Combination of pristimerin and paclitaxel additively induces autophagy in human breast cancer cells via ERK1/2 regulation

Abstract

Pristimerin, a quinonemethide triterpenoid, has demonstrated anticancer activity against a number of types of cancer, including breast cancer. However, its mechanism of action remains unclear. The present study investigated the autophagy‑induced anticancer efficacy of pristimerin on MDA‑MB‑231 human breast cancer cells. Pristimerin inhibited the growth of these cells in a concentration‑dependent manner. Treatment with pristimerin dose‑dependently induced an increase of light chain 3B (LC3‑II), whereas autophagy inhibitor 3‑methyladenine (3‑MA) inhibited pristimerin‑induced LC3‑II accumulation and cytotoxic effects. Autophagy was also activated by paclitaxel as observed by an elevated LC3‑II level. Although 24 µM paclitaxel induced autophagy without cytotoxicity, combined with pristimerin it additively induced cell growth inhibition and autophagy induction. Autophagy induction was measured with an autophagy detection kit and LC3‑II levels were monitored by western blot analysis. Treatment with 3‑MA inhibited LC3‑II accumulation and cell death induced by a combination of paclitaxel and pristimerin. Pristimerin and paclitaxel inhibited extracellular signal‑regulated kinase (ERK)1/2/p90RSK signaling, consistent with autophagy indicators, namely p62 degradation and beclin 1 expression. In addition, ERK activator ceramide C6 treatment suppressed the LC3‑II levels induced by a combination of paclitaxel and pristimerin. These results suggested that exposure to pristimerin induced autophagic cell death, whereas a combination treatment of pristimerin and paclitaxel resulted in an additive effect on ERK‑dependent autophagic cell death.

Figure 1.

Pristimerin-induced cell death is associated with activation of autophagy. (A) Cell viability following treatment with pristimerin (0, 1, 2.5, 5 and 10 µM) in MDA-MB-231 human breast cancer cells. *P<0.05 vs. pristimerin (0 µM). (B) MDA-MB-231 breast cancer cells were treated with 0, 1, 5 and 10 µM pristimerin for 24 h and expression levels of LC3-I and LC3-II were detected by western blot analysis. GAPDH was used as a loading control. Values are expressed as the mean ± standard deviation. *P<0.05 vs. control. (C) Cells were pre-incubated with or without 3-MA (2 mM) for 3 h and then incubated with pristimerin (10 µM) for 24 h, following which LC3-I and LC3-II levels were detected by western blotting analysis. (D) Cells were pre-incubated with or without 3-MA (2 mM) for 3 h and then incubated with 0, 1, 5 and 10 µM pristimerin, and cell viability was detected using a Cell Counting Kit-8 assay. Data are expressed as the mean ± standard deviation. *P<0.05 vs. pristimerin single treatment. 3-MA, 3-methyladenine; LC3-I, light chain 3.

Figure 2.

Cell viability following treatment with paclitaxel in MDA-MB-231 human breast cancer cells. (A) Cell viability following treatment with various concentrations (0, 6, 12, 24 and 30 µM) of paclitaxel. (B) Cells were treated with paclitaxel for 24 h and the expression levels of LC3-II/LC3-I were detected by western blot analysis. GAPDH was used as a loading control. Data are expressed as the mean ± standard deviation. *P<0.05 vs. 0 µM treatment. LC3-I, light chain 3.

Figure 3.

Involvement of autophagy in the additive action of pristimerin and paclitaxel. (A) Cell viability following treatment with pristimerin (0, 1, 2.5, 5 and 10 µM) and paclitaxel (24 µM) in MDA-MB-231 human breast cancer cells. *P<0.05 vs. pristimerin (10 µM) single treatment. (B) Cells were treated with paclitaxel and pristimerin for 24 h and expression levels of LC3-II/LC3-I were detected by western blot analysis. GAPDH was used as a loading control. Data are expressed as the mean ± standard deviation. *P<0.05 vs. paclitaxel and ^&P<0.05 vs. pristimerin. ^#P<0.05 vs. paclitaxel and pristimerin combined treatment. (C) An autophagy detection kit was used for the detection of autophagy in cells following treatment with indicated drugs. (D) Cells were pre-incubated with or without 3-MA (2 mM) for 3 h and then incubated with pristimerin (10 µM), paclitaxel, and a combination of the two for 24 h. Cell viability was then measured. Data are expressed as the mean ± standard deviation. *P<0.05 vs. combined treatment with pristimerin and paclitaxel. ^#P<0.05 vs. pristimerin (10 µM) single treatment. LC3-I, light chain 3; 3-MA, 3-methyladenine.

Figure 4.

Pristimerin-induced autophagy is regulated by ERK1/2 signaling in MDA-MB-231 cells. (A) Cells were treated with pristimerin and paclitaxel for 24 h and expression levels of p-ERK1/2, ERK1/2, p-p90RSK, p90RSK, beclin 1, and p62 were detected by western blot analysis. GAPDH was used as a loading control. *P<0.05 vs. pristimerin (0 µM) (Lane no. 1) and ^#P<0.05 vs. paclitaxel (0 µM) (Lane no. 5). (B) Cells were treated with pristimerin and paclitaxel and expression levels of p-ERK1/2, ERK1/2, LC3-II/LC3-I were detected by western blot analysis. Cells were pretreated with ERK activator ceramide C6 for 3 h prior to combined treatment with pristimerin and paclitaxel. GAPDH was used as a loading control. Data are expressed as the mean ± standard deviation. *P<0.05 vs. paclitaxel (lane no. 3), ^&P<0.05 vs. pristimerin (lane no. 2) and ^##P<0.01 vs. pristimerin and paclitaxel combined treatment without ceramide C6 (lane no. 4). p-ERK, phosphorylated extracellular signal-regulated kinase; LC3, light chain 3; p90RSK, p90 ribosomal S6 kinase.