Cancer Letters

Cancer Letters

Volume 216, Issue 1, 8 December 2004, Pages 9-20
Cancer Letters

Ganoderma lucidum extracts inhibit growth and induce actin polymerization in bladder cancer cells in vitro

https://doi.org/10.1016/j.canlet.2004.06.022Get rights and content

Abstract

This study was conducted to investigate chemopreventive effects of Ganoderma lucidum using a unique in vitro human urothelial cell (HUC) model consisted of HUC-PC cells and MTC-11 cells. Ethanol and water extracts of fruiting bodies and spores of the G. lucidum were used to examine growth inhibition, actin polymerization status, and impact of actin remodeling on cell migration and adhesion. Results showed that ethanol extracts had a stronger growth inhibition effect than water extracts. Cell cycle analysis showed that the growth inhibition effect was associated with G2/M arrest. At non-cytotoxic concentrations (40–80 μg/ml), these extracts induced actin polymerization, which in turn inhibited carcinogen 4-aminobiphenyl induced migration in both cell lines. The increased actin polymerization was associated with increased stress fibers and focal adhesion complex formation, however, expression of matrix metalloproteinase-2 and focal adhesion kinase (total and phospholated) were unchanged, which suggests that other mechanisms may be involved.

Introduction

Ganoderma lucidum (Fr.) Karst. (Polyporaceae) is a medicinal mushroom known to the Chinese as ‘Lingzhi’. Its fruiting bodies have been used for their medicinal properties in traditional Chinese medicine for over 2000 years. This mushroom is described in detail in the Chinese Materia Medica classics, Shen Nung Ben Cao Jin (dated 206 BC–8 AD), and the Compendium of Materia Medica. G. lucidum has been used in traditional Chinese medicine for promotion of vitality and longevity, and more recently used in the treatment of debility and weakness, insomnia, hepatitis, bronchitis and asthma, diabetes, altitude sickness, cardiovascular disease, AIDS, and cancer [1], [2], [3]. In addition to the fruiting bodies, the spores and cultivated mycelia of Ganoderma have been recently consumed as health food and herbal medicines. Chemical investigations on the fruiting bodies, spores, and mycelia of G. lucidum reveal that they contain various bioactive substances. Active constituents of G. lucidum include polysaccharides, proteins, nucleosides, fatty acids, terpenoids, sterols, and cerebrosides [4]. Water or alcohol extracts of G. lucidum have been used to investigate biological activities, as these extracts contain compounds mainly responsible for the immunological and anti-inflammatory properties. Alcohol extracts contain biological compounds that lower blood cholesterol and glucose levels, blood pressure, and inhibit histamine release, while the liver-protective, antiviral and anti-tumor effects can be attributed to both the water-soluble polysaccharides and alcohol-soluble triterpenes [3], [4]. Of particular interest among the reported biological/pharmacological properties of G. lucidum are their anti-tumor activities, including the effects on cell cycle arrest, apoptosis induction, motility inhibition, antiangiogenic, and antimutagenic activities [3], [5], [6], [7], [8], [9], [10].

The primary objective of this study was to evaluate the chemopreventive effects of G. lucidum extracts using our unique in vitro human bladder cancer model consisting of two cell lines, both derived from the same human urothelial clone immortalized by SV-40. In this model, HUC-PC is an untransformed cell line that does not form tumor nodules when inoculated into nude mice, and MTC-11 is a transformed low-grade tumor that forms tumor nodules when inoculated into nude mice [11]. After exposure to carcinogen 4-aminobiphenyl (4-ABP), the HUC-PC cells can be transformed to malignant and the MTC-11 cells induced to form a high-grade tumor [11]. This cell culture system provides a unique model to rapidly test the effect of chemopreventive agents on bladder cancer in association with 4-ABP, a major carcinogenic component found in cigarette smoke. In this study, the growth inhibition effect of various Ganoderma extracts on both cell lines was analyzed by [3H] thymidine incorporation assay and calorimetric tetrazolium (MTT) assay. In addition, dose–response effects on actin polymerization were determined by DNase I inhibition assay analysis. The quantitative relationship between cytoplasmic F- and G-actin, the polymeric filaments and monomeric globules, respectively, reflects cellular differentiation versus dedifferentiation status [12]. In general, cell differentiation is associated with an increased F/G-actin ratio, whereas dedifferentiation and malignant transformation is associated with a decreased F/G-actin ratio [13], [14]. Thus, the ratio of F-actin to G-actin functions as a surrogate marker for cellular differentiation and dedifferentiation [13], [14], [15]. Since, actin is a major cytoskeletal protein involved in migration, the functional signification of actin polymerization on cell motility was examined by ‘wound scratching assay’. Mechanism for growth inhibition was investigated by Laser Scanning Cytometry (LCS) based cell cycle analysis and mechanism for migration inhibition was studied by immunofluorescence analysis of adhesion complex, immunoblot analysis of matrix metalloproteinase-2 (MMP-2) expression, and focal adhesion kinase (FAK) activities.

