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Troglitazone inhibits tumor growth in hepatocellular carcinoma in vitro and in vivo
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.....the observation that treatment of HCC-inoculated mice with troglitazone substantially impaired tumor development and either retarded further growth or effected partial regression of established tumors (depending on the experimental design) is both novel and of potential clinical relevance. This result is supported by a recent study of carcinogen-induced liver cancer in the rat.....
Hepatology Jan 2006
Jun Yu 1, Liang Qiao 1, Lars Zimmermann 2, Matthias P. A. Ebert 3, Hongxia Zhang 1, Wendy Lin 1, Christoph Rocken 4, Peter Malfertheiner 2, Geoffrey C. Farrell 1 *
1Storr Liver Unit, Westmead Millennium Institute, University of Sydney at Westmead Hospital, Westmead, NSW 2145, Australia
2Department of Gastroenterology, Hepatology, and Infectious Diseases, Otto-von-Guericke University, Magdeburg, Germany
3Department of Medicine II, Technical University of Munich, Munich, Germany
4Department of Pathology, Otto-von-Guericke University, Magdeburg, Germany
ABSTRACT
Peroxisome proliferator-activated receptor -gamma (PPAR-gamma) has been implicated in the differentiation and growth inhibition of cancer cells. We examined the effects of PPAR-gamma activation by troglitazone on hepatocellular carcinoma (HCC) cell growth, proliferation, and apoptosis in vitro and in vivo.
We also studied relationships between PPAR-gamma activation and cyclooxygenase-2 (COX-2) expression. Human HCC cell lines Huh7 and Hep3B were cultured in the presence or absence of troglitazone. Cell growth was determined via WST-1 assay, proliferation by cell cycle analysis and proliferating cell nuclear antigen (PCNA) Western blotting, and apoptosis by flow cytometry and TUNEL.
Tumor growth after subcutaneous implantation of Huh7 cells in nude mice was monitored, and the effects of treatment with troglitazone were determined.
In resected HCCs, PPAR-gamma expression was less compared with the histologically normal surrounding liver. In cultures of Hep3B and Huh7 cells, basal expression of PPAR was relatively low, but troglitazone caused dose-dependent induction of PPAR-gamma expression. Cell cycle analysis revealed a decreased proportion of cells in S phase, with arrest at G0/G1. Concomitant downregulation of PCNA and an increase in TUNEL staining, cells were consistent with decreased proliferation and induction of apoptosis by troglitazaone. Troglitazone-mediated PPAR activation also suppressed COX-2 expression and induced p27 in HCC cells.
Administration of troglitazone to Huh7 tumor-bearing mice significantly reduced tumor growth and caused tumor regression. In conclusion, collectively, these results indicate that PPAR-gamma could be a regulator of cell survival and growth in HCC. PPAR-gamma therefore represents a putative molecular target for chemopreventive therapy or inhibition of liver cancer growth.
BACKGROUND
On a global scale, hepatocellular carcinoma (HCC) remains the third leading cause of cancer death due to both high incidence and poor survival. Although prevention of hepatitis B and C virus infections (primary prevention), cleaner water supplies, and more adequate antiviral treatment of advanced-stage hepatitis B and C (secondary prevention) can reduce the risk of HCC, there has been growing interest in the chemoprevention of cancer among high-risk individuals. At least some HCCs develop in a stepwise fashion from dysplastic lesions, through adenomatous proliferation to carcinoma in an analogous manner to colorectal carcinoma. The liver is therefore an attractive target for the identification and use of chemopreventive agents. In addition to DNA mutations, the biological processes relevant to hepatocarcinogenesis include disordered hepatocyte proliferation and operation of cell death pathways, particularly by apoptosis; these may be targets for chemopreventive agents.
