SGK3 is under-expression in tamoxifen-resistant breast cancer cells
MCF-7-R is tamoxifen-resistant breast cancer cells stemmed from MCF-7 parental cells, to generated its resistance, it was treated with 1 µM tamoxifen more than 6 months. To verify it was varied from parental cells in tamoxifen-resistance ability, MTT assay and colony formation experiment were performed to test. The result of MTT assay showed that the cell viability of MCF-7-R were higher than MCF-7 when under same concentration tamoxifen (Fig. 1A). The clone formation results shows that the number of MCF-7-R was more than that in MCF-7 when under same concentrations tamoxifen treatment (Fig. 1B, C). Also, we find that MCF-7-R grown more quickly than parental MCF-7 cells under no tamoxifen treatment.
Figure1. MCF-7 and MCF-7-R (Tamoxifen-resistant MCF-7) were treated with tamoxifen at different concentrations within 72 h. (A) The cell viability analysis using MTT assay of breast cancer cell lines after treatment with various concentrations of tamoxifen or vehicle alone for the indicated time periods. Data are the means of triplicate experiments. The results are expressed as the percentage of vehicle control cells treated with DMSO alone (vehicle). (B) Clone formation assay of MCF-7 and MCF-7-R cells treated with DMSO or tamoxifen. Cells were treated with tamoxifen at varying concentrations (5–10 µM) for approximately 10 days. (Up) The quantification of the number of colonies and the size of the colonies. (Below) Significant differences between the treatment groups were found using a t-test (P < 0.05). Values represent the means ± SD.
Overexpress SGK3 increased tamoxifen sensitivity in MCF-7-Tet-On-SGK3 cells.
We use tetracycline-controlled Tet-On gene expression system to regulate the activity of SGK3 in MCF-7 cells, to analyze the role of SGK3 in the tamoxifen-resistance ability. As the western blotting results, SGK3 can be induced under the doxycycline (DOX) within 48 hours. As shown in MTT and clone assay results, we find that when SGK3 was overexpressed by DOX in MCF7-Tet-On-SGK3 for 24 h, compared with DOX (-) group, the cell viability was higher after tamoxifen-treatment for 48 h (Fig. 2B). At the same time, western blotting results shown that SGK3 expression in MCF-7-R was lower than that in parental MCF-7, and the MCF-7-R has induced the autophagy-related protein LC3-Ⅱ. At the same time, we use immunofluence assay to detect the expression level of LC3B. This indicates that the tamoxifen-resistant ability might has relation with SGK3 expression level, in further induced the change of autophagic flux.
Figure2. SGK3 expression level can influence the autophagy and the sensitivity of MCF-7 cells for the tamoxifen. (A) Western blot assay was used to detect the induced-overexpression of SGK3 in MCF-7-Tet-On cells, while the cells were treated with doxycycline (DOX) for 48 h. (Right) The SGK3 was induced to overexpress under DOX and then treated with concentrations of tamoxifen for 48 h, then the cell viability was detected by MTT assay. (B) The expression level of SGK3 and the autophagy marker LC3 were detected by WB assay and the related expression level was semi-quantitated, significant differences between the treatment groups were found using a t-test (P < 0.05). Values represent the means ± SD.
Overexpress SGK3 can inhibit tamoxifen-induced autophagy level.
To evaluate the level of autophagy induced by tamoxifen, we perform western blotting assay to test, as indicated in the Fig. 3, overexpression of SGK3 can inhibit autophagy, which reduced autophagy caused by tamoxifen. To confirm that SGK3 can change autophagy. We used small interference RNA (siRNA) to knockdown SGK3, as indicated by Fig. 4, reduction SGK3 can obviously reverse autophagic marker.
Figure3. SGK3 inhibits the autophagy level while the tamoxifen induces. (A) The SGK3 expression level in MCF-7-Tet-On cells were induced by DOX treatment in 48 h, and then treated with 5 µM tamoxifen in 48 h, after that, the cells were harvested to detect the autophagy markers by WB assay (B). Representative immunofluorescence
images of LC3B expression in MCF-7-Tet-On cells. Representative images are presented, scale bar, 50 µm.
Inhibition autophagy can increase chemotherapy effect of tamoxifen
To assess the role of autophagy in cell proliferation caused by tamoxifen, specific autophagy inhibitor 3-methyladenine (3-MA) was used to combine tamoxifen to treat cells. As the MTT results shown in Fig. 5, MCF-7 cell viability was significantly decreased when 16 µM tamoxifen combined with 5 mM 3-MA. In addition, tamoxifen treated cells showed a significant decrease in the level of LC3 and Beclin1 expression levels (Fig. 5B). This indicates that autophagy induced by tamoxifen in cells was a kind of protective against death.
Figure4. Inhibition the autophagy enhances the sensitivity of tamoxifen to MCF-7 cells. (A) MCF-7 cells were treated with several concentrations autophagy-inhibitor 3-MA for 48 h, then the cell viability was determined by MTT assay. (A Right)The cells were pre-treated with 5 mM 3-MA or DMSO for 48 h, and then the cells were treated with 5 µM tamoxifen for 48 h, the cell viability was determined by MTT assay (A) or WB. (B) Autophagic proteins were detected by WB assay after the treatment of tamoxifen with or without 3-MA, the related expression level was semi-quantitated, significant differences between the treatment groups were found using a t-test (**P < 0.05). Values represent the means ± SD.
Knockdown of SGK3 sensitizes MCF-7 cells to treatment with tamoxifen.
Next, to in step asses the effect of SGK3 on the tamoxifen-resistant ability. SiRNA was transfected into the MCF-7-Tet-On cells, while WB assay shows that while induction SGK3 inhibits autophagy and knockdown SGK3 induces autophagy (Fig. 5A). The cells were pre-transfected with siNC (negative control) or siSGK3 for 48 h and then cells were treated with varying concentrations of tamoxifen for 48 h, the results shows that inhibition of SGK3 can enhance the resistance of tamoxifen. Above all, we illustrated the pathway diagram SGK3 enhances sensitivity of ER-positive breast cancer cells to tamoxifen (Fig. 6).
Figure 5. Inhibition of SGK3 enhances resistance of tamoxifen in MCF-7 cells. (A) WB assay detected the SGK3 and LC3 expression level. (B) MTT assay detected the relative cell viability of MCF-7 cells under the treatment of tamoxifen with transfected with siNC or siSGK3 for 48 h, significant differences between the treatment groups were found using a t-test (**P < 0.05). Values represent the means ± SD.
Figure6. Mechanism of SGK3 enhances sensitivity of ER positive breast cancer cell to tamoxifen by inhibiting autophagy.