p53 is important for the anti-invasion of ganoderic acid T in human carcinoma cells
Introduction
Ganoderma lucidum (Fr.) Karst (Polyporaceae), a Chinese traditional medicinal mushroom, has been used for centuries in East Asia to prevent and treat various human diseases such as hepatitis, immunological disorders and cancers (Kimura et al., 2002, Liu et al., 2002, Woo et al., 2005, Wu et al., 2006, Jiang et al., 2008, Chen and Zhong, 2009, Chen et al., 2010, Weng and Yen, 2010, Yue et al., 2010, Zhou et al., 2011). Until now, many ganoderma triterpenes have been identified from G. lucidum, but only a few of them exhibited anti-invasion and anti-metastasis bioactivity (Li et al., 2005, Wang et al., 2007, Jiang et al., 2008, Yue et al., 2008, Chen et al., 2008, Chen et al., 2010).
MMP-2/9 can degrade some important proteins of ECM and have been implicated in cancer invasion, metastasis and angiogenesis (Liu et al., 2006, Chen et al., 2010). uPA converts inactive plasminogen to active plasmin, which cleaves several ECM components involved in cell adhesion and migration (Jiang et al., 2008, Chen et al., 2010). NO, an endothelial growth factor, specifically mediates the tumor vascularization to regulate blood flow, and its production by iNOS is associated with suppression of tumor development and inhibition of tumor invasion or metastasis (Jenkins et al. 1995). Taken together, MMP-2/9, uPA and iNOS apparently play an important role in tumor invasion or metastasis.
NF-κB is a transcription factor that regulates the expression of MMP-2/9, uPA and iNOS genes, which were involved in invasion, metastasis or angiogenesis. Therefore, inhibition of NF-κB activation may reduce the risk of cancer development and support chemotherapeutics by inhibiting invasion and metastasis (Sun and Andersson, 2002, Liu et al., 2006).
p53 can transcriptionally regulate a wide variety of target genes including those whose products mediate cell motility, cell adhesion and angiogenesis (O’Farrell et al. 2004). In particular, p53 regulates cell polarization and migration (Alexandrova et al. 2000), modulates uPA, iNOS, MMP-2/9 genes involved in cancer invasion, metastasis and angiogenesis (Akama and Eldik, 2000, Guo et al., 2003, Guo and Zheng, 2004); but in different reports it was different regarding how p53 regulates the cell migration and MMP-2/9 expressions (O’Farrell et al., 2004, Liu et al., 2006). Obviously more investigations are required regarding the role of p53 in the regulation of cell motility, cell adhesion, angiogenesis, invasiveness or metastasis and its involvement in the uPA, MMP-2/9, and iNOS genes expression.
Recently it was documented that Ganoderic acid Me (GA-Me) and GA-T inhibited tumor cell invasion, tumor growth and lung metastasis (Wang et al., 2007, Chen et al., 2008, Chen et al., 2010); GA-T induced 95-D cells apoptosis, G1 phase arrest and induced p53 protein expression (Tang et al. 2006); and GA-Me induces G1 arrest in wild-type p53 cells while G1/S transition arrest in p53-null cells (Chen and Zhong 2009). Here, our hypothesis is that GA-T-induced p53 may modify anti-metastasic effect of GA-T in tumor cells. Until now, there is a lack of reports on the role of p53 induced by ganoderic acids in anti-invasion, especially the role of p53 in anti-invasion of GA-T is yet completely unknown. In this study, we would investigate the role of p53 in anti-invasion effect of GA-T in human carcinoma cells.
Section snippets
Reagents
MTT was obtained from GIBCO Industries, Inc. (Invitrogen, Carlsbad, CA). Matrigel® was obtained from Becton Dickinson Labware (Bedford, MA). McCoy's 5A Modified Medium, DMSO and glutaraldehyde were obtained from Sigma (St. Louis, MO). New bovine serum (NBS) and antibiotics (penicillin and streptomycin) were purchased from Sino American biotechnology Co. (Shanghai, China). TNFα was purchased from ProSpec-Tany TechnoGene Ltd. (Rehovot, Israel). Antibodies for actin, uPA, MMP2, MMP9, p65, IкBα
Effect of GA-T on adhesion, aggregation and migration of tumor cells
To evaluate the effect of p53 on anti-invasiveness of GA-T, we assessed the promotable effect on cell aggregation and the inhibitory effect on the adhesion and migration of cancer cells. The cell aggregation results showed that GA-T promoted spontaneous cell-cell aggregation in a dose-dependent manner in both cell lines (Fig. 2), and the aggregation ratio of control was about 27.5 ± 4.0% (###p < 0.001) when HCT-116 p53+/+ cells were treated with 16.3 μM GA-T for 18 h, whereas the aggregation ratio of
Discussion
The multistep cancer metastasis involved aggregation, adhension, migration and angiogenesis. Consequently, multiple factors (such as MMP-2, MMP-9, uPA, iNOS and induced NO) can affect the individual steps of invasiveness or metastasis (Fukumura and Jain, 1998, Chen et al., 2010). Fig. 2 showed that p53 modified the aggregation effect of GA-T on cancer cells aggregation. The reason may be that p53 deletion suppressed the phosphoinositide 3-kinase activity (Guo et al. 2003), and thereby reducing
Acknowledgements
Financial support from the Shanghai Science & Technology Commission (project no. 054319933 and 08DZ1971900), and the Shanghai Leading Academic Discipline Project (project nos. B203 and B505) is gratefully acknowledged. We thank Dr. B. Vogelstein (The Johns Hopkins University Medical Institutions, Baltimore, USA) for providing HCT-116 colon carcinoma cell lines (HCT-116 p53+/+ and HCT-116 p53−/−).
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