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear receptors that mediate transcriptional regulation of genes involved in the oxidation, transport, and storage of lipids.[1] In addition to this pivotal role in lipid metabolism, PPARs influence such biological processes as inflammation, cell survival, differentiation, cell proliferation, and tumorigenesis.[2] Among the three PPAR isoforms, PPAR-gamma is of particular interest in this respect. PPAR encodes two isoforms, PPAR-gamma1 and PPAR-gamma2; the human liver expresses mostly PPAR-gamma1.[3] In vitro, ligands that activate PPAR inhibit growth and induce differentiation in breast, prostate, colon, gastric, and liver cancer cells.[3][4] It is less clear whether these findings in cell lines can be translated to in vivo biological effects. Thus, there are conflicting data on whether PPAR-gamma ligands promote or oppose tumorigenesis when applied in different animal models of colon cancer.[5-9] The reasons for these discrepant findings is unclear, but it is salient that colorectal cancer and HCC (especially associated with chronic hepatitis C) share the similarity of multistage tumor development. Furthermore, even though PPAR-gamma agonists inhibit cell growth of some other cancers, the molecular mechanisms of their action is not clear.
In this study, we first determined PPAR-gamma expression in clinical samples of human HCC and nontumorous surrounding liver. We used a prototypic thiazolidinedione, troglitazone, to examine the effects of PPAR-gamma activation on PPAR-gamma expression in two human HCC cell lines: Hep3B and Huh7. Having noted PPAR-gamma activation, we then studied the effects on expression of cyclooxygenase-2 (COX-2), another putative epigenetic pathway of tumorigenesis. Troglitazone exerted impressive biological effects in blocking the growth of HCC cells in vitro, as well as their growth as tumors in a nude mouse model. We therefore explored the locus of biological actions of PPAR-gamma activation in HCC cells in terms of cell proliferation and apoptosis.
DISCUSSION
In the present study, we found a significant decrease in expression of PPAR-gamma mRNA and protein in human liver cancers compared with surrounding nontumorous liver. Similar downregulation of PPAR-gamma expression in tumor tissue compared with adjacent nontumorous mucosa has also been observed in human esophageal,[14] breast,[15] and ovarian cancers.[16] Several in vitro and in vivo model systems suggest that PPAR-gamma may behave as a tumor suppressor gene.[5-7][17][18] In the breast, inactivation of PPAR-gamma with induction of COX-2 occurred during the development and progression of human breast carcinoma.[15][19] On the other hand, inhibition of COX-2 and activation of PPAR-gamma prevented mammary carcinogenesis in experimental animals.[20][21] Other authors have reported that PPAR-gamma activation can enhance tumor formation in different murine models.[8][9][22] Given these apparently conflicting data on whether PPAR-gamma activation could be growth-inhibitory or tumor-promoting, a better understanding of the mechanism of action of PPAR-gamma in liver cancer is required.
We analyzed the effects of PPAR-gamma activation in two human HCC cell lines. In both Huh7 and Hep3B cells, the PPAR-gamma agonist, troglitazone, caused a dose-dependent increase in expression of PPAR-gamma. Concomitant with PPAR upregulation, there was dose-dependent growth arrest; similar results have been observed in other HCC cell lines.[23][24] FACS analysis of the effects of troglitazone on the cell cycle in treated HCC cells revealed a dose-dependent decrease in cell proliferation, a concomitant and proportionate increase of cells in G0/G1, and an increase of cells in the sub-G1 fraction. The latter finding indicates increased apoptosis, a finding confirmed by TUNEL staining.
