engleski

Chemosensitisation to microtubule poisons and decreased migration of human melanoma cell lines by silencing of integrin αv

Melanoma is the most aggressive form of skin cancer. Integrin signalling regulates numerous cellular vital processes in tumour cells. The goal of this study was to investigate the impact of integrin αv knockdown on sensitivity to antitumor drugs, cell migration and invasion. Three human metastatic melanoma cell lines A375, RPMI-7951 and MeWo were used. Flow cytometry analysis using integrin-specific antibodies showed that all three cell lines express comparable amounts of integrin subunit αv and heterodimer αvβ5. However, they differ in expression of integrin αvβ3 which is highly expressed in A375 and RPMI-7951 but almost absent in MeWo cells. The siRNA-directed integrin αv knockdown increased sensitivity of RPMI-7951 and MeWo cells to the microtubule- directed antitumor drugs paclitaxel and vincristine, decreased sensitivity of MeWo cells to cisplatin, and neither changed sensitivity of RPMI-7951 cells to cisplatin nor of A375 cells to any of the abovementioned drugs. In vitro transwell migration and invasion assays conducted in the RPMI-7951 cell line upon αv silencing resulted in a drastic inhibition of both, cell migration and invasion. Using the same assay migration could not be assessed in MeWo cells, which is in accordance with literature data showing their low metastatic potential. We have also analysed the involvement of pERK1/2 signalling pathway in integrin αv sensitisation to paclitaxel. The integrin αv knockdown, as compared to control- siRNA transfected cells moderately decreased the total amount of pERK1/2. The lower amount was maintained during 72 hours of paclitaxel exposure. Approximately 60–70% of malignant melanomas have a BRAF mutation that constitutively activates the B-Raf/MEK/ERK1/2 pathway. They are treated with the BRAF inhibitor vemurafenib. The BRAFV600E mutated RPMI-7951 cell line is, however, vemurafenib- resistant due to the overexpression of COT which activates ERK1/2 independent of BRAF. Therefore, we analysed the combined effect of integrin αv knockdown and vemurafenib, and showed that integrin αv knockdown increased sensitivity of RPMI-7951 cells to vemurafenib. Our results identify integrin αv as a potential target for melanomas not responding to vemurafenib therapy such as BRAFV600E mutated but vemurafenib-resistant (RPMI-7951) and BRAF wild type (MeWo) melanoma cell lines to paclitaxel. Also the integrin αv knockdown decreases metastatic potential in the highly metastatic cell model of RPMI-7951 cells.

chemosensitisation ; integrin αV knockdown ; BRAF V600E ; melanoma ; paclitaxel ; vincristine ; vemurafenib ; migration ; invasion

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