Добірка наукової літератури з теми "A375 Melanoma cells, Onconase, PARP inhibitors, BRAF inhibitors"

Onconase (ONC) is an amphibian secretory ribonuclease displaying cytostatic and cytotoxic activities against many mammalian tumors, including melanoma. ONC principally damages tRNA species, but also other non-coding RNAs, although its precise targets are not known. We investigated the ONC ability to modulate the expression of 16 onco-suppressor microRNAs (miRNAs) in the A375 BRAF-mutated melanoma cell line. RT-PCR and immunoblots were used to measure the expression levels of miRNAs and their regulated proteins, respectively. In silico study was carried out to verify the relations between miRNAs and their mRNA targets. A375 cell transfection with miR-20a-3p and miR-34a-5p mimics or inhibitors was performed. The onco-suppressors miR-20a-3p, miR-29a-3p and miR-34a-5p were highly expressed in 48-h ONC-treated A375 cells. The cytostatic effect of ONC in A375 cells was mechanistically explained by the sharp inhibition of cyclins D1 and A2 expression level, as well as by downregulation of retinoblastoma protein and cyclin-dependent-kinase-2 activities. Remarkably, the expression of kinases ERK1/2 and Akt, as well as of the hypoxia inducible factor-1α, was inhibited by ONC. All these proteins control pro-survival pathways. Finally, many crucial proteins involved in migration, invasion and metastatic potential were downregulated by ONC. Results obtained from transfection of miR-20a-3p and miR-34a-5p inhibitors in the presence of ONC show that these miRNAs may participate in the antitumor effects of ONC in the A375 cell line. In conclusion, we identified many intracellular downregulated proteins involved in melanoma cell proliferation, metabolism and progression. All mRNAs coding these proteins may be targets of miR-20a-3p, miR-29a-3p and/or miR-34a-5p, which are in turn upregulated by ONC. Data suggest that several known ONC anti-proliferative and anti-metastatic activities in A375 melanoma cells might depend on the upregulation of onco-suppressor miRNAs. Notably, miRNAs stability depends on the upstream regulation by long-non-coding-RNAs or circular-RNAs that can, in turn, be damaged by ONC ribonucleolytic activity.

Melanoma is a lethal tumor because of its severe metastatic potential, and serine/threonine-protein kinase B-raf inhibitors (BRAFi) are used in patients harboring BRAF-mutation. Unfortunately, BRAFi induce resistance. Therefore, we tested the activity of onconase (ONC), a cytotoxic RNase variant, against BRAFi-resistant cells to re-establish the efficacy of the chemotherapy. To do so, an A375 dabrafenib-resistant (A375DR) melanoma cell subpopulation was selected and its behavior compared with that of parental (A375P) cells by crystal violet, 5-Bromo-2’-deoxyuridine incorporation, and cleaved poly(ADP-ribose) polymerase 1 (PARP1) western blot measurements. Then, nuclear p65 Nuclear Factor kappaB (NF-κB) and IκB kinases-α/β (IKK) phosphorylation levels were measured. Gelatin zymography was performed to evaluate metalloproteinase 2 (MMP2) activity. In addition, assays to measure migration, invasion and soft agar colony formation were performed to examine the tumor cell dissemination propensity. ONC affected the total viability and the proliferation rate of both A375P and A375DR cell subpopulations in a dose-dependent manner and also induced apoptotic cell death. Among its pleiotropic effects, ONC reduced nuclear p65 NF-κB amount and IKK phosphorylation level, as well as MMP2 activity in both cell subpopulations. ONC decreased cell colony formation, migration, and invasion capability. Notably, it induced apoptosis and inhibited colony formation and invasiveness more extensively in A375DR than in A375P cells. In conclusion, ONC successfully counteracts melanoma malignancy especially in BRAFi-resistant cells and could become a tool against melanoma recurrence.

