogenous interaction between SNTA1 and p66Shc protein in MDA-MB-231 cells. Results indicate that SNTA1 and p66Shc interact with RhoA protein under physiological conditions. The ROS generation and RhoA activation were substantially enhanced in cells overexpressing SNTA1 and p66Shc, promoting proliferation and migration in these cells. In addition, we found that loss of SNTA1-p66Shc interaction impaired actin organization, proliferation, and migration in breast cancer cells. Our results demonstrate a novel reciprocal regulatory mechanism between actin modulation and SNTA1/p66Shc/RhoA signaling cascade in human metastatic breast cancer cells.
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Faroque, Habibah, Yi Siang Lau, Chee Xian Yong, Raha Abdul Rahim, Suet Lin Chia, and Sarah Othman. "Bactofection of SW620 cell by Lactococcus lactis M4." Asia Pacific Journal of Molecular Biology and Biotechnology, November 15, 2018, 29–41. http://dx.doi.org/10.35118/apjmbb.2018.026.1.04.
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In this study, a local dairy isolate, L. lactis M4 was investigated for its ability to be developed as a live delivery vector to deliver plasmid DNA into human colon cancer cell line, SW620. L. lactis M4 strain was found to adhere to and internalize SW620 cells optimally after 2 hours of infection period at a multiplicity of infection 250:1, bacteria per cancer cell. Bacteria also managed to survive intracellularly for 7 hours. Entry into SW620 cells was inhibited by Cytochalasin D and Vinblastine, indicating that cell uptake was dependent on microfilament and microtubule stability. Bactofection of SW620 cells by L. lactis M4 was demonstrated through the expression of fluorescent proteins from a novel dual-expression plasmid, pHSR. L. lactis M4 was able to express red fluorescent protein intracellularly of SW620 cells, which were subsequently observed to express green fluorescent protein at 3 hours post-invasion. The expression of fluorescent proteins from pHSR resulted from the bactofection of SW620 cells by L. lactis M4 has proven that this strain can be developed as a vector to deliver plasmid DNA into the cancer cell.
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Buhrmann, Constanze, Aranka Brockmueller, Choudhary Harsha, Ajaikumar B. Kunnumakkara, Peter Kubatka, Bharat B. Aggarwal, and Mehdi Shakibaei. "Evidence That Tumor Microenvironment Initiates Epithelial-To-Mesenchymal Transition and Calebin A can Suppress it in Colorectal Cancer Cells." Frontiers in Pharmacology 12 (July 2, 2021). http://dx.doi.org/10.3389/fphar.2021.699842.
Full textAbstract:
Background: Tumor microenvironment (TME) has a pivotal impact on tumor progression, and epithelial-mesenchymal transition (EMT) is an extremely crucial initial event in the metastatic process in colorectal cancer (CRC) that is not yet fully understood. Calebin A (an ingredient in Curcuma longa) has been shown to repress CRC tumor growth. However, whether Calebin A is able to abrogate TME-induced EMT in CRC was investigated based on the underlying pathways.Methods: CRC cell lines (HCT116, RKO) were exposed with Calebin A and/or a FAK inhibitor, cytochalasin D (CD) to investigate the action of Calebin A in TME-induced EMT-related tumor progression.Results: TME induced viability, proliferation, and increased invasiveness in 3D-alginate CRC cultures. In addition, TME stimulated stabilization of the master EMT-related transcription factor (Slug), which was accompanied by changes in the expression patterns of EMT-associated biomarkers. Moreover, TME resulted in stimulation of NF-κB, TGF-β1, and FAK signaling pathways. However, these effects were dramatically reduced by Calebin A, comparable to FAK inhibitor or CD. Finally, TME induced a functional association between NF-κB and Slug, suggesting that a synergistic interaction between the two transcription factors is required for initiation of EMT and tumor cell invasion, whereas Calebin A strongly inhibited this binding and subsequent CRC cell migration.Conclusion: We propose for the first time that Calebin A modulates TME-induced EMT in CRC cells, at least partially through the NF-κB/Slug axis, TGF-β1, and FAK signaling. Thus, Calebin A appears to be a potential agent for the prevention and management of CRC.
