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Keywords:

  • apoptosis;
  • cell cycle regulation;
  • metastasis;
  • thiazolidinediones;
  • treatment of liver cancer

Abstract

  1. Top of page
  2. Abstract
  3. Hepatocellular carcinoma
  4. PPARs and PPARγ
  5. Expression of PPARγ in HCC
  6. TZDs induce cell cycle arrest and apoptosis in HCC
  7. TZDs can induce growth inhibition independently of PPARγ
  8. PPARγ deficiency induces susceptibility to tumorigenesis
  9. PPARγ overexpression inhibits HCC growth
  10. PPARγ overexpression induces apoptosis
  11. PPARγ induces anti-metastasis effects
  12. TZDs reduces HCC risk among diabetes mellitus patients
  13. Adverse effect of TZDs
  14. Conclusion
  15. References

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Major risk factors of HCC include infection with hepatitis B or C viruses, alcohol and non-alcoholic fatty liver disease. HCC is difficult to diagnose at early stage, and has a very poor survival rate when diagnosed at a late stage. The majority of HCC-related deaths result from local invasion (to cause liver failure) or distant metastases. There is an urgent need to identify effective molecular targets for the treatment of the disease. As the target of an established class of therapeutic agent thiazolidinediones (TZDs), peroxisome-proliferator-activated receptor γ (PPARγ) has been widely studied for its role in the development of HCC. A substantial body of evidence based on in vitro and in vivo models indicates that the activation of PPARγ is able to inhibit HCC cell proliferation and tumor growth through inducing cell cycle arrest and apoptosis via the regulation of a panel of downstream effector molecules. PPARγ activation also induces an inhibitory effect on HCC metastasis. Meanwhile, there is new evidence suggesting that PPARγ inhibition could also be anti-tumorigenic. In the present review, we summarize the available information on the role of PPARγ in HCC development and spread, and discuss whether PPARγ activation by TZDs could play a role in the treatment of HCC, summarizing both in vitro and in vivo. Considering the available data, PPARγ seems to exert beneficial effects against HCC and may therefore represent as a therapeutic target.


Hepatocellular carcinoma

  1. Top of page
  2. Abstract
  3. Hepatocellular carcinoma
  4. PPARs and PPARγ
  5. Expression of PPARγ in HCC
  6. TZDs induce cell cycle arrest and apoptosis in HCC
  7. TZDs can induce growth inhibition independently of PPARγ
  8. PPARγ deficiency induces susceptibility to tumorigenesis
  9. PPARγ overexpression inhibits HCC growth
  10. PPARγ overexpression induces apoptosis
  11. PPARγ induces anti-metastasis effects
  12. TZDs reduces HCC risk among diabetes mellitus patients
  13. Adverse effect of TZDs
  14. Conclusion
  15. References

Primary liver cancer is the second leading cause of cancer death in men and seventh in women. In 2008, there were an estimated 748 000 new cases and 696 000 deaths worldwide and half of the cases occurred in China.1 Rates of liver cancer are two to four times as common in males than in females. Among different types of liver cancers, hepatocellular carcinoma (HCC) accounts for 70–85% of all primary hepatic malignancies. Cholangiocarinomas, which arise mainly from the epithelial lining of bile duct, are relatively rare.2 Major risk factors for HCC include infection with HBV or HCV, alcoholic liver disease, and non-alcoholic fatty liver disease (NAFLD). Most of these risk factors lead to the development of cirrhosis, which is found in 80–90% of HCC patients. The 5-year cumulative risk for the development of HCC in patients with cirrhosis ranges between 5% and 30%, with the highest risk among those infected with HCV, particularly in the presence of other risk factors (obesity, type 2 diabetes, previous heavy alcohol consumption, HIV or HBV coinfection).3

PPARs and PPARγ

  1. Top of page
  2. Abstract
  3. Hepatocellular carcinoma
  4. PPARs and PPARγ
  5. Expression of PPARγ in HCC
  6. TZDs induce cell cycle arrest and apoptosis in HCC
  7. TZDs can induce growth inhibition independently of PPARγ
  8. PPARγ deficiency induces susceptibility to tumorigenesis
  9. PPARγ overexpression inhibits HCC growth
  10. PPARγ overexpression induces apoptosis
  11. PPARγ induces anti-metastasis effects
  12. TZDs reduces HCC risk among diabetes mellitus patients
  13. Adverse effect of TZDs
  14. Conclusion
  15. References

