Several anti-cancer drugs or compounds for cancer treatment have been found in natural products. In this study, we found that aqueous extracts of Chaga mushroom (Inonotus obliquus) caused a growth inhibition of human hepatoma HepG2 cells, and this was closely associated with the arrest of the cell cycle in G0/G1 phase and induction of apoptosis.
The eukaryotic cell cycle is regulated by signal transduction pathways mediated by a series of cell-cycle regulators. Cyclins are positive regulators of cell cycle progression and function by forming a complex with CDKs. CDK inhibitors are negative regulators of the cell cycle and bind to cyclin-CDK complexes and inhibit the activity of those complexes[20
]. Therefore, induction of cell cycle arrest and apoptosis by chemopreventive drugs could be an effective approach to treat uncontrolled cell proliferation and survival in tumor cells.
In recent years, mushroom extracts have been found to have anti-inflammatory, anti-tumor properties[21
]. Although it has been reported that the Chaga mushroom and other mushrooms have therapeutic effects such as anti-tumor, anti-inflammatory and hepatoprotective effects[4
], the mechanisms of anti-inflammatory and anti-tumor effects of the Chaga mushroom have not been clearly elucidated[22
]. In the present investigation, we showed that the water extract of the Chaga mushroom, which has been used in the treatment of cancers and digestive system diseases[2
], significantly inhibited the viability and proliferation, and induced apoptotic cell death in human hepatoma HepG2 cells (Figure ), but this effect was not found in Chang cells, the human immortalized non-tumor cell line (Figure ). These data indicate that Chaga extract has selective cytotoxic effects on human hepatoma cells. This selectivity may be the great advantage of the Chaga extract for therapeutic or preventative use in cancer treatment. Though there were several reports that many natural products induce cell cycle arrest in various cell cycle phases, this study is the first report that reveals the inhibitory effect of Chaga extract on the cell cycle in cancer cell lines. In this study, flow cytometric analysis clearly revealed that HepG2 cells were dose-dependently arrested by Chaga extract at the G0
phase of the cell cycle (Figure ).
The blockade of survival pathways or the induction of apoptosis pathways by anti-cancer agents prevents the proliferation of cancer cells, which may be exploited for cancer therapy[23
]. p53 is a tumor suppressor gene encoding a transcription factor. Its tumor-suppressive activity involves inhibition of cell proliferation through cell cycle arrest and/or apoptosis. Mutation in p53 occurs in more than half of human cancers[24–26
]. Therefore, the activation of p53 by anticancer agents may induce the cell cycle arrest and apoptosis in cancer cells leading to the inhibition of tumor progression. HepG2 has a functional p53 (wt p53), however Hep3B does not have a functional p53 (delete p53)[27
]. In our study, HepG2 cells were more sensitively damaged by Chaga extract than Hep3B cells (Figure ). Therefore, we speculated that wt p53 plays an important role on the apoptosis-inducing effect of Chaga extract in HelpG2 cells. However, our data did not show whether the activation of p53 may mediate the cytotoxicity and cell cycle arrest in Chaga extract-treated HepG2 cells, because wt p53 was down-regulated by Chaga extract (Figure ). Interestingly, Park et al (2006)[28
] showed that 6-gingerol, a major phenolic component in ginger, induced G0
arrest with up-regulation of p21WAF1
expression and down-regulation of p53/pRb expression in pancreatic cancer cells. In the present study, the down-regulation of pRb and p27 protein expression as well as p53 was detected in the Chaga extract-treated HepG2 cells. Our major question remaining to be addressed is that the down-regulation of p53, pRB, and p27 protein, are closely associated with the terminal differentiation or the other signaling pathways levels. However, unfortunately, this question cannot be addressed at the present time.
Eukaryotic cell cycle progression involves sequential activation of Cdks, whose activation is dependent upon their association with cyclins. G1
-phase arrest of cell cycle progression provides an opportunity for cells to either undergo repair mechanisms or proceed by the apoptotic pathway[29
]. It has been suggested that Cdk activities control G1
/S transition in mammalian cells[30
]. Both D- and E-type cyclins are known to be important regulators in G1
/S control, even though some reports raised the possibility that they have other distinct roles[31
]. In our results, we found that treatment with Chaga extract causes a significant decrease in the expression of cyclin D1, D2 and E and Cdk 2, Cdk4, Cdk6 in HepG2 cells. Thus, we speculated that the reduction of cyclin or Cdk expression resulted in the blocking of cyclin/Cdk complex formation and that lowered the levels of pRb (Figure ). When Rb proteins remain in an unphosphorylated form, E2F cannot be activated and the cells fail to enter the S phase[28
]. Based on the data (Figure ), it seems that cyclin D1 and Cdk2, and Cdk6 are responsible for most of the cell cycle arrest observed in response to Chaga extract because these regulators are effectively deceased at the lowest dose of Chaga extract (250 μg/mL).
Taken together, we concluded that Chaga extract induced the growth inhibition, G0/G1-phase arrest, and apoptosis in human hepatoma HepG2 cells, but not in normal Chang liver cells. The present results can provide new hope for chemotherapy of hepatoma cancer. Furthermore, an improved understanding of the interactions between phytochemicals with the genes that are critical to the regulation of cancer cell growths will provide strong armaments to cancer therapy.