Tetrandrine induces mitochondria-mediated apoptosis in human gastric cancer BGC-823 cells

Abstract

Tetrandrine, a bis-benzylisoquinoline alkaloid isolated from the dried root of Hang-Fang-Chi (Stephaniatetrandra S. Moore), has been reported to possess anti-cancer effects on many tumors. In this study, we investigated tetrandrine-induced apoptosis on human gastric cancer BGC-823 cells in vitro and in vivo. The results showed that tetrandrine significantly inhibited cell viability in a dose- and time-dependent manner and induced apoptosis. It increased the apoptosis; upregulation of Bax, Bak, and Bad; and downregulation of Bcl-2 and Bcl-xl in BGC-823 cells. Moreover, tetrandrine increased the activation of caspase-3 and -9, release of cytochrome c, and upregulation of apaf-1, suggesting that tetrandrine-induced apoptosis was related to the mitochondrial pathway. Meanwhile, pretreatment with the pan-caspase inhibitor z-VAD-fmk in BGC-823 cells reduced tetrandrine-induced apoptosis by blocking activation of caspases. Furthermore, tetrandrine effectively inhibited tumor growth via apoptosis induction, which was verified by immunohistochemical analysis in a nude mouse xenograft model. Taken together, we concluded that tetrandrine significantly inhibited the proliferation of gastric cancer BGC-823 cells through mitochondria-dependent apoptosis, which may play a promising role in gastric cancer therapy.

Figure 1. Effect of tetrandrine on the viability of BGC-823 cells.

(A) The chemical structure of tetrandrine. (B) Cell proliferation was determined by an MTT assay, and BGC-823 cells were treated with various concentration of tetrandrine at 24, 48 and 72 h, respectively. The inhibition rate of the control group was set to 0. Data are expressed as means ± SD of three independent experiments performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001 versus the control group, respectively.

Figure 2. Apoptosis of BGC-823 cells induced by tetrandrine.

A PI-Annexin V-FITC binding assay was used to detect apoptosis of BGC-823 cells. (A) Cells treated with tetrandrine for 24 h at 0, 6, 8, and 10 μg/ml. (C) Cells treated with 8 μg/ml tetrandrine for 0, 12, 24, and 48 h. (B, D) Columns represent the means ± SD of apoptotic cells obtained from three independent experiments. **P < 0.01; ***P < 0.001 versus the control group.

Figure 3. The effect of tetrandrine on apoptosis-related proteins in BGC-823 cells.

(A) Western blot analysis of the expression of apoptosis-related proteins in BGC-823 cells treated with 6, 8, and 10 μg/ml tetrandrine for 24 h. (B) Western blot analysis of bcl-2 and bax treated with 8 μg/ml tetrandrine for 12, 24, 48, and 72 h. (C) Effects of tetrandrine on the expression levels of bcl-2, bcl-xl, and bax were determined using real-time PCR (1), control (2) 6 μg/ml (3) 8 μg/ml (4) 10 μg/ml. β-actin expression was used as an internal control. Data are reported as the means ± SD of at least three experiments. *P < 0.05; **P < 0.01; ***P < 0.001 versus the control group.

Figure 4. Tetrandrine-induced apoptosis via a mitochondrial pathway in BGC-823 cells.

(A) Western blot analysis of the expression of procaspase-3, cleaved caspase-3, and cleaved caspase-9 in BGC-823 cells treated with tetrandrine at 6, 8, and 10 μg/ml for 24 h. (B) The cells were pretreated with or without pan-caspase inhibitor z-VAD-fmk (10 μM) for 4 h followed by treatment with tetrandrine at 8 μg/ml for 24 h, and the relative activity of caspase-3 was measured using a caspase-3 activity assay. (C) BGC-823 cells were pretreated with or without z-VAD-fmk (10 μM) for 4 h followed by treatment with tetrandrine at 8 μg/ml for 24 h. A PI-Annexin V-FITC binding assay was used to detect apoptosis of BGC-823 cells. (D) Western blot analysis of the expression of cytoplasmic cytochrome c and Apaf-1 in BGC-823 cells treated with tetrandrine at 6, 8, and 10 μg/ml for 24 h. β-actin expression was used as an internal control. Data are reported as the means ± SD of at least three experiments. *P < 0.05; **P < 0.01; ***P < 0.001 versus the control group.

Figure 5. Tetrandrine inhibits tumor growth in vivo.

BGC-823 cells were subcutaneously inoculated into BALB/c nude mice to establish the xenograft model. (A) Tumor growth was monitored at the indicated time points, and the tumor volume was measured every 3 days. The starting day of drug treatment was defined as day 0. Data represent means ± SD of the relative tumor volume for each group. (B) Tumor weight was measured at the end of the experiment. Data represent means ± SD of the tumor weight for each group. ***P < 0.001 versus the control group.

Figure 6. Immunohistochemical staining for apoptosis-related protein in BGC-823 xenografts.

Immunohistochemical staining for apoptosis-related proteins: Bcl-2, Bcl-xl, Bax, Activated caspase-3, and Activated capase-9. Magnification is 200×. Representative microphotographs of three groups are shown. DAB stained immunoreactive cells (dark brown).