The peroxisome-proliferator-activated receptors (PPARs) are a subfamily of the 48-member nuclear-receptor superfamily. PPARs regulate gene expression in response to ligand binding.4 Various fatty acids serve as endogenous ligands for PPARs. To date, three PPARs have been identified including PPARα, PPARδ (also known as PPARβ), and PPARγ. Upon ligand binding, PPARs undergo specific conformational changes that allow for the recruitment of co-activator proteins.5 Differences in the affinity of ligand binding with co-activators explain the various biologic responses observed.6–8

PPARs regulate gene transcription by transactivation and trans-repression. Transactivation is DNA-dependent and involves binding to PPAR response elements of target genes, as well as heterodimerization with the retinoid X receptor (RXR).5 Trans-repression, on the other hand, involves interfering with other transcription-factor pathways in a DNA-independent way.8

PPAR γ is expressed most abundantly in adipose tissue but low in tissues that predominantly express PPARα, such as the liver, the heart, and skeletal muscle.5 Hepatic PPARγ expression is increased in several animal models of NAFLD, a process termed adipogenic transformation. The discovery of PPARγ as the target for thiazolidinediones (TZDs) led identification of several therapeutic agents, and to clinical trials in type 2 diabetes as well as non-alcoholic steatohepatitis (NASH). The regulatory role of PPARγ in lipid metabolism and insulin sensitizing is clear; thus, its agonists TZDs have been used as an established class of insulin sensitizers in treating type 2 diabetes.

Most of our understanding about a potential role of PPARγ in cancer has been through studying TZDs. In cancer cell lines, xenografts and other animal models, TZDs inhibit the growth of several cancers including lung,9 breast,10 colon,11 and prostate.12 Loss-of-function mutations of PPARγ have also been found in thyroid malignancy and colon carcinomas.13,14 Contrasting effects have also been reported. In one murine model of colon cancer, PPARγ activation by TZDs was found to increase tumor formation.15

Expression of PPARγ in HCC

  1. Top of page
  2. Abstract
  3. Hepatocellular carcinoma
  4. PPARs and PPARγ
  5. Expression of PPARγ in HCC
  6. TZDs induce cell cycle arrest and apoptosis in HCC
  7. TZDs can induce growth inhibition independently of PPARγ
  8. PPARγ deficiency induces susceptibility to tumorigenesis
  9. PPARγ overexpression inhibits HCC growth
  10. PPARγ overexpression induces apoptosis
  11. PPARγ induces anti-metastasis effects
  12. TZDs reduces HCC risk among diabetes mellitus patients
  13. Adverse effect of TZDs
  14. Conclusion
  15. References

Previous reports showed inconsistent results regarding PPARγ level in human HCC tissues. Koga et al. showed in five cirrhotic patients that there was no difference in PPARγ expression between HCC and the surrounding non-tumorous cirrhotic liver.16 In another study, Schaefer et al. showed by immunohistochemistry a constant overexpression of PPARγ protein in 20 HCCs but no expression in benign liver parenchyma remote from the tumor site, regardless of the presence of viral hepatitis (hepatitis B or C).17 More recently, Yu et al. found a significant decrease in PPARγ expression in HCC compared to adjacent liver, as assessed by Western blot.18 Therefore, further studies in a larger sample size are needed before conclusions can be drawn.

TZDs induce cell cycle arrest and apoptosis in HCC

  1. Top of page
  2. Abstract
  3. Hepatocellular carcinoma
  4. PPARs and PPARγ
  5. Expression of PPARγ in HCC
  6. TZDs induce cell cycle arrest and apoptosis in HCC
  7. TZDs can induce growth inhibition independently of PPARγ
  8. PPARγ deficiency induces susceptibility to tumorigenesis
  9. PPARγ overexpression inhibits HCC growth
  10. PPARγ overexpression induces apoptosis
  11. PPARγ induces anti-metastasis effects
  12. TZDs reduces HCC risk among diabetes mellitus patients
  13. Adverse effect of TZDs
  14. Conclusion
  15. References

Koga et al. were among the first group to demonstrate the inhibitory effect of PPARγ activation on HCC cell growth.16 They showed that troglitazone, a potent PPARγ agonist, induced a dose-dependent inhibition of cell growth in HCC cell lines including HLF, HAK-1A, HAK-1B and HAK-5. At 50 µM, troglitazone induced G0/G1 cell cycle arrest associated with upregulation of p21, p27, and p18. However, the HCC cell line Huh7, which lacks p21, did not show apparent G0/G1 arrest.

