To assess the putative position of AQP3 in cell volume regulation in response to genotoxic agents, we measured adjustments from the cell diameter right after nucleoside analog remedy in non transfected, negative handle siRNA transfected and AQP3 siRNA transfected cells. Cells have been incubated for 90 min with 50 DFUR or gemcitabine, and cell diameters measured soon after 48 h. As shown previously, the two medication induced a marked raise in cell diameter. Inhibition of AQP3 expression considerably reduced but did not totally avert the maximize in cell volume triggered by the nucleoside derived drugs in MCF7 and HT29 cells. Both nucleosides also exerted dramatic results on cell viability as determined by measuring the number of cells just after 48 h of treatment. Similarly to cell vol ume modifications, AQP3 silencing resulted in considerable reversion of nucleoside induced cell growth inhibition during the breast cancer cell line MCF7, and to a lesser extent in the colon cancer cell line HT29 after treatment with 50 DFUR.
Even so, the cell development arrest induced by gemcitabine in HT29 was not blocked through the inhibition of AQP3 expression. Interestingly, equivalent effects have been selleck at first obtained upon blocking the activity of AQP3 with CuSO4 in MCF7 cells. Copper salts are helpful AQP3 inhibi tors but additionally can show toxicity, and independ ently exert various effects on cell responses to DNA injury. So, inhibition of AQP3 action supports the information obtained when silencing AQP3 expression. AQP3 silencing partially reverses cell cycle arrest triggered by nucleoside derived medicines and up regulation of transcriptional targets Therapy of cells with 50 DFUR and gemcitabine induced cell cycle arrest at the G1 S phase in MCF7 cells, whereas cisplatin promoted accumulation of cells with the S G2 phase, fact that had previously been reported.
Interestingly, AQP3 siRNA drastically blocked cell cycle arrest induced by each nucleoside analogs in MCF7 cells. Similarly to the reversion of cell growth inhibition in HT29 cell line, only the cell cycle arrest trig gered by 50 DFUR was reversed, but not the a single trig gered by gemcitabine. To eliminate the chance that cell cycle dependent regulation of AQP3 expression interferes with these phenomena, MCF7 cells additional hints were synchronized by serum depletion, and AQP3 connected mRNA amounts analyzed in the course of cell cycle progres sion. Underneath these conditions, we observed no distinctions in AQP3 mRNA levels. 50 DFUR and gemcitabine up regulate a variety of genes, typically in a p53 dependent manner. We analyzed regardless of whether AQP3 knockdown impacts the tran scriptional response connected with drug treatment method in MCF7, cell line in which we observed the clearest effects on cell cycle.