Melanoma is the most prevalent type of skin cancer with high mortality rates. This study demonstrates the in vitro and in vivo anticancer properties of chalcone flavokawain B (FKB) induced ROS-mediated apoptosis and autophagy in human melanoma (human epithelial melanoma cell line A375 and/or human skin lymph node derived melanoma cell line A2058) cells. Cell viability was calculated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the expression patterns of various apoptosis, autophagy-associated proteins were determined by Western blot methods. Annexin V was detected by flow cytometry, whereas acidic vesicular organelles (AVOs) and intracellular ROS levels were measured by fluorescence microscopy. The in vivo anticancer properties of FKB were evaluated by xenografting the A375 cells into nude mice. The results convey that FKB inhibited cell viability, B-Raf proto-oncogene, serine/threonine kinase (BRAF)/extracellular signal-regulated kinase (ERK) expression in human melanoma cells. Caspase-3 activation, poly (ADP-ribose) polymerase (PARP) cleavage pathway, and Bcl2 associated X (Bax)/B-cell lymphoma 2 (Bcl-2) dysregulation were involved in the execution of apoptosis. Moreover, FKB-induced autophagy was observed through increased microtubule-associated protein 1A/1B-light chain 3B (LC3-II) accumulation and AVOs formation, which was also associated with an increase in sequestosome 1 (SQSTM1/p62), decreased protein kinase B (AKT)/mammalian target of rapamycin (mTOR) expressions, and dysregulated Beclin-1/Bcl-2 levels. Autophagy inhibitors [3-methyladenine (3-MA)/chloroquine (CQ)] and LC3 silencing suppressed FKB-induced apoptosis by decreasing caspase-3 in melanoma cells. The antioxidant N-acetylcysteine (NAC) diminished FKB-induced apoptotic and autophagic cell death. However, the inhibition of apoptosis decreased FKB-induced autophagy (LC3-I/II). The in vivo study confirmed that FKB inhibited melanoma growth in A375-xenografted nude mice. This study concluded that FKB is critically associated with the execution and generation of ROS-modulated apoptotic and autophagic cell death of melanoma cells. FKB also repressed tumor growth in xenografted nude mice. Therefore, flavokawain B might be a potential anti-tumor agent in human melanoma treatment.

Melanoma is one of the most aggressive form of skin cancer, characterized by high mortality rate due to the metastatic potential of its cells. Several therapies have been approved during last few years. Recently, the melanoma molecular characterization led to development of drugs acting against specific targets. For instance, BRAF inhibitors (BRAFi) have been tested against BRAF-mutated melanoma. Unfortunately, all chemotherapy strategies failed for the resistance acquired by tumor cells. Poly (ADP-ribose) polymerase (PARP) enzymes are crucial in the DNA damage response and the PARP inhibition causes the accumulation of unrepaired DNA, inducing cell death. PARP inhibitors (PARPi) are commonly used in chemotherapy. The most used PARPi in cancer therapy is olaparib, although it can be quickly throwing outside the cells by the P-glycoprotein drug efflux transporter. For this reason, a new PARPi generation has been designed, such as AZD2461 that demonstrated a lower binding affinity to the P-glycoprotein than olaparib. In vitro and in vivo studies showed that the protein onconase (ONC) exerted an antitumor effect in different cancer types, due to its ribonuclease activity. Therefore, in this work, the ONC effects on A375 human melanoma cell line that harbors a mutation in the BRAF kinase gene, has been evaluated. A reduction in A375 cell viability has been observed with ONC treatment, while no cytotoxicity was registered in normal human melanocytes. In A375 cells a reduction in BrdU incorporation and a decrease in Ki67 protein expression occurred, suggesting that ONC elicits a cytostatic effect. In addition, an increase of both Annexin V-FITC fluorescence and cleaved PARP1 expression level were observed, suggesting that ONC induces apoptotic cell death. In order to investigate the ONC intracellular targets, changes in the proteome profile have been investigated using mass spectrometry. A decreased expression level of proteins involved in telomere elongation, in the mesenchymal cell development and in the ribosomal subunit biogenesis has been obtained. A decrease in protein synthesis in ONC-treated cells was also demonstrated. Besides ONC, AZD2461 PARPi can reduce A375 cell viability, conversely, no strong benefits were obtained when ONC was administered in combination with AZD2461, in comparison with each single drug treatment. ONC and AZD2461 have displayed an inhibitory effect on both TNF-α gene transcription and NF-κB DNA binding activity, but no additive effect was observed when they were used in combination. In order to obtain A375 cells resistant to AZD2461, cells were treated for two months with AZD2461, but this long treatment did not induce resistance. Nevertheless, the AZD2461 long-time treated cells resulted more responsive to the ONC pro-apoptotic action, if compared to the parental ones. Subsequently, resistance against the BRAFi dabrafenib has been induced in A375 since long time treatment with increasing drug concentrations of dabrafenib induced a sorting of an A375 resistant cell (A375DR) subpopulation. A375DR cells displayed activation of the cancer stem cells markers CD133 and NANOG and increase expression of epithelial-mesenchymal transition-related proteins N-cadherin and nuclear β-catenin, in comparison with A375 parental (A375P) cells. In addition, A375DR showed an increase in the ERK1/2 phosphorylation level, suggesting a reactivation of the MAPK pathway. By comparing A375P and A375DR, ONC treatment can inhibit the total viability and the proliferation rate in both cell subpopulations and induce apoptotic cell death. Moreover, among its pleiotropic effects, ONC reduced nuclear p65 NF-κB amount and IκB kinase phosphorylation level, as well as metalloproteinase-2 activity in both cell subpopulations. Finally, ONC decreased cell colony formation, migration and invasion capability more extensively in A375DR than in A375P cells. In conclusion, ONC successfully counteracts malignant phenotype of A375 cells especially in BRAFi resistant cells and could become a helpful tool for therapy of melanoma recurrence.