Ruanjian Sanjie decoction exhibits antitumor activity by inducing cell apoptosis in breast cancer

Abstract

Traditional Chinese medicine, based on theories developed and practiced for >2,000 years, is one of the most common complementary and alternative types of medicine currently used in the treatment of patients with breast cancer. Ruanjian Sanjie (RJSJ) decoction, is composed of four herbs, including Ban xia (Pinellia ternata), Xia ku cao (Prunella vulgaris), Shan ci gu (Cremastra appendiculata) and Hai zao (Sargassum pallidum), and has traditionally been used for softening hard lumps and resolving hard tissue masses. However, the active compounds and mechanisms of action of RJSJ remain unknown. The present study demonstrated the antitumor activity of RJSJ against Ehrlich ascites carcinoma in Swiss albino mice and breast cancer xenografts in nude mice. Notably, RJSJ does not induce body weight loss, immune function toxicity or myelosuppression in mice, indicating that it is safe and well tolerated. In addition, RJSJ shows potent cytotoxicity against breast cancer cells in vitro by the suppression of the anti-apoptotic proteins B-cell lymphoma 2 and survivin, leading to the activation of caspase-3/7 and caspase-9, and the apoptotic cascade. These findings provide a clear rationale to explore the therapeutic strategy of using RJSJ alone or in combination with chemotherapeutic agents for breast cancer patients and the characterization of its active principles.

Keywords: Ehrlich ascites carcinoma; Ruanjian Sanjie decoction; antitumor; apoptosis; breast cancer.

Figure 1.

RJSJ treatment is effective and well tolerated in EAC tumor models. (A) Tumor size and (B) body weight of EAC-bearing mice subsequent to treatment with PBS (control), 5-Fu, RJSJ or 5-Fu plus RJSJ. (C) Effect of different drug treatments on RBC count, left panel, and Hb content, right panel. (D) Effect of different drug treatments on the spleen, left panel, and thymus, right panel, indexes. (E) Effect of different drug treatments on WBC, left panel, and neutrophil, right panel, counts. The results presented are the mean ± standard deviation of 12 mice per group. *P<0.05. RBC, red blood cell; WBC, white blood cell; RJSJ, Ruanjian Sanjie; EAC, Ehrlich ascites carcinoma; Hb, hemoglobin; 5-Fu, 5-fluorouracil.

Figure 2.

RJSJ is cytotoxic to breast cancer cells. (A) Dose-response curves used to calculate the IC₅₀ of RJSJ for MCF-7 and MDA-MB-231 cells. (B) IC₅₀ values of RJSJ of MCF-7 and MDA-MB-231 cells. (C) Cells were treated with different concentrations of RJSJ in 6-well dishes for 7 days and then stained with crystal violet. Pictorial data are representative images of wells from 3 independent replicates, left panel. The dye was solubilized and the OD at 592 nm was measured, right panel. Numerical data represent the mean ± standard deviation of 3 independent experiments. *P<0.05. RJSJ, Ruanjian Sanjie; IC₅₀, 50% inhibitory concentration; OD, optical density.

Figure 3.

RJSJ induces apoptosis in breast cancer cells. (A) Representative fluorescence images of MCF-7 cells, upper panel, and MDA-MB-231 cells, lower panel, stained with Hoechst 33258 subsequent to treatment with RJSJ for 24 h; red arrows indicate cell shrinkage and nuclear fragmentation (magnification, ×200). (B) Cells were treated with RJSJ at different concentrations for 48 h. Annexin V/propidium iodide staining was detected with flow cytometry. Representative plots of 3 independent experiments are shown. Quantitative data show the average percentage of Annexin V-positive cells in early apoptosis, lower right quadrant, and late apoptosis, upper right quadrant, of 3 independent experiments, right panel. (C) Caspase-3/7 and caspase-9 activities in MCF-7, left histograms, and MDA-MB-231, right histograms, cells subsequent to RJSJ treatment. (D and E) Bcl-2 and survivin expression levels in MCF-7 and MDA-MB-231 cells determined using (D) reverse transcription-quantitative polymerase chain reaction and (E) western blot analysis subsequent to RJSJ treatment for 48 h. Pictorial data show representative images of 3 independent experiments. Numerical data represent the mean ± standard deviation of 3 independent experiments. *P<0.05. RJSJ, Ruanjian Sanjie; Bcl-2, B-cell lymphoma 2; mRNA, messenger RNA.

Figure 4.

Antitumor activity of RJSJ in MDA-MB-231 advanced xenograft tumors. (A) Tumor size and (B) body weight of nude mice subsequent to treatment with PBS (control), DOX, RJSJ or DOX plus RJSJ. Data are presented as the mean tumor size ± standard deviation of 6 mice per group. (C) RNA was isolated from tumors 28 days post-implantation, and Bcl-2 and survivin messenger RNA expression was determined by quantitative polymerase chain reaction. The individual tumor expression data, dots, and the mean values, lines, are indicated. *P<0.05. RJSJ, Ruanjian Sanjie; Bcl-2, B-cell lymphoma 2; DOX, doxorubicin.