Antrodia camphorata extract induces replicative senescence in superficial TCC, and inhibits the absolute migration capability in invasive bladder carcinoma cells

https://doi.org/10.1016/j.jep.2006.07.009Get rights and content

Abstract

The Antrodia camphorata crude extract (ACCE), an extract obtained from a precious traditional Chinese folkloric herbal medicine Zhan-Ku (a camphor tree mushroom) since the 18th century, has showed rather significant inhibitory effects on the growth and proliferation of the transitional cell carcinomas (TCC) cell lines RT4, TSGH-8301, and T24. On treatment with ACCE at 100 μg/mL, the p53-independent overexpression of p21 with simultaneous down alteration of pRb was observed in RT4, which was thus speculative of proceeding through a mechanism of replicative senescence. On the contrary treatment with ACCE, at 50 μg/mL, resulting in simultaneous down-regulations of Cdc2 and Cyclin B1, with suppression of the absolute migrating capability of the two cell lines TSGH-8301 and T24, and eventually the cell deaths. We conclude that ACCE can be rather effective and beneficial in suppression of both the superficial cancer cell line RT4 and the metastatic cell lines (TSGH-8301 and T24) through different mechanisms.

Introduction

Bladder cancer (BC) is a heterogeneous neoplasm, presenting as either primary superficial low grade tumors confined to the superficial mucosa (80%) or deeply muscle-invasive carcinomas (20%) (Knowles, 1999). Consisting of a broad spectrum of tumors including transitional cell carcinoma (TCC), BC has been identified to be a neoplastic lesion caused by environmental and/or occupational factors (Doll and Peto, 1981). Recently it has become the fourth and the eighth most common cancer in men and women in the United States (Jemal et al., 2003), as well as the seventh most common cancer in men with rising incidence and prevalence in Taiwan. In all genitourinary tumors, TCC is the second most common cause of death. Although of superficial low grade, yet with high recurrence rate (Skinner and Lieskovsky, 1988), only a small proportion of BC progresses to invasive diseases, while distant invasive metastases TCC only seldom develop, yet has caused the majority of deaths with TCC being resistant to chemotherapy. Many molecular and genetic changes in TCC of the bladder have exploded, which include (1) chromosomal alternations leading to carcinogenesis, e.g. mutations (point and insertional/deletional), translocation, and loss of alleles, each insult may effect the translated protein products; oncogenes such as c-H-ras, c-myc, and c-erB-2 are believed to be categorized in this event, (2) loss of cell cycle regulation accounting for tumor cell proliferation, several tumor suppressor genes (TSGs) acting at the G0/G1 check point of the cell cycle are now recognized, and their protein products – p53, pRb, p16 and p14 – are vital for preventing cell cycle progression in bladder tumors: inactivation of the Rb gene or increased p53 immunoreactivity has been found in higher grade and stage bladder cancers, which is associated with the disease progression, and the overall and disease-specific survival rates, but these two proteins act in an independent yet synergistic manner in patients with bladder cancers, and (3) metastasis guided by events such as angiogenesis and loss of cell adhesion (Williams and Stein, 2004). This varied presentation results in widely divergent clinical outcomes. Detailed information, which identifies and characterizes the biological potential of various bladder cancers may be employed to dictate suitable treatments for TCC. One approach to control bladder cancers is through growth inhibition by which the disease can be prevented, slowed-down, or reversed substantially. This has been accomplished by the administration of one or more non-toxic naturally occurring or synthetic agents (Gee et al., 2002), among which naturally occurring compound Antrodia camphorata, formerly named as Antrodia cinnamomea Chang & Chou, sp. nov. Polyporaceae, commonly named “Zhan Ku,” or “Zhan Chi”, has come to practical applications (Tsai, 1982).

Antrodia camphorata belongs to the following taxonomy: Kingdom, Mycoteae; Division, Amastigomycota; Subdivision, Basidiomytina; Class, Hymenomycetes; Order, Aphyllophorales; Family, Polyporaceae; Genus, Taiwanofungus; Species, camphorautus (Peng et al., in press).

