HPV E6 down-regulation and apoptosis induction of human cervical cancer cells by a novel lipid-soluble extract (PE) from Pinellia pedatisecta Schott in vitro

Abstract

Aim of the study

To evaluate the cytotoxicity and apoptosis induction effects of a novel lipid-soluble extract (PE) from Pinellia pedatisecta Schott on CaSki, HeLa and HBL-100 cells. Particularly, the effect of PE on HPV E6 gene expression was tested, and the mechanism of its apoptosis induction effect was also studied.

Materials and methods

Cell viability was measured by the MTT assay. DAPI staining and flow cytometric analysis (FCM) were used to identify apoptotic cells in PE-treated CaSki, HeLa, and HBL-100 cells. Expression of the HPV E6 gene in CaSki and HeLa cells was detected by real-time RT-PCR and western blot analysis. Apoptosis-associated genes were examined by RT-PCR and western blot analysis in CaSki cells.

Results

PE inhibited the growth of CaSki and HeLa cells in a time- and dose-dependent manner, but it had no obvious inhibiting effect on HBL-100 cells except at a relatively high dose (500 μg/mL). PE could induce apoptosis in CaSki and HeLa cells in a time-dependent manner but not in HBL-100 cells. HPV E6 mRNA and protein were decreased significantly by PE. Caspase-8, caspase-3, Bax, P53 and P21 mRNAs as well as proteins were increased while Bcl-2 mRNA and protein were decreased significantly by 24 h of PE treatment.

Conclusions

PE can function as a tumor suppressor by inducing apoptosis in human cervical cancer cells but it has little side effect on normal cells. It probably acts via mitochondria-dependent and death receptor-dependent apoptotic pathways. HPV E6 may be the key target of its action.

Introduction

Cervical cancer is one of the most common cancers among women worldwide, second only to breast cancer, and disproportionately affects women in developing countries (Schoell et al., 1999). In 2000, approximately 468,000 new cases were diagnosed worldwide, of which 80% were in developing countries (Parkin, 2001). An estimated, 100,000 new cases are diagnosed annually, with an overall incidence rate of over 27 per 100,000 women (Parkin et al., 1999). Furthermore, the number of cervical cancer mortality cases in China is among the highest in the world, with over 25,000 women dying in 2002 (Ferlay et al., 2004).

Infection with high-risk human papillomavirus (HR-HPV) is known to be directly associated with the development of cervical cancer, and more than 99% of cervical cancer can be detected with HR-HPV infection (Bosch et al., 2002, Münger et al., 2004). As the prevalence of HPV infection increased, the incidence of cervical cancer increased rapidly (Munoz, 2000, Bosch and De, 2007). Among more than 100 kinds of HPVs, HPV16 is the most common type associated with cervical cancer worldwide, with little geographic variation. It is responsible for about 50% of HPV-associated cancers, and HPV18 is responsible for about 20% of these cancers (Munoz et al., 2003, Kraus et al., 2006). Therefore, HPV has become a target for cervical cancer prevention and treatment. Blocking the infection of HR-HPV will block the development of cervical cancer in a great degree. Vaccines against two main stains of HPV (16 and 18) produced by large pharmaceutical companies are great success in prevention and treatment of the disease.

Pedate Pinellia Rhizome, the rhizome of Pinellia pedatisecta Schott, is a traditional Chinese medicine (TCM) that has long been used to cure thanatophidia bite, nameless swelling and toxicum, and cancer. In TCM books, it was recorded to possess efficacy in dispelling wind and relieving convulsion, drying dampness to eliminate phlegm, and eliminating stagnation. In the 1970s, Pedate Pinellia Rhizome was used in our hospital to treat 247 cases of cervical cancer and the total effective rate was 81.5% (Li et al., 1981). Women in all phase of cervical cancer exhibited beneficial results from the treatment with Pinellia pedatisecta, but women in Phase I were most effectively treated. Dr. Li's team also did some initial studies on the chemistry and pharmacology of Pinellia pedatisecta. They initial in vitro studies on the active constituents of the plant, and found the best activity in the lipid-soluble fraction of the plant. Chemical analysis of the lipid-soluble fraction indicated it contained alkaloids, fatty acids and β-sitosterol. They hypothesized that β-sitosterol was the anticancer agent in Pinellia pedatisecta. Nevertheless, we think further study is needed since identifying β-sitosterol as the sole anticancer constituent of the extract is not reasonable for that compound is widespread in plants. Besides, the pharmacological mechanism of action by which Pinellia pedatisecta has the therapeutic effect has not been identified. In this study, PE, a novel lipid-soluble extract obtained from the rhizomes of Pinellia pedatisecta was selected to evaluate in cervical cancer cells. Its effects on the growth of the tumor cells and on the expression of HPV E6, as well as the corresponding mechanism of action, were studied. The results will make for better understanding of the anticancer efficacy of the plant and the active constituents in it.