Koga et al. further showed that troglitazone-induced p27 was associated with downregulation of Skp2 in HCC. Skp 2, an F-box protein component of the SCF ubiquitin-ligase complex, was found to be universally upregulated in surgically resected human HCC tissues.19 Troglitazone downregulated Skp2 at the mRNA level. But when Skp2 was ectopically overexpressed, troglitazone induced G0/G1 arrest was attenuated.

Yu et al. confirmed the growth inhibition induced by troglitazone in another two HCC cell lines (Hep3B and Huh7).18 At a concentration of 100 µM, troglitazone caused G0/G1 arrest in both cell lines as evidenced by flow cytometry, and induced cell apoptosis as evidenced by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. p27 was demonstrated to play a major role in G0/G1 arrest instead of p21. Yu et al. also showed reciprocity in expression between PPARγ and COX-2, another potential mediator in hepatic tumorigenesis. Using a xenograft model in which Huh7 cells were subcutaneously implanted into nude mice, feeding with 200 ppm troglitazone was found to inhibit growth of the xenografted tumor compared with control chow.18 A diagram of the molecular basis of PPARγ activation by TZDs in regulating cell cycle arrest is shown in Figure 1.

image

Figure 1. Schematic diagram showing the pathways that peroxisome-proliferator-activated receptor γ (PPARγ) activation triggers, leading to cell cycle arrest, apoptosis, and the inhibition of cell proliferation and cell metastasis in hepatocellular carcinoma (HCC).

Download figure to PowerPoint

TZDs can induce growth inhibition independently of PPARγ

  1. Top of page
  2. Abstract
  3. Hepatocellular carcinoma
  4. PPARs and PPARγ
  5. Expression of PPARγ in HCC
  6. TZDs induce cell cycle arrest and apoptosis in HCC
  7. TZDs can induce growth inhibition independently of PPARγ
  8. PPARγ deficiency induces susceptibility to tumorigenesis
  9. PPARγ overexpression inhibits HCC growth
  10. PPARγ overexpression induces apoptosis
  11. PPARγ induces anti-metastasis effects
  12. TZDs reduces HCC risk among diabetes mellitus patients
  13. Adverse effect of TZDs
  14. Conclusion
  15. References

Although an inhibitory role of troglitazone on HCC cells growth is evident, there have been reports suggesting the effect could be PPARγ independent. Palakurthi et al. demonstrated that two TZDs, troglitazone and ciglitazone, were able to inhibit cell growth in both PPARγ−/− and PPARγ+/+ mouse embryonic stem cells in a dose-dependent manner by blocking G1-S transition.20 Evidence showed the effect was induced by inhibiting translation initiation. There were subsequent reports supporting the PPARγ-independent anti-cancer effect of troglitazone.21,22 One study featured on the HCC developed in HBV-transgenic mice showed the TZD anti-tumorigenic effects, such as reduced tumor incidence, anti-proliferation and apoptosis, were more profound in PPARγ-deficient mice compared with control animals with normal PPARγ expression.23

PPARγ deficiency induces susceptibility to tumorigenesis

  1. Top of page
  2. Abstract
  3. Hepatocellular carcinoma
  4. PPARs and PPARγ
  5. Expression of PPARγ in HCC
  6. TZDs induce cell cycle arrest and apoptosis in HCC
  7. TZDs can induce growth inhibition independently of PPARγ
  8. PPARγ deficiency induces susceptibility to tumorigenesis
  9. PPARγ overexpression inhibits HCC growth
  10. PPARγ overexpression induces apoptosis
  11. PPARγ induces anti-metastasis effects
  12. TZDs reduces HCC risk among diabetes mellitus patients
  13. Adverse effect of TZDs
  14. Conclusion
  15. References

