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The peroxisome proliferator-activated receptor-γ (PPARγ) is a ligand-activated transcription factor belonging to the nuclear receptor superfamily. Peroxisome proliferator-activated receptor-γ ligands can inhibit cell growth and increase apoptosis of cancer cell lines, suggesting a potential role for PPARγ as a tumor suppressor. Whereas the related studies between PPARγ and cancer cell invasion are still poor. Our previous study indicates that β-estradiol (E2) suppresses hepatocellular carcinoma (HCC) cell invasion. We report here that E2 can activate PPARγ of HCC cells, and activated PPARγ suppresses cell invasion by upregulating the expression level of plasminogen activator inhibitor-1 (PAI-1). We found that PPARγ plays an important role in the E2-induced HCC cell invasion process. Using PPARγ agonist GW1929, a reduced invasion effect was found in HCC cell lines, and this inhibition of cell invasion was dosage-dependent. However, cell invasion was restored by treatment with PPARγ antagonist GW9662. The activated PPARγ upregulated the expression of cell migration-related protein PAI-1. Furthermore, knockdown of PPARγ in HCC cells decreased the level of PAI-1 and advanced cell invasion in response to GW1929. On the contrary, overexpression of PPARγ in HCC cells elevated the level of PAI-1 and inhibited cell invasion. These findings suggest that PPARγ activation inhibits HCC cell invasion via the upregulation of PAI-1 and implicate that PPARγ is a target for the treatment and prevention of HCC cell invasion.
Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third most common cause of cancer-related death in the world. An estimated 748 300 new liver cancer cases and 695 900 cancer deaths occurred worldwide in 2008. Half of these cases and deaths were estimated to occur in China. Hepatitis B and C virus infection, aflatoxin B1 (AFB) exposure, alcohol-related cirrhosis and insulin resistance syndrome mainly account for the liver cancer. The high mortality rate is due to its diagnosis at a stage when the disease is already incurable. To date, surgical resection is clinically the most effective treatment for HCC. In addition, sorafenib is the only targeted therapy approved by the US Food and Drug Administration to therapy HCC. However, sorafenib is associated with some side-effects such as liver cirrhosis with impaired metabolic function and dose-limiting toxicities. Thus, there is still a compelling need for a novel strategy that will improve the treatment of HCC and ultimately increase the survival of patients with HCC.
The nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) is a ligand-activated transcription factor that functions as an obligate heterodimer with RXRs. Peroxisome proliferator-activated receptor-γ has been suggested to behave as a tumor suppressor gene. Peroxisome proliferator-activated receptor-γ−deficient (PPARγ+/−) mice were more susceptible to diethylnitrosamine (DEN) -induced HCC than wild-type (PPARγ+/+) mice. Clinical evidence shows that PPARγ protects against colorectal cancer in human. Patients with PPARγ-positive tumors have significantly lower overall mortality than patients with PPARγ-negative tumors.
The 15-deoxy-Δ12,14-prostaglandin J2 is the most potent endogenous ligand for PPARγ. In addition, some unsaturated fatty acids also are natural ligands of the PPARγ. Synthetic PPARγ ligands, known as antidiabetic drugs thiazolidinediones (TZDs), include rosiglitazone, pioglitazone, and troglitazone. Peroxisome proliferator-activated receptor-γ ligands inhibit the growth of HCC cells and induce apoptosis. Peroxisome proliferator-activated receptor-γ could be a regulator of cell survival and growth in HCC.[9, 10] Peroxisome proliferator-activated receptor-γ activation not only inhibits tumor cells growth, but also represses invasion and metastasis. Studies on breast cancer indicate that PPARγ is expressed at significant levels in metastatic tumor cell, but ligand activation of PPARγ causes less malignant state of cells. The metastasis of non-small-cell lung cancer cells (NSCLC) that overexpress PPARγ decreases apparently, and survival rate of tumor-bearing animals also is improved.
In the process of cancer development, direct invasion is a very important mode. This process can make the mass larger, which causes many severe complications. Degradation of the extracellular matrix (ECM) components is important for tumor from invasion to metastasis. Glycosidase, matrix metalloproteinases, cathepsin B and plasminogen activation system play a critical role in this process. Thus, research on catabolic enzymes of ECM has been a focal point in tumor field. Plasminogen activator inhibitor-1 (PAI-1), a member of the serpin family of serine protease inhibitors, inactivates urokinase-type plasminogen activator (uPA) and inhibits degradation of extracellular matrix. There is increasing evidence for involvement of PAI-1 in cell migration, tumor invasion, and metastasis. Growth of some tumors can be attenuated by PAI-1. In addition, Sawai have found that activation of PPARγ by ligands decreases pancreatic cancer cell invasion through increasing PAI-1 and decreasing the uPA level. The results reveal that PAI-1 can be the downstream gene of nuclear receptor PPARγ. Therefore, our interests are attracted by the function of PPARγ in HCC cell invasion and regulation of PPARγ activation on PAI-1 expression.
In the present study, we demonstrate the plasminogen activator system plays a critical role in the HCC cell invasion. We observe that PPARγ activation decreases liver cancer cell invasion by specific PPARγ-dependent regulation of PAI-1. Our data also suggest the potential role of PPARγ agonists therapeutic agents in HCC.
