Eriocalyxin B Induces Apoptosis and Autophagy Involving Akt/Mammalian Target of Rapamycin (mTOR) Pathway in Prostate Cancer Cells

Abstract

BACKGROUND Eriocalyxin B (EriB), a diterpenoid isolated from the plant Isodon eriocalyx, has been shown to possess anti-tumor properties. However, few systematic studies of the mechanism underlying the anti-tumor activity of Eriocalyxin B in prostate cancer cells have been published. The aim of this study was to investigate the effect of Eriocalyxin B on prostate cancer cells. MATERIAL AND METHODS In the present study, the PC-3 (androgen-independent) and 22RV1 (androgen-dependent) human prostate cancer cell lines were cultured with and without increasing doses of Eriocalyxin B. MTT assay was used to measure cell viability. Western blotting was performed to measure levels of proteins associated with apoptosis and autophagy. Flow cytometry was used to assess changes in cell apoptosis and cycle. Fluorescence microscopy was used to capture images of autophagy-related proteins. RESULTS Treatment of human prostate cancer cells with Eriocalyxin B resulted in apoptosis in a dose- and time-dependent manner. Eriocalyxin B also induced autophagy, with elevated LC3B-II protein expression and punctuate patterns. Additionally, autophagy protected prostate cancer cells from apoptosis induced by Eriocalyxin B, which was demonstrated by addition of chloroquine (CQ). Moreover, the results indicated that Eriocalyxin B could inhibit the phosphorylation of Akt and mTOR. Eriocalyxin B induced apoptosis and autophagy by inhibition of the Akt/mTOR pathway. CONCLUSIONS Eriocalyxin B induces apoptosis and autophagy involving the Akt/mTOR pathway in prostate cancer cells in vitro. These findings provide evidence for Eriocalyxin B as a potent therapeutic for the treatment of prostate cancer.

Figure 1

Inhibitory effect induced by EriB on cell viability of human prostate cancer cell lines. (A) Eriocalyxin B chemical structure. (B) Various concentrations (0.25–8.0 μM) of EriB-treated PC-3 and 22RV1 cells for 24 and 48 h, after which MTT assay was used to measure cell proliferation rate.

Figure 2

Apoptosis induced by EriB alone or cotreatment with autophagy inhibitor. EriB-treated PC-3 cells (0.5 μM, 48 h) and 22RV1 cells (2 μM, 48 h). We added 50 μM CQ to culture medium for the last 6 h. Cell apoptosis was assessed by flow cytometry with Annexin V/PI staining.

Figure 3

Apoptosis induced by EriB in human prostate cancer cell lines. PC-3 and 22RV1 cells treated with 0.5 and 2 μM of EriB, respectively, for 24 and 48 h. We assessed caspase-3, caspase-8, and PARP levels by Western blot.

Figure 4

Effect of EriB on the cell cycle distribution of human prostate cancer cell lines. PC-3 and 22RV1 cells treated with 0.5 and 2 μM of EriB, respectively, for 48 h. Various phases of the cell cycle were analyzed by flow cytometry.

Figure 5

Regulation by EriB alone or EriB with autophagy inhibitor on the expression of LC3B in human prostate cancer cell lines. (A) PC-3 and 22RV1 cells treated with 0.5 and 2 μM of EriB, respectively, for 24 and 48 h. The proteins of LC3B were detected by Western blot. (B) After staining endogenous LC3 proteins and nucleus with anti-LC3 antibody (FITC, green) and DAPI (blue) respectively. The images of LC3 distribution were captured under a fluorescence microscope. (C) PC-3 and 22RV1 cells were treated with 0.5 and 2 μM, respectively, of EriB for 48 h. We added 50 μM CQ or 5 MM 3-MA to culture medium for the last 6 h. The proteins of LC3B were detected by Western blot.

Figure 6

Regulation of EriB-induced apoptosis in human prostate cancer cell lines by autophagy inhibitor. (A) EriB-treated PC-3 cells (0.5 μM, 48 h) and 22RV1 cells (2 μM, 48 h). We added 50 μM CQ to culture medium for the last 6 h, after which MTT assay was used to measure cell viability. (B) The proteins of caspase 3, caspase 8, and PARP were detected by Western blot.

Figure 7

Inhibition of Akt/mTOR signal pathway by EriB. EriB-treated PC-3 cells (0.5 μM, 24 h) and 22RV1 cells (2 μM, 48 h). mTOR, p-mTOR, Akt, and p-Akt were detected by Western blot.