Gotowa bibliografia na temat „Formation of vincristine”

Vincristine is used in the clinical treatment of colon cancer, especially in patients diagnosed in the advanced phase of cancer development. Unfortunately, similar to other agents used during antitumor therapy, vincristine might induce chemoresistance. Studies of this process focus mainly on the analysis of the molecular mechanisms within cancer, usually ignoring the role of stromal cells. Our present findings confirm that vincristine stimulates the secretion of tumor growth factors class beta and interleukin-6 from cancer-associated fibroblasts as a result of paracrine stimulation by cancer cells. Based on alterations in morphology, modulation of capillary formation, and changes in endothelial and mesenchymal marker profile, our findings demonstrate that higher levels of tumor growth factor-βs and interleukin-6 enhance cancer-associated fibroblast-like cell formation through endothelial–mesenchymal transition and that nonsteroidal anti-inflammatory drug treatment (aspirin and ibuprofen) is able to inhibit this phenomenon. The process appears to be regulated by the rate of microtubule polymerization, depending on β-tubulin composition. While higher levels of tubulin-β2 and tubulin-β4 caused slowed polymerization and reduced the level of factors secreted to the extracellular matrix, tubulin-β3 induced the opposite effect. We conclude that nonsteroidal anti-inflammatory drugs should be considered for use during vincristine monotherapy in the treatment of patients diagnosed with colorectal cancer.

Cancers treated with Vincristine and vinblastine include: acute leukemia, Hodgkin's and non- Hodgkin's lymphoma, neuroblastoma, rhabdomyosarcoma, Ewing's sarcoma, Wilms' tumor, multiple myeloma, chronic leukemias, thyroid cancer, brain tumors, non-small cell lung cancer, bladder cancer, melanoma, and testicular cancer andIt is also used to treat some blood disorders. It is given by injection into a vein. Vincristine and vinblastine exhibit differential activity against tumors and normal tissues. In this work, a number of cultured cell lines were assayed for their sensitivity to the antiproliferative and cytotoxic effects of the two drugs following short-term (4 hr) or during continuous exposures. Differential activity was not seen when cells were subjected to continuous exposures. The concentrations of Vincristine and vinblastine, respectively, that inhibited growth rates by 50% were: mouse leukemia L1210 cells, 4.4 and 4.0 nw; mouse lymphoma S49 cells, 5 and 3.5 nM; mouse neuroblastoma cells, 33 and 15 nw; HeLa cells, 1.4 and 2.6 nw; and human leukemia HL-60 cells, 4.1 and 5.3 nM. In contrast, differential toxicity was seen when cells were subjected to 4-hr exposures and transferred to drug-free medium: the 50% growth-inhibitory concentrations for Vincristine and vinblastine, respectively, for inhibition (a) of proliferation of L1210 cells were 100 and 380 nM and of HL-60 cells were 23 and 900 nM and (b) of colony formation of L1210 cells were 6 and >600 nM and of HeLa cells were 33 and 62 nM. Uptake and release of [3H]- vincristine and [3H]vinblastine were examined in L1210 cells under the conditions of growth experiments. Uptake of both drugs was dependent on the pH of culture media, and signifi cantly greater amounts of [3H]vinblastine than of [3H]vincristine were associated with cells after 4-hr exposures to equal concen trations of either drug. When cells were transferred to drug-free medium after 4-hr exposures, vinblastine was released much more rapidly from cells than was Vincristine, and by 0.5 hr after resuspension of cells, the amount of Vincristine associated with the cells was greater than the amount of vinblastine and remained so for up to at least 6 hr.