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Apart from the established metabolic actions, PPARγ also plays a critical role in the treatment of cancer. Emerging evidence suggests that activation of PPARγ suppresses tumorigenesis. In this study, we show that PPARγ activation can inhibit HCC cell invasion by upregulating PAI-1. E2 suppresses HCC cell invasion, and this inhibition is associated with PPARγ activity. GW1929 is selected as PPARγ agonist. Peroxisome proliferator-activated receptor-γ activation significantly attenuates HCC cell invasion, and PPARγ overexpression or siRNA also further demonstrates the relationship between PPARγ and HCC cell invasion. In addition, PPARγ activation modulates the expression of PAI-1, which is an inhibitor of uPA. Finally, we show that PAI-1 level rises and uPA activity lowers during the inhibition of HCC cell invasion.
Nevertheless, the function of PPARγ in tumor development is still controversial. Some evidence suggests that activating PPARγ suppresses tumorigenesis, but some studies indicate that activating PPARγ promotes tumorigenesis. The role of PPARγ in the onset and treatment of cancer has been the focus of recent attention. Most research to date indicates that PPARγ agonists can promote terminal differentiation, inhibit cell growth and increase apoptosis of human cancer cell lines, as well as inhibit tumorigenesis in animal models of cancer. Genetic studies in mice demonstrate that loss of one allele of PPARγ gene predisposes mice to cancer. Our results have showed that PPARγ agonist GW1929 can not inhibit HCC cell proliferation (data not shown), but can suppress HCC cell invasion in a dose-dependent manner.
By now, the studies on relationship between PPARγ activation and HCC mainly focus on growth and apoptosis of HCC cells.[9, 10] Koga et al. showed that human hepatoma cell lines exist a concentration-dependent inhibition of cellular growth and arrest of the cell cycle in G0/1 cell after treatment with troglitazone. Peroxisome proliferator-activated receptor-γ agonist inhibits the growth of HCC by inducing apoptosis through caspase 3 activation. However, few studies pay attention to HCC invasion or metastasis. Annicotte et al. reported that PPARγ activation increases the expression of E-cadherin and inhibits invasion of prostate cancer. E-cadherin is one of the major factors that inhibit metastasis and invasion of prostate cancer cells. The studies ultimately showed that E-cadherin is a bona fide PPARγ target gene. Thus, one possibility to explain the inhibitory of PPARγ agonist on HCC cell invasion is by regulating invasion and metastasis-related proteins.
Our studies have showed that when PPARγ is activated by specific agonist GW1929, invasiveness of different HCC cells is significantly inhibited. Some evidence suggests that anticancer activity of PPARγ ligands could occur independently of PPARγ. Thiazolidinediones, which are widely used as insulin-sensitizing agents for the treatment of type 2 diabetes, are synthetic PPARγ ligands. Studies have indicated that TZD inhibits invasiveness of pancreatic cancer cells via PPARγ-independent (“off-target”) mechanisms. How PPARγ ligands can act independent of PPARγ is under study. To address the issue of PPARγ-dependent versus PPARγ-independent mechanisms of action, we used PPARγ overexpression and RNA interference experiments. The results demonstrated that HCC cell invasion is mediated by PPARγ- dependent pathway in our studies. On the basis of the above results we continued to explore the associated effector molecules of PPARγ downstream in the invasion.
As is known, invasion and metastasis are critical determinants of cancer mortality. Breaking through the barrier of ECM is the first step of tumor cell invasion. Matrix metalloproteinase and plasminogen activation system play an extensive role in this process. Therefore, we detect the intracellular content of related molecules in two enzyme systems. The results showed that the effect of PPARγ activation on MMP family is not significant, but the effect on inhibitor PAI-1 of plasminogen activation system is significant. Because PAI-1 is the main inhibitory factor of uPA in vivo, it can suppress ECM degradation. Peroxisome proliferator-activated receptor-γ activation upregulates mRNA and protein levels of PAI-1, and introduction of PPARγ antagonist reverse this effect. Similarly, PPARγ overexpression and RNA interference tests also have indicated PPARγ activation increases PAI-1 expression. Our results are consistent with associated research results of human pancreatic cancer, which were performed by Sawai et al., who inferred PAI-1 may be downstream target gene of PPARγ regulation.
In view of the above results and related articles,[25, 26] we construct a promoter-receptor vector of PAI-1 to further analyze the relationship between PPARγ and PAI-1. The results confirm our conjecture that activation degree of PAI-1 promoter-receptor gene rose along with the increasing concentration of PPARγ agonist. This indicated that PAI-1 can be the downstream gene of PPARγ. It is well known that extracellular PAI-1 reacts rapidly with uPA forming a stable complex with a 1:1 stoichiometry, thus inhibits activity of uPA. We further validated the effect of increasing PAI-1 on inhibition of uPA activity by using enzymatic activity analysis assay. Taken together, we draw some conclusions: PPARγ activation upregulates the expression levels of PAI-1. Increasing secretion of PAI-1 inhibits uPA activity, and thus suppresses the whole plasminogen activation system, ultimately causing the descent of HCC cell invasion.
In conclusion, the present study demonstrated PPARγ agonist upregulates expression levels of PAI-1 by activating PPARγ, increased PAI-1 inhibits extracellular uPA activity, and ultimately HCC cell invasion is suppressed. In addition to classical PPARγ target genes, our findings suggest that PAI-1 can be another target gene of PPARγ. Although some clinical studies show that administration of PPARγ agonists is accompanied by some side-effects, whether these side-effects represent agonist-specific or off-target effects, remains uncertain. This study may provide a molecular mechanism for the development of ‘non-agonist’ modulators of PPARγ, which can target PPARγ without causing the side-effects.