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Sun, Xiaowei, Yujie Liu, Shuheng Zhou, Li Wang, Jinzi Wei, Rui Hua, Zhongyang Shen, and Sei Yoshida. "Circular dorsal ruffles disturb the growth factor-induced PI3K-AKT pathway in hepatocellular carcinoma Hep3B cells." Cell Communication and Signaling 20, no. 1 (July 7, 2022). http://dx.doi.org/10.1186/s12964-022-00911-6.
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Abstract Background Circular dorsal ruffles (CDRs) are rounded membrane ruffles induced on the dorsal surfaces of cells stimulated by growth factors (GF). They can serve as signal platforms to activate AKT protein kinase. After GF stimulation, phosphatidylinositol 3-kinase (PI3K) generates phosphatidylinositol (3,4,5)-triphosphate (PIP3) in the plasma membrane. PIP3 accumulates inside CDRs, recruits AKT into the structures, and phosphorylates them (pAKT). Given the importance of the PI3K-AKT pathway in GF signaling, CDRs are likely involved in cell growth. Interestingly, some cancer cell lines express CDRs. We hypothesized that CDRs contribute to carcinogenesis by modulating the AKT pathway. In the present study, we identified CDR-expressing cancer cell lines and investigated their cellular functions. Methods CDR formation was examined in six cancer cell lines in response to epidermal growth factor (EGF) and insulin. The morphology of the CDRs was characterized, and the related signaling molecules were observed using confocal and scanning electron microscopy. The role of CDRs in the AKT pathway was studied using biochemical analysis. The actin inhibitor cytochalasin D (Cyto D) and the PI3K inhibitor TGX221 were used to block CDRs. Results GF treatment induced CDRs in the hepatocellular carcinoma (HCC) Hep3B cell line, but not in others, including HCC cell lines HepG2 and Huh7, and the LO2 hepatocyte cell line. Confocal microscopy and western blot analysis showed that the PI3K-PIP3-AKT pathway was activated at the CDRs and that receptor proteins were recruited to the structures. Cyto D and TGX221 completely blocked CDRs and partially attenuated GF-induced pAKT. These results indicate that CDRs regulate the receptor-mediated PI3K-AKT pathway in Hep3B cells and the existence of CDR-independent pAKT mechanisms. Conclusions Our results showed that CDRs modulate the AKT pathway in Hep3B cells. Since CDRs were not observed in other HCC and hepatocyte cell lines, we propose that CDRs in Hep3B would determine the carcinoma characteristic of the cell by aberrantly triggering the AKT pathway. Signaling molecules involved in CDR formation are promising therapeutic targets for some types of HCC.
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Keurhorst, Dennis, Ivan Liashkovich, Fabian Frontzek, Svenja Nitzlaff, Verena Hofschröer, Rita Dreier, and Christian Stock. "MMP3 activity rather than cortical stiffness determines NHE1-dependent invasiveness of melanoma cells." Cancer Cell International 19, no. 1 (November 9, 2019). http://dx.doi.org/10.1186/s12935-019-1015-7.
