Synergistic cytotoxic effects of a combined treatment of a Pinellia pedatisecta lipid-soluble extract and cisplatin on human cervical carcinoma in vivo

Abstract

Herbal medicines are known to have numerous benefits, including lower toxicity and fewer side effects than traditional chemotherapeutic drugs. In traditional Chinese medicine, the rhizome of Pinellia pedatisecta (PE) Schott has long been used to treat cancer, undiagnosed swelling and erythema toxicum. However, its medical benefits lack support from scientific evidence. A novel lipid-soluble extract from PE has been previously verified to enhance the cytotoxicity of cis-dichlorodiammineplatinum-II (CDDP) against human cervical cancer cells in vitro. The present study evaluated the synergistic cytotoxic effects of PE and CDDP against human cervical cancer. Combination therapy of PE with CDDP exhibited synergistic cytotoxicity towards CaSki cell growth in mouse xenograft tumors. PE exhibited a cytotoxic effect on tumor size and weight, although the inhibitory ratio of tumor weight was only 26.3% in the PE-treated group. However, when mice were co-treated with PE and CDDP, the inhibitory ratio was higher than that of mice treated with CDDP alone (50.8 vs. 68.4%, respectively). The potential synergistic mechanism was likely via inhibiting the signaling E6/p53 pathway, restoring p53 function and inducing downstream tumor suppressor chain effects on apoptosis. Western blot analysis and immunohistochemistry indicated thatE6protein expression was significantly decreased upon treatment with combined PE and CDDP. The expression of p53 was increased in the combined PE and CDDP treatment group. Upregulation of p53-dependent apoptosis-associated proteins, including Bcl-2-associated X protein and cleaved caspases-9 and -3, was observed in the combined PE and CDDP treatment group. Our results present a molecular basis for the future application of the combination of PE and CDDP in the treatment of cervical cancer as a novel and pharmacologically safe chemotherapeutic strategy.

Keywords: Pinelliapedatisecta Schott; cervical cancer; cisplatin; cytotoxicity; synergistic.

Figure 1.

Changes in the body weight of nude mice following treatments. Eight mice were used in each group. (A) The changes in trend of mean body weight of mice in different groups following treatment. (B) The mean body weight of mice in different groups when treatment was over. The CDDP-treated mice had a remarkable body-weight loss compared with that observed in the PE-treated and control groups (P<0.001). The combined group has less body-weight loss than the CDDP-treated group, with no statistical significance. Bars represent the upper 95% confidence interval. ***P<0.001, for comparisons between mice treated with the combined treatment and mice treated with CDDP. PE, Pinelliapedatisecta; CDDP, cis-dichlorodiammineplatinum-II.

Figure 2.

Effect of PE and CDDP alone and in combination on (A) tumor volume, (B) tumor weight and (C) tumor weight inhibitory ratio in subcutaneously CaSki cervical cancer xenografts of nude mice. Eight mice were used in each group. (A and B) The tumor burden was reduced significantly in the CDDP group, but not in the PE group. However, when PE was combined with CDDP, either tumor volume or tumor weight had a remarkable reduction compared with that displayed by the CDDP group (P<0.05 and P<0.01, respectively). (C) The tumor weight inhibitory ratio of the combined group was larger than that of the CDDP (P<0.05) or the control (P<0.01) groups. (D) Images of the tumors from the subcutaneous nude-mouse model for the different groups. PE, Pinelliapedatisecta; CDDP, cis-dichlorodiammineplatinum-II; SD, standard deviation.

Figure 3.

Survival curve of the mice in the four groups. Each group has 7 mice. Kaplan-Meier survival curve showing improved survival in tumor bearing mice treated with CDDP (P<0.001) and the combination of PE and CDDP (P<0.001) compared with that exhibited by the untreated control group. However, compared with the CDDP-treated group, the combination group had no significantly longer lifespan (P>0.05). N.S., no significance; PE, Pinelliapedatisecta; CDDP, cis-dichlorodiammineplatinum-II.

Figure 4.

Western blot analysis of subcutaneously transplanted tumors. Western blot analysis of human papilloma virus E6, p53, p21, p27 Apaf-1and apoptosis-associated proteins expressed in xenografts from mice treated with PE and CDDP alone or in combination. GAPDH was used as a reference. PE, Pinelliapedatisecta; CDDP, cis-dichlorodiammineplatinum-II; Apaf-1, apoptosis protease activating factor 1; Bcl-2, B cell lymphoma/leukemia-2; Bax, Bcl-2-associated X protein.

Figure 5.

Expression of Ki-67 and p53 proteins in xenografts of mice (magnification, ×400). Ki-67 immunohistochemistry revealed the level of cellular proliferation in the xenografts of mice treated with PE and CDDP alone or in combination. Scale bar=50 µm. PE, Pinelliapedatisecta; CDDP, cis-dichlorodiammineplatinum-II.