Figure 9

Effect of HIF-1α and/or BCRP siRNA on mammosphere formation and cell proliferation in LTLTCa cells. A) LTLTCa cells were treated with either negative control siRNA or HIF-1α siRNA for 48 hours. Total mRNA was extracted and HIF-1α, BCRP, GAPDH, Nanog, BMI-1 and TWIST mRNA, and 18S rRNA were analyzed by real-time RT-PCR. Real-time results are expressed as the fold-change in mRNA levels compared with negative control after normalization to 18S rRNA (mean ± SD, n = 6 independent cell samples/group; *versus vehicle; P = 0.0.132 HIF-1; P = 0.0058 GAPDH, P = 0.0377 BCRP, P = 0.0612 TWIST, P = 0.058 Nanog, P = 0.0214 BMI-1; two-sided t-test). B) LTLTCa cells were plated in passage media and then treated with negative control siRNA, HIF-1α siRNA, BCRP siRNA, or 100 μM CoCl₂ for 48 hours. Cells were then collected and resuspended in mammosphere media on low-attachment cell culture wells. Results are expressed as number of mammospheres counted per 20,000 cells plated (mean ± SD, n = 6 independent cell samples/group; *versus negative control, P <0.001; † versus CoCl₂, P < 0.001; overall P <0.0001, one-way ANOVA). BCRP siRNA confirmed to decrease BCRP expression (0.35- and 0.15-fold versus negative control, P <0.01, one-way ANOVA; data not shown). C) Viability of the cells was measured by the MTT assay after 48 hours treatment with negative control or HIF-1 alpha siRNA and subsequently 6 day treatment with increasing doses of letrozole. Results are expressed as percent of 0 μM letrozole (vehicle) (mean ± SD, n = 4 independent cell samples/group; *versus 0 μM letrozole, P <0.001; overall P <0.0001 one way ANOVA). ANOVA, analysis of variance; BCRP, breast cancer resistant protein; HIF-1α, hypoxia inducible factor 1 α subunit; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; n, number; SD, standard deviation.