Using a genetically engineered PPARγ heterozygous deficient mouse model, Yu et al. demonstrated PPARγ deficiency resulted in significantly enhanced liver carcinogenesis, as induced by diethylnithrosamine (DEN) compared with wild-type littermates.24 Further, rosglitazone treatment was effective in reducing tumor size in wild-type mice but not in such PPARγ deficient mice. Using cDNA array and chromatin immunoprecipitation (CHIP) assay, Yu et al. also identified GDF15 as a direct target gene of PPARγ. GDF-15 is inducible by both rosiglitazone and adenoviral vector carrying PPARγ overexpression. As a distant member of the transforming growth factor (TGF)-β, GDF-15 is overexpressed in multiple types of cancer as an anti-tumorigenic response (reviewed by Bauskin et al. 2006).25 Consistently, ectopic overexpression of GDF-15 was able to reduce cell viability and enhance apoptosis in Hep3B.

PPARγ overexpression inhibits HCC growth

  1. Top of page
  2. Abstract
  3. Hepatocellular carcinoma
  4. PPARs and PPARγ
  5. Expression of PPARγ in HCC
  6. TZDs induce cell cycle arrest and apoptosis in HCC
  7. TZDs can induce growth inhibition independently of PPARγ
  8. PPARγ deficiency induces susceptibility to tumorigenesis
  9. PPARγ overexpression inhibits HCC growth
  10. PPARγ overexpression induces apoptosis
  11. PPARγ induces anti-metastasis effects
  12. TZDs reduces HCC risk among diabetes mellitus patients
  13. Adverse effect of TZDs
  14. Conclusion
  15. References

More recently, Yu et al. demonstrated direct effects of PPARγ overexpression in HCC cells.24 PPARγ overexpression mediated by adenoviral vector in Hep3B cells inhibited cell growth in a time- and dose-dependent manner. Cell cycle analysis revealed PPARγ overexpressing cells were often arrested in G2/M phase, in contrast to the G0/G1 arrest by troglitazone as reported previously. The G2/M phase arrest was found to associate with Cdc25C phosphatase activation by Ser216 phosphorylation. Phosphorylated Cdc25C is known to bind to members of the 14-3-3 proteins, sequestering it into the cytoplasm and prevents premature mitosis.26 In the presence of both PPARγ overexpression and rosiglitazone, a more pronounced decrease in Hep3B cell viability was observed, attributable to the synergistic effect of the two agents.

PPARγ overexpression induces apoptosis

  1. Top of page
  2. Abstract
  3. Hepatocellular carcinoma
  4. PPARs and PPARγ
  5. Expression of PPARγ in HCC
  6. TZDs induce cell cycle arrest and apoptosis in HCC
  7. TZDs can induce growth inhibition independently of PPARγ
  8. PPARγ deficiency induces susceptibility to tumorigenesis
  9. PPARγ overexpression inhibits HCC growth
  10. PPARγ overexpression induces apoptosis
  11. PPARγ induces anti-metastasis effects
  12. TZDs reduces HCC risk among diabetes mellitus patients
  13. Adverse effect of TZDs
  14. Conclusion
  15. References

Yu et al.24 have also reported evidence that PPARγ-stimulation induces apoptosis through both an intrinsic mitochondrial caspase-dependent pathway and the extrinsic death receptor-triggered apoptotic pathway. Overexpression of PPARγ in Hep3B cells induced Fas and tumor necrosis factor α (TNFα), which activates the extrinsic apoptosis pathway through Fas-associated death domain, as mediated by caspase-8, an initiator caspase, followed by the cleavage of downstream effector caspases.24 Intrinsically, PPARγ overexpression activates the transcription of Bax and the release of caspase-activating proteins into the cytosol, resulting in the activation of the apoptotic protease activating factor 1 (APAF-1), to form an activation complex with caspase-9. Activated caspase-9 then triggers downstream effector caspases, including caspases-3 and -7, initiating the caspase-dependent cascade leading to apoptosis (Fig. 1). In contrast, a study demonstrated that PPARγ inhibition could induce anchorage dependent apoptosis (anoikis).17 In this study, though activator-induced apoptosis was evident, PPARγ inhibition was found to induce a more pronounced apoptotic effect at a low dosage, and through a different mechanism. PPARγ inhibitor T000907 or PPARγ-specific siRNAs reduced HCC cell adherence to the extracellular matrix by reducing phosphorylation of focal adhesion kinase (FAK), and thereby induced caspase-dependent apoptosis. The authors also found that PPARγ was overexpressed in HCC.