Because downstream genes regulated by PPAR-gamma have not been well characterized, it is difficult to speculate about the underlying mechanisms for the antiproliferative effects caused by PPAR-gamma signaling in liver cancer cells. On the basis of the immunoblot analysis of known cell cycle inhibitors, G1 arrest by troglitazone was associated with induction of p27 but not p21. In agreement with a recent report,[25] this indicates that hepatocyte cell growth arrest can be mediated by p27 when p21 is downregulated. In the present study, the induction of p27 by troglitazone in Hep3B cells, which lack p53, as well as in Huh7 cells, in which p53 is mutated, indictes that the mechanism of PPAR-mediated p27 induction is likely to be independent of p53. p27 is a potent inhibitor of cyclin D/cyclin-dependent kinase (Cdk) 4 and cyclin E/Cdk2, which kinases govern cell cycle progression at the restriction and late transition points of G1, respectively. The role of p27 as a major player in G1 arrest has been well accepted, and p27 is generally expressed at high levels in quiescent cells.[11][26][27] It is also known that p53, acting through p21, also plays an important role in replicative senescence.[11-13] It was therefore of interest in the present study to observe that the troglitazone-induced p53 in Huh7 cells was nonfunctional in terms of p21 induction, although troglitazone induced cell arrest. The reason for the inability of enhanced p53 to induce p21 in Huh7 cells could be explained by mutation of p53. Furthermore, p53-independent replicative senescence has been demonstrated in some studies in cells that lack p53.[28][29] Other investigations have shown that PPAR activation can cause G1 cell cycle arrest through a mechanism that involves downregulation of protein phosphatase 2A,[30] cyclin D1, and cyclin E.[31]
In the present work, we showed that the growth inhibitory effect of PPAR-gamma activation by troglitazone was also related to induction of apoptosis. In addition to the results in cultured HCC cell lines, treatment with troglitazone increased apoptosis in xenografted tumors in nude mice. Rumi et al.[23] and Koga et al.[24] have also reported that troglitazone induces apoptosis of human HCC cells. However, others have indicated that apoptosis may not be the predominant mechanism through which troglitazone inhibits growth of lung cancer cells.[31][32] Others have suggested that the mechanisms through which troglitazone can induce apoptosis in breast cancer cells include reduced expression of Bcl-2[33] or activation of TRAIL,[34] both resulting in caspase 3 activation.[33]
In the present study, there appeared to be a striking reciprocity between the downregulation of PPAR-gamma we detected in liver cancer and the increased expression of COX-2, which we and others have found to be enhanced in the majority of HCCs.[24][35] A reciprocal relationship between PPAR activity and COX-2 expression has also been observed in colon,[36] cervical,[37] breast,[38] and tongue cancer cells.[39] These findings raise the possibility that high expression of COX-2 might result from the removal of regulatory suppression by PPAR-gamma; the inverse relationship between PPAR expression and COX-2 expression levels in human HCC tumors noted in the present study is consistent with this proposal. We also observed that troglitazone induced a dose-dependent increase in expression of PPAR-gamma with concomitant downregulation of COX-2 in HCC cells. The same effect was also observed in Huh7 xenograft tumors in nude mice, suggesting that COX-2 expression in HCC could be regulated through PPAR-gamma. In other tissues, PPAR signaling has been implicated in the control of COX-2 expression.[36][40][41] It is therefore possible that the inhibitory properties of troglitazone against liver cancer cell growth observed in this study could be explained, at least in part, by the resultant downregulation of COX-2 expression, but this requires further study.
Having observed substantial suppression of HCC cell growth by PPAR-gamma agonist treatment in vitro, we conducted experiments designed to test the potential for PPAR agonists to exert chemopreventive effects against HCC in vivo. It is recognized that the model used in this study - nude mice injected with human HCC cells subcutaneously - is an artificial one and is not necessarily equivalent to the stepwise development of hepatic carcinogenesis during chronic liver disease. Nonetheless, the observation that treatment of HCC-inoculated mice with troglitazone substantially impaired tumor development and either retarded further growth or effected partial regression of established tumors (depending on the experimental design) is both novel and of potential clinical relevance. This result is supported by a recent study of carcinogen-induced liver cancer in the rat.[42] The observation of decreased mitotic rates, decreased PCNA expression, increased apoptosis, and increased P27 induced by troglitazone in vivo was entirely consistent with the in vitro effects, adding further weight to the potential significance of these findings. Because only two selected human HCC cell lines were tested here, the generalizability of these results remain unclear. However, the observation from the human samples that HCC seems to be associated with the underexpression of PPAR - and that such underexpression can be corrected or reversed through exposure to pharmacological doses of a PPAR ligand - adds weight to the potential efficacy of phenolizidinediones against HCC. However, the precise mechanisms for the inhibition of growth by PPAR- ligands needs to be investigated further.
In conclusion, we report that human HCCs underexpress PPAR-gamma in relation to surrounding liver tissue. In HCC cell lines, the PPAR-gamma ligand troglitazone induces PPAR-gamma expression and inhibits cell growth both in vitro and in vivo. The mechanisms appear to involve both inhibition of cell proliferation and induction of apoptosis. We also provide further evidence that ligand-mediated PPAR activation suppressed COX-2 expression in liver cancer cells. The possibility that PPAR-gamma ligands may be effective chemopreventive agents against liver carcinogenesis or novel chemotherapeutic agents is raised by these studies.
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