Elsevier

Journal of Ethnopharmacology

Volume 115, Issue 2, 17 January 2008, Pages 209-216
Journal of Ethnopharmacology

Trichosanthes kirilowii tuber extract induces G2/M phase arrest via inhibition of tubulin polymerization in HepG2 cells

https://doi.org/10.1016/j.jep.2007.09.030Get rights and content

Abstract

Trichosanthes kirilowii tuber is one of most popular herbal plant of East Asia, which has been prescribed for patients with diabetes, rigorous coughing, breast abscesses, and cancer-related symptoms.

Aim of the study

To investigated the anticancer properties of the methanol extract of Trichosanthes kirilowii tuber (TKE), focusing on cell cycle arrest and microtubule instability in HepG2 cells.

Materials and Methods

Cell growth and death were checked using a CCK-8 assay and a LDH release assay respectively. Cell cycle was analyzed by FACS after PI staining. Immunofluorece, western blot, real-time PCR for tubulin were performed.

Results

TKE treatment inhibited cell growth at around 25 μg/mL of IC50 in a CCK-8 assay and a LDH release assay, but did not result in cell death. We found that TKE induced cell cycle arrest at the G2/M phase in a time-dependent manner. However, an immunofluorescence assessment of beta-tubulin revealed a dramatically reduced amount of polymerized tubulin after TKE treatment. Furthermore, TKE treatment radically decreased the polymerized portion of soluble tubulin in a dose-dependent manner, as did colchicine; the effects, however, were opposite to those of paclitaxel in comparative analysis of polymerized to soluble tubulin. We also found that TKE treatment moderately affected α-tubulin protein production, but not that of β-tubulin and its gene expression using a Western assay and real-time PCR.

Conclusions

Anticancer mechanisms of TKE linked to the inhibition of tubulin polymerization, through which it exerts cell cycle arrest at the G2/M phase in the HepG2 cell line.

Introduction

Many medical plants have served as anticancer pharmaceutical resources, and over 60% of current anticancer drugs such as vinblastine, topotecan, etoposide, and paclitaxel were originally plant-derived compounds (Newman et al., 2003, Cragg and Newman, 2005). Trichosanthes kirilowii tuber, an herbal plant of East Asia, traditionally has been prescribed for patients with diabetes, rigorous coughing, breast abscesses, and cancer-related symptoms. In the 1980s, a ribosome-inactivating protein, trichosanthin (TCS), was isolated from the tuber of this plant; it has been of interest because of its multiple pharmacological properties such as abortifacient, anti-HIV, immunoregulatory, and antitumor functions (Leung et al., 1986, Zheng et al., 1995, Zheng et al., 2000, Lu et al., 2001, Zhao et al., 2006). These TCS-derived pharmaceutical activities, including antitumor effects, were assumed to be connected to its biochemical activities, disrupting the binding of elongation factors to the P-complex as well as N-glycosidase activity for ribosome inactivation (Zhang and Liu, 1992, Chan et al., 2001, Shaw et al., 2005).

Thus far, many studies have focused on TCS, but seldom on other components of Trichosanthes kirilowii tuber as anticancer pharmaceutical resources. We screened numerous herbal plants and found that the methanol extract of Trichosanthes kirilowii tuber lacking TCS has interesting bioactivity related to the inhibition of cancer cell growth. In addition, a recent study demonstrated that the extract of Trichosanthes kirilowii tubers was superior to TCS in antitumor activity (Dou and Li, 2004).

We investigated the pharmaceutical effect of a 50% methanol extract of Trichosanthes kirilowii tubers on anti-cell growth and anti-tubulin polymerization causing M/G2 arrest in HepG2 cells.

Section snippets

Extraction of Trichosanthes kirilowii tuber and fingerprinting analysis

Dried and sliced Trichosanthes kirilowii tuber was provided from Dunsan Oriental Hospital of Daejeon University and identified by professor, SI Yim of Daejeon University. This herb (Voucher specimen number, TK-2006-01-Tu) was stocked at our laboratory for future reference. One hundred grams of powdered Trichosanthes kirilowii tuber was mixed with 1 L of 50% methanol and shaken for 48 h at 37 °C. The extract was filtered, dried out with a vacuum evaporator to remove methanol, redissolved with

Inhibition of cell proliferation without mortality

We first evaluated the antigrowth activity of TKE by determining the HepG2 cell growth rate during 48 h within media treated with various concentrations of TKE and 5-FU as a positive control. As shown in Fig. 2A, proliferation of HepG2 cells was significantly inhibited via treatment by TKE in a dose-dependent manner. The estimated IC50 of TKE in this cell line was approximately 25 μg/mL. Next, to determine whether these cells die or just stop growing from the treatment with TKE, we measured the

Discussion and conclusions

The tubers and fruits of Trichosanthes kirilowii have been a traditional herbal remedy and an attractive medical resource due to their novel pharmaceutical components (Oh et al., 2002, Fei et al., 2004, Kondo et al., 2004). Here, we found that Trichosanthes kirilowii methanol extract (TKE) induces cell cycle arrest at the G2/M phase and has activity inhibiting tubulin polymerization in HepG2 cells. This is the first study to report the pharmaceutical mechanisms of Trichosanthes kirilowii tubers

Acknowledgement

This study was supported by a grant from the Oriental Medicine R&D Project (B050018), Ministry of Health and Welfare, Republic of Korea.

References (30)

  • B. Carlson et al.

    Down-regulation of cyclin D1 by transcriptional repression in MCF-7 human breast cancer cells induced by flavopiridol

    Cancer Research

    (1999)
  • S.H. Chan et al.

    Trichosanthin interacts with acidic ribosomal proteins P0 and P1 and mitotic checkpoint protein MAD2B

    European Journal of Biochemistry

    (2001)
  • C.M. Dou et al.

    Effect of extracts of Trichosanthes root tubers on HepA-H cells and HeLa cells

    World Journal of Gastroenterology

    (2004)
  • X. Fei et al.

    Isolation of a putative ribosome inactivating protein from dried roots of Trichosanthes kirilowii used in traditional Chinese medicine

    Planta Medica

    (2004)
  • Y. Hirose et al.

    Abrogation of the Chk1 mediated G2 checkpoint pathway potentiates temozolomide-induced toxicity in a p53-independent manner in human glioblastoma cells

    Cancer Research

    (2001)
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