PPARγ induces anti-metastasis effects

  1. Top of page
  2. Abstract
  3. Hepatocellular carcinoma
  4. PPARs and PPARγ
  5. Expression of PPARγ in HCC
  6. TZDs induce cell cycle arrest and apoptosis in HCC
  7. TZDs can induce growth inhibition independently of PPARγ
  8. PPARγ deficiency induces susceptibility to tumorigenesis
  9. PPARγ overexpression inhibits HCC growth
  10. PPARγ overexpression induces apoptosis
  11. PPARγ induces anti-metastasis effects
  12. TZDs reduces HCC risk among diabetes mellitus patients
  13. Adverse effect of TZDs
  14. Conclusion
  15. References

More than 90% of HCC-related deaths are the result of advanced local disease with liver failure or are linked to distant metastatic disease. In two HCC cell lines, MHCC97L and BEL-7404, Shen et al. showed PPARγ or its agonist, rosiglitazone inhibited metastatic activity in vitro, as demonstrated by wound healing, cell migration, and invasion assays.27 Using an orthotopic HCC metastasis model that implants xenograftic tumors into the left liver lobes of nude mice, rosiglitazone treatment significantly reduced lung metastases (three out of nine cases) compared to control treatment (seven out of eight) cases. Complementary DNA profile of PPARγ overexpressed MHCC97L cells was also investigated. Notably, matrix metallopeptidases (MMP) 9 and 13 and heparanase (HPSE) were downregulated, while TIMP3 and E-cadherin were upregulated.

Matrix metallopeptidases 9 and 13 mediate the turnover of extracellular matrix and degradation of cell surface molecules such as E-cadherin; their function contributes essentially to cancer cell migration. Upregulation of HPSE has been associated with increased lymph node, as well as distant metastases,28 and reduced postoperative survival of cancer patients.28,29 Among these important metastatic genes, CHIP assay identified direct interactions between PPARγ and its binding sites in the promoters of TIMP3, MMP9, MMP13 and HPSE, inferring that these genes could be direct targets of PPARγ in liver cancer cells (Fig. 1).

TZDs reduces HCC risk among diabetes mellitus patients

  1. Top of page
  2. Abstract
  3. Hepatocellular carcinoma
  4. PPARs and PPARγ
  5. Expression of PPARγ in HCC
  6. TZDs induce cell cycle arrest and apoptosis in HCC
  7. TZDs can induce growth inhibition independently of PPARγ
  8. PPARγ deficiency induces susceptibility to tumorigenesis
  9. PPARγ overexpression inhibits HCC growth
  10. PPARγ overexpression induces apoptosis
  11. PPARγ induces anti-metastasis effects
  12. TZDs reduces HCC risk among diabetes mellitus patients
  13. Adverse effect of TZDs
  14. Conclusion
  15. References

Although PPARγ activation has been demonstrated to exert inhibitory roles on HCC biology in in vitro and in vivo models, there are no clinical data supporting the use of TZDs as potential HCC chemopreventives to reduced HCC incidence in those at risk or for HCC treatment. The only clinical insight that can be gleaned is based on studying diabetes patients. Clinical data show that people with type 2 diabetes are more prone to cancer, among which the most frequent (at least in Japanese studies) is HCC. In a population-based case-control study conducted in the US that studied 2061 HCC patients, and 6183 healthy controls, diabetes patients were found to have two- to three-fold increased risk of HCC regardless of the presence of other HCC risk factors including HBV, HCV infection, or alcoholic liver disease.30 A population study from Taiwan further confirmed the increased risk of HCC among patients with diabetes, and found that use of either metformin or TZDs was associated with apparently reduced HCC risk.31 It is not known whether the reduced HCC risk was an effect of ameliorated metabolic syndrome, or also an effect of PPARγ activation induced anti-tumorigenesis effect. An extensive body of observations on metformin use, an anti-diabetic drug whose action is independent to PPARγ, may be interpreted in favor of non-PPARγ metabolic effects, but it remains possible that these indirectly alter hepatocyte PPARγ expression. Further human studies are required to establish whether this is the case.

