Antrodia camphorata extract induces replicative senescence in superficial TCC, and inhibits the absolute migration capability in invasive bladder carcinoma cells
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.
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These authors have contributed equally.