Triptolide induces autophagy and apoptosis through ERK activation in human breast cancer MCF-7 cells

Abstract

To investigate the effects of triptolide (TPI) on proliferation, autophagy and death in human breast cancer MCF-7 cells, and to elucidate the associated molecular mechanisms, intracellular alterations were analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays. The results of the MTT assay revealed that TPI significantly reduced the MCF-7 cell survival rate when the concentration was >10 nmol/l. TPI activated a caspase cascade reaction by regulating Bcl-2-associated X protein (Bax), caspase-3 and B-cell lymphoma 2 expression, and promoted programmed cell death via the mitochondrial pathway. The results demonstrated that TPI significantly reduced the cell proliferation rate and viability in a time- and dose-dependent manner, which was confirmed by western blotting and immunofluorescent staining. TPI induced autophagy and influenced p38 mitogen-activated protein kinases, extracellular signal-regulated kinase (Erk)1/2, and mammalian target of rapamycin (mTOR) phosphorylation, which resulted in apoptosis. When cells were treated with a combination of TPI and the Erk1/2 inhibitor U0126, the downregulation of P62 and upregulation of Bax were inhibited, which demonstrated that the inhibition of Erk1/2 reversed the autophagy changes induced by TPI. The results indicated that Erk1/2 activation may be a novel mechanism by which TPI induces autophagy and apoptosis in MCF-7 breast cancer cells. In conclusion, TPI affects the proliferation and apoptosis of MCF-7 cells, potentially via autophagy and p38/Erk/mTOR phosphorylation. The present study offers a novel view of the mechanisms by which TPI regulates cell death.

Keywords: apoptosis; autophagy; breast cancer; extracellular signal-regulated kinase; mechanism; triptolide.

Figure 1.

TPI induced apoptosis in human breast cancer MCF-7 cells. (A) Cytotoxicity was studied by MTT assay and the average was presented as a percent of viability. MCF-7 cells were treated with different concentrations of TPI (0, 10, 20, 50, 100, 200 and 400 nmol/l) for 12, 24 and 48 h. *P<0.05, **P<0.01 and ***P<0.001 vs. 0 nmol/l, and as indicated. Representative fluorescent images labeled with Annexin V-FITC/PI following treat with (B) 0 nmol/l, (C) 100 nmol/l and (D) 200 nmol/l TPI for 24 h. Flow cytometric assay was performed and the statistical results were presented in (E). (F) Expression of apoptosis-associated proteins following treatment with 0, 10, 20, 50 and 100 nmol/l TPI. Data are representative of three independent experiments. *P<0.05 and ***P<0.001 vs. 0 nmol/l (control). TPI, triptolide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; FITC, fluorescein isothiocyanate; PI, propidium iodide; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.

Figure 2.

TPI induced apoptosis in human breast cancer MCF-7 cells. Cells were treated with 0, 50 and 200 nmol/l TPI for 24 h, respectively, and examined by (A) optical microscopy. Cells were treated with (B) Hoechst 33258 and (C) Annexin V-fluorescein isothiocyanate/propidium iodide, then analyzed by fluorescence microscopy. Magnification, ×400 in each field. Images are representative of three independent experiments. TPI, triptolide.

Figure 3.

TPI mediated autophagy induction in MCF-7 cells when treated with the (A) indicated concentration (0, 10, 20, 50 and 100 nmol/l) for (B) 12, 24 and 48 h. The protein expression levels for LC3B I/LC3B II, P62 and Beclin-1 were analyzed according to the (C) concentration and (D) duration of TPI treatment. Data are representative of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. 0 nmol/l TPI. TPI, triptolide; LC3B, light chain 3B.

Figure 4.

Detection of autophagy induction and p-Erk1/2, p38 and mTOR protein in different concentrations of TPI by western blot analysis. (A) Representative immunoblot and the quantified protein expression levels of p-P-38, p-Erk 1/2 and p-mTOR following treatment with increasing concentrations of TPI, presented as p-p38/p38, p-Erk/Erk and p-mTOR/mTOR expression ratios. (B) The expression of Bax, Bcl-2 and p62 were affected by the Erk inhibitor U0126 (20 µmol/l) and TPI (50 nmol/l). (C) Quantitative analyses for Bax protein expression. Data are representative of three independent experiments. *P<0.05 and ***P<0.001 vs. 0 nmol/l TPI. TPI, triptolide; p-, phosphorylated; Erk, extracellular signal-regulated kinase; mTOR, mammalian target of rapamycin; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.

Figure 5.

Schematic representation of triptolide induction of MCF-7 cell apoptosis. LC3B, light chain 3B; p-, phosphorylated; Erk, extracellular signal-regulated kinase; mTOR, mammalian target of rapamycin; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.