Adverse effect of TZDs

  1. Top of page
  2. Abstract
  3. Hepatocellular carcinoma
  4. PPARs and PPARγ
  5. Expression of PPARγ in HCC
  6. TZDs induce cell cycle arrest and apoptosis in HCC
  7. TZDs can induce growth inhibition independently of PPARγ
  8. PPARγ deficiency induces susceptibility to tumorigenesis
  9. PPARγ overexpression inhibits HCC growth
  10. PPARγ overexpression induces apoptosis
  11. PPARγ induces anti-metastasis effects
  12. TZDs reduces HCC risk among diabetes mellitus patients
  13. Adverse effect of TZDs
  14. Conclusion
  15. References

As reviewed above there is substantial evidence demonstrating the anti-tumorigenic effect of TZDs on HCC and other types of cancer based on in vitro and in vivo studies. Clinically, the use of TZDs has been proven to be effective in reducing HCC risk among diabetic patients, possibly through ameliorating metabolic syndromes.30,31 However, the use of TZDs has only been approved in type 2 diabetes but never in any type of cancer. Moreover, the safety of TZDs remains controversial, as many of its members, especially the most studied ones, were found to induce various adverse effects. Troglitazone was the first approved TZD for treatment of type 2 diabetes mellitus, but by the year 2000 it had been withdrawn due to rare but severe instances of drug-induced liver injury.32 In human hepatocytes, troglitazone induced reactive oxygen species production accompanied by decrease in mitochondrial trans-membrane potential and mitochondrial ultra-structural changes, leading to disrupted mitochondrial function.33 Rosiglitazone, meanwhile, was found to increase the risk of myocardial infarction and was withdrawn in Europe and Australia in 2010 and restricted in the United States. The low-density lipoprotein (LDL) elevation caused by rosiglitazone may be a contributing factor of the suspected adverse cardiac effects.34 More recently, pioglitazone exposure was found to be associated with increased risk of bladder cancer,35 and the debate about use of these agents (which also cause weight gain and have been implicated in osteoporosis and fluid retention) is on-going.

Conclusion

  1. Top of page
  2. Abstract
  3. Hepatocellular carcinoma
  4. PPARs and PPARγ
  5. Expression of PPARγ in HCC
  6. TZDs induce cell cycle arrest and apoptosis in HCC
  7. TZDs can induce growth inhibition independently of PPARγ
  8. PPARγ deficiency induces susceptibility to tumorigenesis
  9. PPARγ overexpression inhibits HCC growth
  10. PPARγ overexpression induces apoptosis
  11. PPARγ induces anti-metastasis effects
  12. TZDs reduces HCC risk among diabetes mellitus patients
  13. Adverse effect of TZDs
  14. Conclusion
  15. References

The role of PPARγ in HCC has been widely studied but conflicting data exist regarding the PPARγ expression level in HCC tissue and whether PPARγ activation actually promotes or inhibits HCC growth and viability. The majority of evidence tends to support PPARγ as an anti-tumorigenic factor in HCC, particularly evident by its inhibitory role in tumor growth and metastasis. Most of these conclusions have been drawn based on studying the effect of TZDs. But TZDs, which had only been approved in treating type 2 diabetes (and most have now been withdrawn), are not yet ready to extend their use in cancer because of concern about adverse effects. If PPARγ does prove to be a worthwhile target for chemoprevention and/or treatment of HCC among the millions so affected, attention may need to be directed at non-pharmacological modulation of hepatocyte PPARγ expression.

References

  1. Top of page
  2. Abstract
  3. Hepatocellular carcinoma
  4. PPARs and PPARγ
  5. Expression of PPARγ in HCC
  6. TZDs induce cell cycle arrest and apoptosis in HCC
  7. TZDs can induce growth inhibition independently of PPARγ
  8. PPARγ deficiency induces susceptibility to tumorigenesis
  9. PPARγ overexpression inhibits HCC growth
  10. PPARγ overexpression induces apoptosis
  11. PPARγ induces anti-metastasis effects
  12. TZDs reduces HCC risk among diabetes mellitus patients
  13. Adverse effect of TZDs
  14. Conclusion
  15. References