engleski

Chemosensitisation of human breast carcinoma cell line MDA-MB-435s to vincristine and paclitaxel by silencing integrin αv or α4

Integrin signalling regulates numerous cellular processes in tumor cells including tumor cell adhesion, migration, invasion and survival. Integrins play an important role in breast cancer metastasis. In addition, metastatic cancer cells are highly resistant to antitumor drugs. The aim of this study was to investigate the role of integrins αvβ3, αvβ5, α3β1 and α4β1 expressed on MDA-MB-435S cells in sensitivity to different antitumor drugs and cell migration, one of the necessary requirements for efficient cell invasion and metastasis. For silencing of integrin subunits β3, β5, αv, α3 and α4 in human breast carcinoma MDA-MB-435S cell line, the siRNA sequences specific for β3, β5, αv, α3 and α4 were transfected using Lipofectamine RNAiMAX Reagent. Flow cytometry analysis was used to analyse the expression of integrins αvβ3, αvβ5, αv, α3β1 and α4. Confocal microscopy was used to visualise paxillin, phospho-paxillin or F-actin fibers using specific antibodies or phalloidin-FITC, respectively. Cell survival was assessed using MTT assay. Boyden chambers (pore size 8 μm) were used to measure migration. While decreased expression of integrins αvβ3, αvβ5 or α3β1, achieved by β3, β5, αv or α3-specific siRNA transfection, reduced sensitivity of MDA-MB-435S cells to cisplatin, no change was observed in MDA-MB-435S cells upon knockdown of α4β1 using α4 siRNA transfection. Conversely, decreased expression of integrins αvβ5 or α4β1 upon integrin subunit β5, αv or α4 siRNA transfection sensitized MDA-MB-435S cells to vincristine or paclitaxel, but there was no change in cell survival upon knockdown of integrins α3β1 or αvβ3 upon α3 or β3 siRNA transfection. The cytotoxic activity of camptothecin and doxorubicin was not altered upon transfection of any of analysed siRNAs. The down-regulation of integrins αvβ3 and αvβ5 achieved by αv siRNA transfection strongly decreased migration, amount of stress fibers and focal adhesions, whereas the down-regulation of integrin α4β1 upon α4 siRNA transfection moderately increased cell migration with simultaneous slight increase in amount of stress fibers and focal adhesions. We suggest that transfection of at least some breast carcinoma cells with integrin αv siRNA could enhance activity of vincristine or paclitaxel and simultaneously inhibit their metastatic potential. These new insights should be useful when devising strategies for more efficient treatment.

integrins ; silencing ; tumor cell resistance ; anticancer drugs ; migration

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