Cidan inhibits liver cancer cell growth by reducing COX-2 and VEGF expression and cell cycle arrest

Abstract

Cidan is a traditional Chinese medicine formula that has been used for >10 years as an antitumor drug. In the present study, the antitumor effect of cidan on hepatocellular carcinoma (HCC) and the underlying molecular mechanisms were investigated. A total of 372 patients with primary HCC, as confirmed by pathological examination in the Eastern Hepatobiliary Surgery Hospital and Beijing Oncology Hospital of Weida TCM, were prospectively enrolled in the study. In total, 92 patients were treated with cidan capsules for three months postoperatively, while 280 patients served as controls. The efficacy of cidan was analyzed by monitoring associated symptoms and liver function tests, including measuring the levels of α-1-fetoprotein, α-L-fucosidase, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase and γ-glutamyl transferase. In addition, in vivo analysis was performed using mice Hepa1-6 xenograft models, while in vitro studies were performed with SMMC-7721 and CSQT-1 cells; this included cidan-dependent cell viability and migration assays, cell cycle analyses and the evaluation of cidan effects on cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) mRNA transcription rates using quantitative polymerase chain reaction. The postoperative two-year overall survival (77 and 58% for the cidan and control groups, respectively; P<0.01) and disease-free survival (36 and 24% for the cidan and control groups, respectively; P<0.01) rates were superior in the cidan-treated group when compared with the control. In addition, the size and weight of the tumor xenografts in the C57BL/6 mice were significantly reduced in a time- and dose-dependent manner following cidan treatment (P<0.01). Cidan significantly reduced the cell viability of SMMC-7721 and CSQT-1 cells after four and five days when compared with the control (P<0.01). Furthermore, COX-2 and VEGF mRNA expression levels decreased following cidan treatment (P<0.01), and cidan treatment resulted in enhanced G1 and G2/M cell cycle arrest of CSQT-1 cells. Therefore, cidan effectively inhibited cell proliferation, reduced cell viability and downregulated COX-2 and VEGF expression levels in hepatoma cells.

Keywords: Rhizoma Curcumae; cidan; cyclooxygenase-2; primary hepatocellular carcinoma; vascular endothelial growth factor.

Figure 1.

Comparison of the postoperative OS rates of patients with HCC treated with liver-protection drugs only (control group) or with liver-protection drugs and cidan (treatment group). HCC, hepatocellular carcinoma; OS, overall survival.

Figure 2.

Comparison of the postoperative DFS times of patients with HCC treated with liver-protection drugs only (control group) or with liver-protection drugs and cidan (treatment group). HCC, hepatocellular carcinoma; DFS, disease-free survival.

Figure 3.

COX-2 and VEGF mRNA expression levels in SMMC-7721 cells following treatment with cidan. ***P<0.001 and *P<0.05, vs. control group (saline). COX-2, cyclooxygenase-2; VEGF, vascular endothelial growth factor.

Figure 4.

In vitro cytotoxic activity of cidan in human tumor cell lines. **P<0.01, vs. Cidan group (saline).

Figure 5.

Inhibition of Matrigel cell invasion. SMMC-7721 and CSQT-1 cells were incubated with cidan-containing medium for 12 h. **P<0.01 and ***P<0.001, cidan treatment group vs. control group (saline).

Figure 6.

Flow cytometric analysis of CSQT-1 cells following 24 h incubation with 40 µg/ml cidan or saline (control). (A) Representative flow cytometry images; (B) percentage chart and (C) numerical analysis of the cells in the various cell cycle stages. **P<0.01 and ***P<0.001, vs. control cells (saline treatment).