Although PXR has been recognised for its involvement in xenobiotic/drug metabolism through its role as a ligand-dependent transcription factor in the regulation of detoxifying enzymes such as CYP3A4, UGT1A1, GST and transporters (Handschin and Meyer, 2003
), recent research reveals unexpected novel physiological functions, many of which seem to be rooted at its fundamental role as the sensor and effector guarding the organism against mutagenic insults from xenobiotics and endobiotics. Interestingly, in addition to reducing mutagenic DNA damages, the physiological functions of PXR may have further evolved to protect cells from unregulated proliferation, which is a critical step for tumourigenesis. In earlier studies, we observed that many cancer cells lines derived from GI tract, such as HepG2, HT29 and HCT116, showed loss of PXR expression (Xie et al, 2009
; data not shown). Upon ectopic expression of PXR through transfection, the PXR function could be restored and cells showed significant reduced proliferation, suggesting its role in controlling tumour cell proliferation. It has been reported that certain polymorphisms of PXR
were identified to be strongly associated with the susceptibility to IBD (Dring et al, 2006
), which is a significant risk factor for colon cancer. In patients with IBD, decreased expressions of PXR and its target genes have been observed (Langmann et al, 2004
; Martinez et al, 2007
). On the basis of collective evidence, we hypothesised that PXR has tumour suppressor activity and investigated the effects of PXR expression in PXR-negative colon cancer cells in vitro
and in vivo
(Workman et al, 1998
In this study, we have shown that transfected PXR significantly suppressed HT29 cancer cell proliferation, anchorage-independent growth and tumourigenicity in xenograft assays. In tumour xenograft model, the PXR expression was retained in the HT29 cells 2 weeks after xenograft transplantation into nude mice and the tumour size was markedly suppressed by PXR. Interestingly, results of immunofluorescence double staining suggest that PXR and Ki-67 expressions are mutually exclusive (), suggesting at cellular level the presence of PXR is inhibitory for the colon cancer cell growth. At organ/tissue levels however, PXR may regulate the growth of cell/tissues through different mechanisms. Wan and colleagues reported the mouse lacking PXR showed decreased liver regeneration after partial hepatectomy and the PXR-regulated lipid homoeostasis, which fuels the liver regeneration, may have a role in the suppressed liver growth (Dai et al, 2008
). Verma et al (2009)
reported that PXR activation induced apoptosis in breast cancer cell line and PXR is anti-proliferative and the effect is mechanistically dependant on the local production of NO and NO-dependent upregulation of p53. As the HT29 cells have mutated p53, the mechanism of PXR-regulated inhibitory effects on HT29 growth may differ from the NO-dependent upregulation of p53 reported. Interestingly, Gupta et al (2008)
reported that in ovarian cancer cell lines certain PXR target genes such as CYP3A4
were inducible by rifampicin and the ligand promoted the proliferation of the ovarian cancer cell line in culture and xenograft model. The underlying mechanisms for these differences in the roles of PXR in regulating proliferation and apoptosis of colon, breast and ovarian cancer cells are not clear and warrant further investigation. The important questions that await further investigation are what is the physiological level of PXR expression in various tissues, what are the factors that influence its expression/functions, and ultimately, what is the mechanism by which PXR regulates cell cycle.
To investigate the role of PXR in regulation of cell cycle, we first performed flow cytometry analysis of the effects of PXR on the cell cycles and our study results showed that PXR caused G0
cell-cycle arrest. Cyclin-dependent kinase (CDK) inhibitors p21Cip1/Waf are important as the negative regulator of cell-cycle progression (Gartel and Radhakrishnan, 2005
). Normally p21WAF1/CIP1
has an inhibitory role regarding cyclin D–CDK4 complex phosphorylation of Rb and is regulated principally by the tumour suppressor p53. The p53
gene is mutated in HT29 cell line and is transcriptionally inactive. Our study results showed that p21WAF1/CIP1
expression is almost completely absent in Vector-HT29 cancer cells but highly expressed in PXR-HT29 cells determined by immunohistochemistry analysis and western blot analysis (). Interestingly, the expression of E2F1 was negatively correlated with the p21 expression, suggesting its expression is downregulated by the presence of PXR. It has been shown that control of the p53–p21CIP1
axis by E2F family genes is essential for G1
/S progression and cellular transformation (Sharma et al, 2006
). It is possible that the negative interaction between p21 and E2F1 was lost in certain colon cancer cells and restored on PXR expression, the mechanism of reduction of E2F1 remains to be investigated. Comparison of the apoptotic cells in the xenograft tumour tissues by TUNNEL staining shows no significant difference between the PXR-HT29 and parental HT29 tumours. However, flow cytometry analysis with the Annexin V antibody staining in cell culture showed slight increases in apoptosis in PXR-HT29 cells (data not shown). Therefore, both cell-cycle regulation and apoptosis may have a role in PXR-regulated suppression of colon cancer growth, however, overall our study results shows that the regulation of cell-cycle appears to be a primary mechanism by which PXR inhibits tumour growth.
In conclusion, our study results suggest that PXR has a novel anti-proliferative function in vitro and in vivo, beyond its known role in control of metabolism. This function appears to be exerted at least in part through p21WAF1/CIP1 control of the E2F/Rb pathway in HT29 cells. These novel results suggest potential applications for new therapies for colon cancer treatment based on modulation of PXR function.