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Abstract Background Both cell adhesion and matrix metalloproteinase (MMP) activity depend on pH at the cell surface. By regulating extracellular juxtamembrane pH, the Na+/H+ exchanger NHE1 plays a significant part in human melanoma (MV3) cell migration and invasion. Because NHE1, besides its pH-regulatory transport function, also serves as a structural element tying the cortical actin cytoskeleton to the plasma membrane, we investigated whether NHE1 affects cortical stiffness of MV3 cells, and how this makes an impact on their invasiveness. Methods NHE1 overexpressing MV3 cells were compared to the corresponding mock-transfected control cells. NHE1 expression was verified by Western blotting, cariporide (HOE642) was used to inhibit NHE1 activity, cell stiffness was determined by atomic force microscopy, and F-actin was visualized by phalloidin-staining. Migration on, and invasion of, native and glutaraldehyde-fixed collagen I substrates were analyzed using time-lapse video microscopy and Boyden-chamber assays, respectively. MMP secretion and activity were detected by Western blot and zymography, respectively. MMP activity was inhibited with NNGH. Results The cortical, but not the bulk stiffness, was significantly higher in NHE1 overexpressing cells. This increase in cortical stiffness was accompanied by a reorganization of the cortical cytoskeleton, i.e. a condensation of F-actin underneath and along the plasma membrane. However, it was not affected by NHE1 inhibition. Nevertheless, actin dynamics is required for cell invasion as demonstrated with the application of cytochalasin D. NHE1 overexpression was associated with an elevated MMP3 secretion and an increase in the invasion of a native matrix. This increase in invasiveness could be antagonized by the MMP inhibitor NNGH. Transmigration through a glutaraldehyde-fixed, indigestible substrate was not affected by NHE1 overexpression. Conclusion NHE1, as a structural element and independently of its transport activity, contributes to the organization of the cortical F-actin meshwork and thus impacts cortical stiffness. Since NHE1 overexpression stimulates MMP3 secretion but does not change transmigration through a fixed substrate, MV3 cell invasion of a native substrate depends on MMP activity rather than on a modifiable cortical stiffness.
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Shen, Jing, Ji-Min Wu, Guo-Min Hu, Ming-Zhe Li, Wen-Wen Cong, Ye-Nan Feng, Shuai-Xing Wang, et al. "Membrane nanotubes facilitate the propagation of inflammatory injury in the heart upon overactivation of the β-adrenergic receptor." Cell Death & Disease 11, no. 11 (November 2020). http://dx.doi.org/10.1038/s41419-020-03157-7.
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Abstract Acute sympathetic stress quickly induces cardiac inflammation and injury, suggesting that pathogenic signals rapidly spread among cardiac cells and that cell-to-cell communication may play an important role in the subsequent cardiac injury. However, the underlying mechanism of this response is unknown. Our previous study demonstrated that acute β-adrenergic receptor (β-AR) signaling activates inflammasomes in the heart, which triggers the inflammatory cascade. In the present study, β-AR overactivation induced inflammasome activation in both the cardiomyocytes and cardiac fibroblasts (CFs) of mice hearts following a subcutaneous injection of isoproterenol (ISO, 5 mg/kg body weight), a selective agonist of β-AR. In isolated cardiac cells, ISO treatment only activated the inflammasomes in the cardiomyocytes but not the CFs. These results demonstrated that inflammasome activation was propagated from cardiomyocytes to CFs in the mice hearts. Further investigation revealed that the inflammasomes were activated in the cocultured CFs that connected with cardiomyocytes via membrane nanotubes (MNTs), a novel membrane structure that mediates distant intercellular connections and communication. Disruption of the MNTs with the microfilament polymerization inhibitor cytochalasin D (Cyto D) attenuated the inflammasome activation in the cocultured CFs. In addition, the MNT-mediated inflammasome activation in the CFs was blocked by deficiency of the inflammasome component NOD-like receptor protein 3 (NLRP3) in the cardiomyocytes, but not NLRP3 deficiency in the CFs. Moreover, ISO induced pyroptosis in the CFs cocultured with cardiomyocytes, and this process was inhibited by disruption of the MNTs with Cyto D or by the NLRP3 inhibitor MCC950 and the caspase-1 inhibitor Z-YVAD-FMK (FMK). Our study revealed that MNTs facilitate the rapid propagation of inflammasome activation among cardiac cells to promote pyroptosis in the early phase of β-adrenergic insult. Therefore, preventing inflammasome transfer is a potential therapeutic strategy to alleviate acute β-AR overactivation-induced cardiac injury.