Abstract Introduction: Multiple myeloma is still incurable and optimize existing chemotherapeutic strategies and development of novel agents are necessary to improve the outcome of patients. Cotylenin A, a fusicoccane diterpene glycoside with a complex sugar moiety, was isolated as a plant-growth regulator. Cotylenin A modulates the 14-3-3 intracellular signaling pathway and has been shown to inhibit the growth of several cancer cells. Herein, we examined the antitumor effects of cotylenin A to develop a novel treatment against myeloma. Methods: Five human myeloma cell lines, RPMI8226, KMS-11, KMS-26, KMS-12 PE and KMS-12 BM were cultured with cotylenin A alone or in combination with several anticancer drugs. To measure the effects of various drugs on the growth of myeloma cells, the number of viable cells was determined by the MTT assay after 6 days of exposure to various concentrations of drugs with or without 2 μg/ml cotylenin A. The growth-inhibiting effects of the drugs were examined by determining the concentrations of drugs required to reduce the cell number to one-half of that in untreated cells (IC50). Xenografts in six-week-old female (Fox Chase SCID C.B-17/Icr-scid Jcl) mice were used to determine the in vivoefficacy of cotylenin A. Results: Cotylenin A alone inhibited the growth of myeloma cells in dose dependent manner, but the effect of growth inhibition was relatively weak. Cotylenin A and vincristine synergistically inhibited the growth and induced apoptosis in myeloma cells. While other microtubule-disturbing agents also showed co-operative effects with cotylenin A, other anticancer agents, such as doxorubicin, cisplatin, camptothecin, methotrexate, gemcitabine and 5-fluorouracil, did not show such co-operation with cotylenin A (Table 1). Cotylenin A had synergistic effects with vincristine and the results were confirmed by an isobologram analysis. These differences might be attributed to the effects on autophagic responses. Combined treatment with cotylenin A and vincristine induced autophagy (formation of LC3-II and degradation of p62 protein). However, doxorubicin did not enhance the autophagy induced by cotylenin A. The induction of apoptosis was confirmed by an analysis of the DNA histogram and the expression of annexin V. The expression of cleaved caspase-3 was increased in treatment with 2 ng/ml vincristine and enhanced by 2 μg/ml cotylenin A by western blot analysis. An invasion assay using transwell chamber revealed that low concentration cotylenin A (0.6 μg/ml) effectively inhibited the invasive activity of RPMI 8226 myeloma cells without inhibiting growth. A colony-forming assay indicated that 2 μg/ml cotylenin A preferentially inhibited the formation of large colonies, which have high self-renewal activity. The combined treatment with 3 μg/ml cotylenin A and 3 ng/ml vincristine more effectively suppressed the formation of large colonies than vincristine alone. Expression of pluripotency-associated transcription factor Sox2 mRNA in RPMI 8226 myeloma cells was significantly suppressed by treatment with 4 μg/ml cotylenin A. Combined treatment with 5 mg/kg cotylenin A and 0.5 mg/kg vincristine significantly inhibited the growth of KMS-26 myeloma cells as xenografts. In addition, mice with cotylenin A and vincristine showed the reduction of body weight loss comparing with those of mice by vincristine alone. This result supported that the combination of cotylenin A and vincristine might be safe. Conclusions: Cotylenin A and vincristine synergistically inhibited the growth of myeloma cells in vitro and in vivo, and the invasion of myeloma cells. Our results suggest that the combination of cotylenin A and vincristine may have therapeutic value for myeloma. Table 1. Potentiation of the growth-inhibitory activities of various anticancer agents in RPMI8226 myeloma cells by cotylenin A. Growth inhibition (IC50) Anticancer agent (ng/ml) - Cotylenin A + Cotylenin A Ratio(-/+) Doxorubicin 5.5 ± 0.6 5.3 ± 0.5 1.03 Cisplatin 246 ± 30.2 237 ± 28.4 1.03 Camptotecin 1.42 ± 0.16 1.38 ± 0.14 1.03 Methotrexate 2.76 ± 0.3 1.81 ± 0.2 1.52 Gemcitabine 4.3 ± 0.4 3.1 ± 0.3 1.39 5-Fluorouracil 56.5 ± 5.1 57.8 ± 6.3 0.98 Vincristine 6.3 ± 0.6 1.6 ± 0.1 3.94 Vinblastine 0.91 ± 0.11 0.42 ± 0.05 2.17 Paclitaxel 16.4 ± 1.9 7.7 ± 0.9 2.13 Ratio (-/+), IC50 without cotylenin A: IC50 with cotylenin A. The values are the mean ± SD of four determinations. Disclosures No relevant conflicts of interest to declare.

