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Objective To determine the effects of deoxyelephantopin on mTOR and its related target molecules (Akt/mTOR/P70S6K) in the ER-positive breast cancer cell line. Materials and Methods Primary in silico simulations were determined, and the effects of deoxyelephantopin on the phosphorylation of the Akt/mTOR/P70S6K molecules were evaluated using AlphaScreen-based assays and western blot analysis, respectively. Results Based on the estimated FEB and Ki values, deoxyelephantopin appeared to have a stronger affinity toward P70S6K as compared with Akt and mTOR. Both deoxyelephantopin and control inhibitors were observed to form hydrogen bonds with the same key residue, Leu175 of the P70S6K molecule. Deoxyelephantopin downregulated the p P70S6K protein expression significantly from 18 µM (p < 0.05) and onward. Based on the AlphaScreen assay, deoxyelephantopin produced a concentration-dependent inhibition on the phosphorylation of P70S6K with an IC50 value of 7.13 µM. Conclusion Deoxyelephantopin induced cell death in MCF-7 cells possibly via DNA fragmentation, inhibition of the phosphorylation of P70SK6, and downregulation of the relative p-p70S6K protein expression levels.
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Objective To investigate the effects of metformin on the radiosensitivity of breast carcinoma cells with different estrogen receptors stasus (MCF-7 and MDA-MB-231) and to explore the underlying mechanisms.Methods Two cell lines,MCF-7 and MDA-MB-231 in logarithmic phase were divided into four groups:control group,drug group (mefformin),irradiation group and experimental group (irradiation plus metformin).MTT assay and the clonogenic assay were performed to evaluate the effects of metformin on the proliferation and survival of breast carcinoma cell lines,respectively.The change of cell cycle distribution and apoptosis rates were measured by propidium iodide (PI) and Hoechst 33342 staining analysis repectively.Western blot was used to detect the expression of p-AMPK and p-mTOR.Resutls Metformin could obviously inhibit the proliferation of the two breast carcinoma cell lines in a dose dependent manner.The cloning formation capacity was decreased in the group of metformin plus irradiation,which displayed the values of Dq,D0 and SF2 significantly lower than those of irradiation alone group (MCF-7:t =9.305,14.528,13.708,P ＜0.05;MDA-MB-231:t =19.560,16.893,36.048,P ＜0.05),and the sensitizing enhance rate (SER) of D0 were 1.29 and 1.21 for MCF-7 and MDA-MB-231 cell lines,respectively.Compared with irradiation alone,metformin plus irradiation obviously increased the proportion of cells in the G2/M phase in both cell lines (t =6.103,38.431,P ＜ 0.05).Metformin plus irradiation also enhanced radiation-induced apoptosis in both cell lines so that the apoptosis rates were higher than that in the metformin group or irradiation alone group (t =9.143,14.561,P ＜ 0.05).In MCF-7 cell lines,the expression of p-AMPK in the metformin combined with irradiation group was significantly higher than other treatment groups (t =35.194,8.647,10.316,P ＜ 0.05),but no significant changes of p-AMPK expression in MDA-MB-231 cell lines was observed (P ＞ 0.05).While inhibition of p-mTOR by metformin was observed in both cell lines (MCF-7:t =80.133,31.820,11.308,P＜0.05;MDA-MB-231:t=12.436,15.757,8.402,P＜0.05).Conclusions This study suggests that metformin possessed a strong radiosensitizing potential in both breast carcinoma cell lines of MCF-7 (ER positive) and MDA-MB-231 (ER negative).This radiosensitizing effect may result from the activation of AMPK or AMPK-independent pathway,inhibition of mTOR signaling pathway,and the enhancement of radiation-induced G2/M phase arrest and cell apoptosis after metformin treatment.
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Expression of epidermal growth factor receptor (EGFR) in SK BR 3 cells, a human breast carcinoma cell line,was analysed with immunohistochemistry. Inhibitory effects of TP40 on SK BR 3 cells growth and protein synthesis were analysed with crystal violet staining and 3 H leucine incorporation. Competitive assays were performed by the addition of excess of EGF. The results showed that the SK BR 3 cells exhibited large amounts of brown immunoperoxidase reaction indicative of EGFR. When the concentration of TP40 was in the range of 1 100?g/L,TP40 inhibited SK BR 3 cell growth and protein synthsis in a dose dependent form. An excess of EGF could completely block inhibitory effects of TP40. The results suggested that the human breast carcinoma SK BR 3 cells express EGFR at a high level. TP40 could significantly inhibit the growth of SK BR 3 cells. The cytotoxic effects of TP40 were specifically mediated by EGFR.