Taiwanofungus camphoratus Sheng H. Wu, Z.H. Yu, Y.C. Dai & C.H. Su is proposed as a new genus of lignicolous family Polyporaceae (Wu et al., 2004). Originally, Ganoderma comphoratum M. Zang & CH. Su was once designated as its generic type (Zang and Su, 1990), in fact it did not belong to the genus of Ganoderma due to a careless misidentification from the contaminated specimen of Ganoderma spores, hence later renamed as Antrodia cinnamomea T.T. Chang & W.N. Chou, sp. nov. Polyporaceae (Chang and Chou, 1995). Recently, two new species of Genus Taiwanofungus have been identified, viz. Taiwanofungus camphoratus and Taiwanofungus salmoneus (T.T. Chang & W.N. Chou) Sheng H. Wu, Z.H. Yu, Y.C. Dai & C.H. Su. In folkloric remedies, it is popularly and commonly called “Zhan Ku”, “Zhan Chi”, “niu chang ku” or “jang-jy”, which in Chinese means “The mushroom from the camphor tree Cinnamomum kanehirai Hay Lauraceae in Taiwan” (Su, 2002), further re-named as Antrodia camphorata (Zang & Su) Sheng H. Wu, Ryvarden & T.T. Chang (Wu et al., 1997). Although the latter has been used for years and recently has been further assigned with a novel Latin name Taiwanofungus camphoratus (Wu et al., 2004), still it is not very popular (Peng et al., in press). In this manuscript, we are determined to use this traditional old name Antrodia camphorata in description of such a unique Formosan (Taiwanese) mushroom.

A. camphorata had long been popularly used as an amazing folkloric medicine by the Taiwan aborigine long before 1773 (Su, 2002) for the treatment of twisted tendon and muscle damages, terrified mental state, influenza and cold, headache, fever, and many internally affiliated diseases. In fact, since the eighteenth century, it has gradually become a more and more popular folkloric medicine with various effects as a carminative, a tonic, a laxative, an antidote, an anti-bacterial, a sedative, an analgesic, for the treatment of malignant tumors, viral infection, stomachitis, diabetes mellitus, nephritis, proteinuria, hepatoma, influenza, and motion-sickness (Su, 2002). Recently, it has been further investigated for its apoptotic (Song et al., 2005, Hsu et al., 2005), anti-oxidative (Hsiao et al., 2003), and anti-inflammatory (Shen et al., 2004) effects on human hepatocellular carcinomas or leukocytes. Yet, no documented effects of A. camphorata in treatment of bladder cancers have been described. In this study, we try to investigate the effect of the A. camphorata crude extract (ACCE) on three bladder cancer cell lines (RT4, TSGH-8301, and T24), which have been used as models of non-invasive (RT4), the moderately invasive (TSGH-8301), and the invasive (T24) bladder cancer, respectively. Through such an investigation, much clinical benefits can be expected in finding the molecular determinants of various bladder tumor cell sensitivity or resistance to A. camphorata.

Section snippets

Antrodia camphorata crude extract (ACCE)

The A. camphorata used in this study was supplied by Well-Shine Biotechnology Development Co., Ltd. (Taipei, Taiwan), whereas ACCE was prepared as previously described (Hsiao et al., 2003). The ACCE was dissolved in 100% EtOH at a concentration of 80 mg/mL and stored at −20 °C. The concentrations of ACCE used were 10–200 μg/mL, respectively.

Separation of fat fraction and ergosterol

The extraction method for fatty acids was according to Folch et al. (1957) and the extraction solvent used was a mixture of methanol and chloroform. Methods

ACCE is enriched in ergosterol and unsaturated fatty acids

The fatty acid content obtained in the extract of A. camphorata fruiting bodies (ACCE) was 1.46, 14.74, 6.20, 14.34, 10.78, and 12.69 mg/g for C14:0, C16:0, C17:0, C18:0, C18:1 and C18:2, respectively, whereas the ergosterol content was 0.71 ± 0.24 μg/g (Table 1).