Abstract Introduction Vincristine, a vinca alkaloid, is one of the most widely used and effective medications for acute lymphoblastic leukemia (ALL). Vincristine exerts its cytotoxic effects by interfering with microtubule formation and mitotic spindle dynamics, leading to mitotic arrest and cell death. However its use often causes neuropathy characterized by abdominal pain, sensory and motor dysfunctions. To date, various candidate gene studies have failed to identify consistent genetic variants associated with an increased risk of vincristine-induced neuropathy. Methods Vincristine-induced neuropathy was assessed in children enrolled on SJCRH protocol study XIIIB for the treatment of newly diagnosed ALL and a subset of children with relapsed ALL enrolled on a Children’s Oncology Group protocol (COG AALL0433). Neuropathy was graded using CTCAE v1.0 (XIIIB) or a modified (“Balis”) pediatric scale (AALL0433). We performed a genome wide analysis of germline SNPs using the Affymetrix 500K or 6.0 array and imputed SNPs. Weighted logistic regression was used to test the association between genotypes and vincristine neuropathy in patients experiencing multiple episodes of grade 2-4 vincristine-induced neuropathy during their treatment. We also performed a meta-analysis that estimated p values across the two cohorts using Fisher’s method. Results Genome -wide SNP analysis and vincristine-induced neuropathy were assessed in 321 patients (222 on SJCRH protocol and 99 on COG protocol). The meta-analysis identified several loci that were significant in both patient cohorts in the same direction related to the risk of multiple episodes of neuropathy, with Rs924607 reaching genome-wide significance (p<10-8). After adjusting for genetically defined race and for vincristine dose, Rs924607 (localized in chromosome 5, near the CEP72 gene) remained significant (p = 4.68 x10-8). The cumulative incidence of developing multiple episodes of Vincristine-induced neuropathy differed among the three genotypes (p<0.0001) for this SNP, and this SNP was also associated with low expression of the CEP72 mRNA in the HapMap cells (CEU); the rs924607 risk allele (T) was associated with lower expression of CEP72 compared to the C allele. CEP72 regulates the localization of key centrosomal proteins and proper bipolar spindle formation, making it plausible that this SNP alters the pharmacologic effects of vincristine. We subsequently confirmed that this SNP was associated with lower expression of CEP72 in a luciferase reporter construct containing 2612 bp of the CEP72 promoter region, including the Rs924607 SNP (comparing constructs containing either the C allele or the T allele of Rs924607). The T allele of this SNP creates a DNA binding sequence for the NKX family of transcription repressors. Molecular dynamics and binding free energy calculations indicated that NKX-6.3 bound with a higher affinity (the binding free energy of NKX-6.3 bound to T allele is 7.67 kcal/mol lower than bound to C allele, which is equivalent to approximately 400000 fold decrease of kd) to the T allele than the C allele, consistent with our finding of lower expression of CEP72 in the presence of the T allele. When expressed in SHSY5Y or NALM6 cells, the construct containing the T allele had significantly lower expression than the construct containing the C allele, and knockdown of NKX-6.3 rescued the low expression of the construct with the T allele. When CEP72 was knocked down in human ALL cells, there was increased accumulation of the cells at G2M after vincristine treatment, consistent with augmented vincristine effects. Thus, these findings have revealed a new and plausible inherited SNP in the CEP72 promoter region that is linked to the risk of vincristine-induced neuropathy in children with ALL. Conclusions Our genome-wide analysis has revealed a novel genomic region that is associated with increased risk of vincristine-induced neuropathy, providing new insights into this treatment complication and a means to identify patients at highest risk. Disclosures: No relevant conflicts of interest to declare.

During apoptosis, the cell actively dismantles itself and reduces cell size by the formation and pinching off of portions of cytoplasm and nucleus as “apoptotic bodies.” We have combined our previously established quantitative assay relating the amount of release of [3H]-membrane lipid to the degree of apoptosis with electron microscopy (EM) at a series of timepoints to study apoptosis of lymphoid cells exposed to vincristine or etoposide. We find that the [3H]-membrane lipid release assay correlates well with EM studies showing the formation and release of apoptotic bodies and cell death, and both processes are regulated in parallel by inducers or inhibitors of apoptosis. Overexpression of Bcl-2 or inhibition of caspases by DEVD inhibited equally well the activation of caspases as indicated by PARP cleavage. They also inhibited [3H]-membrane lipid release and release of apoptotic bodies. EM showed that cells overexpressing Bcl-2 displayed near-normal morphology and viability in response to vincristine or etoposide. In contrast, DEVD did not prevent cell death. Although DEVD inhibited the chromatin condensation, PARP cleavage, release of apoptotic bodies, and release of labeled lipid, DEVD-treated cells showed accumulation of heterogeneous vesicles trapped in the condensed cytoplasm. These results suggest that inhibition of caspases arrested the maturation and release of apoptotic bodies. Our results also imply that Bcl-2 regulates processes in addition to caspase activation.