Section snippets

Materials

G. lucidum spore powder was obtained from Zhongke Capsule (Nanjing, China), and G. lucidum fruiting body powder was provided by Pharmanex Inc. (Provo, UT). All solvents used for extraction are HPLC grade and were purchased from Fisher Scientific. 4-Aminobiphenyl (4-ABP) was purchased from Sigma (St Louis, MO).

Extract preparation

Extraction was performed on both spores and fruiting bodies. Briefly, powder from spores or fruiting bodies was first extracted with 95% ethanol and then with water by successive

Growth inhibition and cell cycle analysis of Ganoderma lucidum extracts on HUC-PC and MTC-11 cells

To examine the growth inhibition of G. lucidum extracts, HUC-PC and MTC-11 cells were exposed to various concentrations of the extracts for 24 h and then subjected to [3H] thymidine incorporation assay (Fig. 1a). Both cells were exposed to incremental concentrations of four extracts (15–1000 μg/ml) and incubated for 24 h. Cell survival was calculated as the percentage of [3H] thymidine incorporation assay (% surviva1=mean experimental absorbance/mean control absorbance×100). Overall, the growth

Discussion

In this study we characterized the cell growth, migration and actin polymerization effects of medicinal plant G. lucidum aqueous and alcohol extracts on an in vitro urothelial cell model. The purified, dietary supplements of both fruiting bodies and spores were extracted with ethanol and water to obtain triterpenoid- and polysaccharide-enriched fractions, respectively. Our results show that G. lucidum inhibits cell growth, and at non-cytotoxic concentrations it inhibits 4-ABP-induced migration

Acknowledgements

Research supported by a research grant from the UCLA Center for Dietary Supplement Research in Botanicals (NIH Grant No. U01 Ca96116 and AT00151), UCLA Lung Cancer SPORE (NIH Grant No. P50 CA90833) and the Starr Foundation. Frederick P. Li, MD is a Harry and Elsa Jiler American Cancer Society Clinical Research Professor. We are grateful to Pharmanex Inc. for providing us with the source of fruiting bodies.

References (41)

  • W.H. Yeung et al.

    VLW Go Chemical and Biochemical basis of the potential anti-tumor properties of Ganoderma lucidum

    Curr. Top. Nutraceutical Res.

    (2004)
  • H. Hu et al.

    Ganoderma lucidum extract induces cell cycle arrest and apoptosis in MCF-7 human breast cancer cell

    Int. J. Cancer

    (2002)
  • M.A. Ghafar et al.

    Regression of prostate cancer following administration of genistein combined polysaccharide (GCP™), a nutritional supplement: a case report

    J. Altern. Complement. Med.

    (2002)
  • Y. Gao et al.

    Effects of ganopoly (a Ganoderma lucidum polysaccharide extract) on the immune functions in advanced-stage cancer patients

    Immunol. Invest.

    (2003)
  • B. Lakshmi et al.

    Antiperoxidative, anti-inflammatory, and antimutagenic activities of ethanol extract of the mycelium of Ganoderma lucidum occurring in South India

    Teratog Carcinog Mutagen

    (2003)
  • E.A. Bookland et al.

    Tumorigenic transformation and neoplastic progression of human uroepithelial cells after exposure in vitro to 4-aminobiphenyl or its metabolites

    Cancer Res.

    (1992)
  • J.Y. Rao et al.

    Quantitative changes in cytoskeletal and nuclear actins during cellular transformation

    Int. J. Cancer

    (1997)
  • J.Y. Rao et al.

    Cellular F-actin levels as a marker for cellular transformation: relationship to cell division and differentiation

    Cancer Res.

    (1990)
  • J.Y. Rao et al.

    Cellular F-actin levels as a marker for cellular transformation: correlation with bladder cancer risk

    Cancer Res.

    (1991)
  • J.Y. Rao et al.

    Alterations in phenotypic biochemical markers in bladder epithelium during tumorigenesis

    Proc. Natl Acad. Sci. USA

    (1993)
  • Cited by (0)

    View full text