Cell growth characters affected by ACCE are related with the status of the cell differentiation

On addition of ACCE (50 μg/mL) to TSGH-8301 and T24, respectively, and to RT4, with ACCE at 100 μg/mL, the macroscopic growth appearances after incubated for 72 h were all distinctly affected. As can be seen in Fig. 1, for RT4, the cell

Conclusion

In conclusion, ACCE has showed rather different significant inhibitory effects on the growth and proliferation of TCC cell lines, RT4, TSGH-8301, and T24. In terms of the cell cycle regulatory protein expressions, RT4 proceeds most probably through the mechanism of replicative senescence, as evidenced by the p53-independent overexpression of p21 with simultaneous down alteration of pRb. On the contrary, growth inhibition of TSGH-8301 and T24 as affected by simultaneous down-regulations of Cdc2

Acknowledgement

The authors are indebt to Mr. Jr-Hung Yan for his valuable and helpful assistance in the experimentations. Special thanks due for the research grant offered by NSC-94-2311-B003-001.

References (39)

  • J. Bubenik et al.

    Established cell line of urinary-bladder carcinoma (T24) containing tumor-specific antigen

    International Journal of Cancer

    (1973)
  • S.J. Chatterjee et al.

    Combined effects of p53, p21, and pRb expression in the progression of bladder transitional cell carcinoma

    Journal of Clinical Oncology

    (2004)
  • R.J. Cote et al.

    Elevated and absent pRb expression is associated with bladder cancer progression and has cooperative effects with p53

    Cancer Research

    (1998)
  • M.B. Datto et al.

    Transforming growth factor beta induces the cyclin-dependent kinase inhibitor p21 through a p53-independent mechanism

    Proceedings of the National Academy of Sciences of the United States of America

    (1995)
  • R. Doll et al.

    The causes of cancer: quantitative estimates of avoidable risks of cancer in the United States today

    Journal of National Cancer Institute

    (1981)
  • L. Fang et al.

    p21Waf1/Cip1/Sdi1 induces permanent growth arrest with markers of replicative senescence in human tumor cells lacking functional p53

    Oncogene

    (1999)
  • G. Hsiao et al.

    Antioxidative and hepatoprotective effects of Antrodia camphorata extract

    Journal of Agricultural and Food Chemistry

    (2003)
  • A. Jemal et al.

    Cancer statistics

    CA: A Cancer Journal for Clinicians

    (2003)
  • T. Kawasaki et al.

    Abrogation of apoptosis induced b DNA-damaging agents in human bladder-cancer cell lines with p21/WAF1/CIP1 and/or p53 gene alterations

    International Journal of Cancer

    (1996)
  • Cited by (60)

    • Antrodia cinnamomea ameliorates neointimal formation by inhibiting inflammatory cell infiltration through downregulation of adhesion molecule expression in vitro and in vivo

      2021, Food Science and Human Wellness
      Citation Excerpt :

      The inflammatory responds, oxidative stress and abnormal proliferation are the common pathologic basis of cancer and vascular injury. A. cinnamomea has been shown to harbor a broad spectrum of antitumor efficacy, such as liver, prostate, bladder, lung and breast through its antiflammatory and antioxidant activity [12,13,32,33]. In addition, polysaccharides isolated from A. cinnamomea inhibit angiogenesis in endothelial cells [34,35].

    • Fungal secondary metabolites: A potential source of anticancer compounds

      2020, New and Future Developments in Microbial Biotechnology and Bioengineering: Recent Advances in Application of Fungi and Fungal Metabolites: Applications in Healthcare
    • Antrodia cinnamomea produces anti-angiogenic effects by inhibiting the VEGFR2 signaling pathway

      2018, Journal of Ethnopharmacology
      Citation Excerpt :

      Whole extracts and active compounds of A. cinnamomea produce various biological activities, such as reduction of inflammation (Huang et al., 2014), liver protection (Ao et al., 2009), immunomodulation (Chen et al., 2008) and antioxidant effects (Song and Yen, 2002). Previous studies have shown that A. cinnamomea promotes anticancerogenic activity in cancer of the liver, prostate, bladder, lung and breast (Chiang et al., 2010; Chen et al., 2007; Peng et al., 2007; Yang et al., 2006; Huang et al., submitted for publication). In addition, polysaccharides isolated from A. cinnamomea inhibit angiogenesis in bovine endothelial cells (Cheng et al., 2005) and human endothelial cells (Yang et al., 2009; Cheng et al., 2011).

    View all citing articles on Scopus
    1

    These authors have contributed equally.

    View full text