Abstract BACKGROUND: Despite improvements in front-line therapy for adult ALL, most patients eventually relapse and do not tolerate or respond to reinduction therapy. Novel targeted therapies are needed that have both activity against adult ALL and a toxicity profile distinct from conventional chemotherapy. MAb216 is a naturally occurring human IgM monoclonal antibody derived from the VH4-34 (variable heavy chain) gene. It has shown promise as a novel therapy for B-ALL in preclinical studies. In vitro, mAb216 specifically binds and is cytotoxic to normal human B-lymphocytes and B-progenitor lymphoblasts from patients with ALL. Binding of mAb216 to its linear lactosamine ligand leads to formation of large membrane pores resulting in cell lysis. This non-classical apoptosis occurs in the absence of complement fixation but the cytotoxicity is enhanced by complement. The membrane pores may also facilitate entry of chemotherapeutic drugs into the leukemic blast providing an explanation for the enhanced cell killing that is seen when mAb216 is combined with vincristine. METHODS: The primary aims of the study are to determine the maximum-tolerated dose, and dose-limiting toxicities of mAb216 as a single agent and in combination with vincristine in patients with relapsed or refractory B-ALL. Secondary aims are to characterize the pharmacokinetic behavior of mAb216 and to preliminarily assess clinical efficacy. Binding of mAb216 to the patient’s leukemic blasts is confirmed prior to enrollment. Two treatment courses of mAb216 are given with the same dose of antibody administered on days 0 and 7. In case of a suboptimal response (< 75% reduction in peripheral blood blasts) after the first dose of mAb216, the second dose is given in combination with vincristine. Five mAb216 dose levels are planned using a starting dose of 1.25 mg/kg and a standard 3+3 dose-escalation design. Clinical response is assessed by measuring the peripheral blood blast count weekly and by bone marrow biopsy if no blasts are present in the peripheral blood. RESULTS: Nine of 10 patients screened exhibited binding of mAb216 to their blasts and were enrolled in the study (median age 27; range 10–73). Four patients had relapsed after allogeneic bone marrow transplantation, four patients had relapsed after a median of four (range 2–5) prior therapies, and one patient had primary refractory disease. In these patients, doses up to 2.5 mg/kg of mAb216 have been well tolerated. At a dose of 1.25 mg/kg one patient experienced vomiting and grade 3 epistaxis three days after the infusion. At 2.5 mg/kg one patient developed hives during the infusion that resolved with diphenhydramine and hydrocortisone. MAb216 has not induced immune complex formation. The median half-life is 1.76 h (range 0.78–9.3 h) and strongly correlates with the binding affinity of mAb216 to the blasts. 7/9 patients experienced a reduction of their peripheral blast count between 8–61% after infusion of mAb216 alone. 7/9 patients received the second dose of mAb216 in combination with vincristine on day 7 or earlier. All seven patients, who had failed previous treatment with regimens containing vincristine, achieved a reduction of their peripheral blast count of 50–100%. One patient had a hypocellular bone marrow with no residual blasts on day 21. CONCLUSIONS: These results indicate that mAb216 alone and in combination with vincristine is a promising treatment for relapsed/refractory B-ALL. Patient recruitment and dose-escalation is ongoing.

Abstract BACKGROUND: Acute myeloid leukemia (AML) is an aggressive leukemia with 5-year overall survival of 20-25%. The major reason for treatment failure in AML is resistance to chemotherapy. Thus, there is an urgent need for identification of novel therapeutic agents for AML. Neoplastic cells, including AML, have dysfunctional redox regulation that results in increased reactive oxygen species (ROS). Accumulation of ROS leads to oxidation of free and incorporated nucleotides, leading to DNA damage and cell death. MTH1 is a nudix family hydrolase that sanitizes the oxidized nucleotide pool to prevent incorporation of these damaged bases in the DNA. MTH1 is thought to be non-essential for normal cells but crucial for neoplastic cells in order to avoid incorporation of oxidized dNTPs into DNA, thereby evading DNA damage and cell death. Whether MTH1 inhibitors have any activity against AML is not known. METHODS: Neoplastic myeloid cell lines HL-60, HEL, K562, KG1A, ML1, MV-4-11, SET2, and U937 were treated with varying concentrations of TH588 for a total of 48 hours. In experiments using the pan-caspase inhibitor Q-VD-OPh (Qvd), cells were pre-treated with 5µM Qvd for 1 hour followed by TH588. Cells were washed and stained with annexin, propidium iodide (PI), or MitoTracker (Life Technologies, Carlsbad, CA) for flow cytometry. To evaluate the potential impact of MTH1 inhibition on chemorefractory AML, HL-60/VCR cells were treated with vehicle control or TH588 in culture medium with or without vincristine (1µg/ml). Percentage apoptosis was calculated by normalizing to vehicle only control. With IRB approval, bone marrow aspirate samples were obtained from patients with untreated AML or healthy controls. Mononuclear cells were analyzed using colony-forming unit (CFU) assays. The total number of erythroid (CFU-E) and myeloid (CFU-G, CFU-GM) colonies containing ≥50 cells were read on day 14 and reported as percentage colonies compared to vehicle control. RESULTS: TH588 induced dose-dependent cell death in each of the neoplastic cell lines tested except HEL. In particular, treatment with TH588 resulted in a dose-dependent increase in the number of cells undergoing apoptosis as indicated by annexin V and/or PI staining (IC50 3.1-21.3µM, Figure 1). Pre-treatment with Qvd significantly inhibited TH588-induced cell death in all the cell lines studied except KG1A and SET2, suggesting a caspase-dependent mechanism of cell death. In further studies, cells treated with TH588 exhibited decreased MitoTracker staining; and Qvd pretreatment increased the number of MitoTrackerLow cells at the same time apoptotic cells decreased, suggesting that mitochondrial damage is upstream of caspase activation in TH588-induced apoptosis. Treatment with TH588 not only induced apoptosis in HL-60/VCR cells, but also facilitated further apoptosis in cells co-treated with vincristine and TH588 (Figure 2). Treatment with TH588 also diminished colony formation in a primary AML sample (IC50 6µM, Figure 3). Analysis of additional primary AML samples is ongoing. DISCUSSION: Our results show that the MTH1 inhibitor TH588 induces apoptosis in most neoplastic myeloid cells. MTH1 causes mitochondrial damage that, in turn, leads to caspase-dependent apoptosis in these cells. In HL-60/VCR cells representing chemorefractory phenotype, TH588 induces apoptosis as a single agent and resensitizes cells to vincristine. Moreover, TH588 significantly diminished colony formation in primary AML ex vivo. Further preclinical and possible clinical study of this class of agent appears warranted. Figure 1. Induction of cell death by MTH1 inhibitor TH588 in neoplastic myeloid cell lines. Figure 1. Induction of cell death by MTH1 inhibitor TH588 in neoplastic myeloid cell lines. Figure 2. TH588 induces apoptosis in HL-60/VCR cells and resensitizes cells to vincristine. Figure 2. TH588 induces apoptosis in HL-60/VCR cells and resensitizes cells to vincristine. Figure 3. TH588 significantly diminished colony formation in primary AML ex vivo indose-dependent manner. Figure 3. TH588 significantly diminished colony formation in primary AML ex vivo indose-dependent manner. Disclosures No relevant conflicts of interest to declare.

Calcium channel blockers (CCBs) such as verapamil and nitrendipine are capable of increasing drug sensitivity in resistant murine and human tumor cells. This finding has potential value in the treatment of acquired drug resistance in human malignancies. Thus, we tested the ability of CCBs of two different structural classes to enhance the toxicity of doxorubicin (DOX), vinblastine (VBL), and vincristine (VCR) for normal myeloid and macrophage colony formation (marrow colony forming units-granulocyte-monocyte [CFU-GM]). Drug effects on colony formation from 35 normal volunteer marrows and from seven patient marrows in the recovery phase after cytotoxic chemotherapy were determined. No enhancement of toxicity was mediated by verapamil or nitrendipine when these drugs were co-incubated with the cytotoxic drugs for one hour or 24 hours before plating marrow cells in a semisolid agar system.

Characterization of the role and extent of pericyclic reactions in natural product biosynthetic pathways has dramatically increased over the past decade. By definition, pericyclic transformations involve the concerted conversion of a substrate to product without any detectable intermediates. Examples of four major types: electrocyclizations (e.g. 7-dehydrocholesterol to pre-vitamin D3), cycloadditions (e.g. Diels–Alder [4+2]-cyclizations in conversion of trienes to decalins), sigmatropic shifts (e.g. Cope rearrangements), and Alder-ene reactions have been validated to occur in biologic systems. Diels–Alderases have been found to catalyze intramolecular decalin formations and spiro-tetronate/spiro-tetramate cyclizations, as well as intermolecular cyclizations. Pericyclase-catalyzed heteroatom cyclizations include pyridine ring formation in thiazole-containing nonribosomal thiazolopeptide antibiotics. In the biosynthetic route to the vinca anticancer alkaloids vinblastine and vincristine, a metabolic precursor stemmadenine has been shown to undergo variants of enzyme-catalyzed [4+2]-cyclizations to give catharanthine and tabersonine frameworks.