PI3K/Akt Signaling Pathway In The PPAR-γ Agonist-induced Cell Cycle Arrest And Apoptosis Of Hepatocellular Carcinoma Cell Line HepG2

Purpose: Hepatocellular carcinoma (HCC) is a significant public health problem and the fifth common cancer and the third leading cause of cancer related death in world. Due to the high morbidity rate and a huge population base, incidence rate of HCC has been becoming the most in China, where HCC is the second leading cause of cancer death now. Due to the onset occult disease, rapid progress, and ineffective treatment, 60%-70% of patients are usually diagnosed with advanced cancer and not available for the surgical treatment when they go to hospital. They have to suffer from a palliative symptomatic treatment finally. Since HCC has the lower radiotherapy and chemotherapy sensitivity and results in the higher multi-drug resistant and toxic side effects, it is very important to develop new treatments strategy and medicines for the patients with HCC to achieve a good survival rates and quality of life. Therefore, understanding the mechanism of pathogenesis of HCC might contribute the achievements of new therapeutic targets and effective drugs. As we knew, the imbalance between cell proliferation and death is considered to be an early and important event in carcinogenic process, so it is desirable to develop new strategy to induce apoptosis, proliferation inhibition, and cell cycle arrest in tumor cells.Peroxisome proliferator-activated receptor gamma (PPAR-γ) is a member of the nuclear receptor superfamily. Recent studies have shown that PPAR-γis associated with differentiation and apoptosis in various human cancers. PPAR-γis capable of being activated by Thiazolidinediones (TZDs) and the activated PPAR-γhas been reported to inhibit cancer cell growth and lead to G1 cell cycle arrest. In lung, prostate, colon, thyroid, gastric and pancreatic cancers, PPAR-γactivation inhibited cell growth in a dose-dependent manner. However, the mechanism by which PPAR-γaffects cell proliferation or differentiation remains unclear.Some studies showed that PPAR-γagonists may play anticancer role by up-regulating PTEN. PTEN gene is found to the first tumor suppressor genes with a new type of phospholipase activity, which serves as a negative regulator of PI3K/Akt signaling pathway in inhibiting cell proliferation and differentiation and promoting apoptosis. Recent studies found that down-regulation of PTEN showed PTEN/Akt-dependent regulation of Skp2 and P27^kip1. Skp2 is the main determinant in the PI3K/Akt-dependent regulation of P27^kip1 and plays a key role in the degradation of P27^kip1. P27^kip1 belongs to cell cycle inhibitory proteins, which negatively regulates cell cycle progression and induce apoptosis of tumor cells through cyclin-CDKI complex. Taken together, it is possible that PPAR-γactivation regulating PTEN/PI3K/Akt/Skp2/P27^kip1 signaling pathway plays an important role in the anti-tumor effects, such as inducing cell cycle arrest and apoptosis.In this study, we investigated the PPAR-γ, PTEN, Akt, pAkt, Skp2 and P27^kip1 protein expression in human liver tumor tissue, analyzed the relationship between their expression and clinical pathological characteristics, and explored the prognostic significance of those in HCC. In order to test the hypothesis that the molecular mechanism of anticancer role of PPAR-γagonist is due to induce HepG2 cell cycle arrest and apoptosis through regulating PTEN/PI3K/Akt signaling pathway, in vitro and in vivo studies were processed. In vitro studies have been carried out to observe the effects of PPAR-γagonist Rosiglitazone(ROZ) and antagonist GW9662, as well as PI3K/Akt signaling pathway inhibitor Wortmannin on proliferation, apoptosis and cell cycle of HepG2 cell line and detect the activation of PTEN/PI3K/Akt signaling pathway and P27^kip1 and Skp2 expression. In vivo growth of implanted HepG2 cells in nude mice was monitored after oral treatment with ROZ to further confirm the antitumor effect the PPAR-γagonist. This study might provide insight the possible mechanism of PPAR-γagonists in the clinical treatment of HCC. Method:1 To explore the significance of PPAR-γ, PTEN, Akt, pAkt, Skp2 and P27^kip1 expression on the carcinogenesis and prognosis of HCCSamples were obtained from 78 HCC patients who had undergone operations at the Fourth Hospital of Hebei Medical University, between 2004 and 2007. The patients were diagnosed histologically and follow-up information intact. The length of the patients'follow-up time ranged from 1 to 61.3 months, and median survival at last follow-up was 31.2 months. None of the patients had received any chemotherapy before surgery. Specimens removed from the body were immediately fixed in 10% neutral formaldehyde solution, embedded in paraffin. Immunohistochemestry was used to assess the expression of PPAR-γ, PTEN, Akt, pAkt, Skp2 and P27^kip1 in 78 cases of HCC and 21 cases of normal liver tissue. The relationship between the expression of key proteins related to PTEN/PI3K/Akt/Skp2/P27^kip1 signaling pathway and the clinicpathological parameters in HCC was also analyzed. The prognostic significance combined with follow-up information was statistically explored with Kaplan-Meier.2 Effects of PI3K inhibitor Wortmannin on cell cycle arrest and apoptosis in hepatocellular carcinoma cell line.Hepatocellular carcinoma cell line HepG2 cultured in non-FBS RPMI-1640 medium were incubated with different concentrations of Wortmannin (0, 10, 50, 100 and 200nmol/L) for 3h, 10h and 24h. MTT was explored to evaluate cell proliferation. Alterations of cell cycle and apoptosis were detected by FCM. Expression of PPAR-γ, PTEN, Akt, pAkt, Skp2 and P27^kip1 was detected by Western blot and RT-PCR.3 Effects of ROZ on cell cycle arrest and apoptosis in HepG2 and the activation of PPAR-γ-PTEN-PI3K/Akt-Skp2-P27^kip1 signaling pathway.HepG2 cultured in 10%FBS RPMI-1640 medium were incubated with different concentrations of ROZ (0, 1, 10, 50 and 100μmol/L) for 24h, 48h and 72h. Alterations of cell cycle and apoptosis were detected by FCM. Expression of PPAR-γ, PTEN, Akt, pAkt, Skp2 and P27^kip1 was detected by Western blot and RT-PCR.HepG2 were incubated with both GW9662 and ROZ (0, 5μmol/L GW9662 and 50μmol/L ROZ, 1μmol/L GW9662 and 50μmol/L ROZ, 50μmol/L ROZ) for 48h. Then the same experiments were repeated as above.4 Effect of PPAR-γagonist ROZ on tumor growth in vivo A HepG2 cell suspension (4×106 cells suspended in 0.2ml of media) was injected subcutaneously into the left scapular of each BALB/c-nu/nu nude mouse. Animals were randomly divided into two groups (5 animals per group): one group received ROZ (30mg/kg/day) and the other received vehicle only. All study medications were given by oral gavages once a day starting from day 5 after injection to the end of week 5. Tumor size was measured once 3 days with callipers in two dimensions: length (a) and width (b). Tumor volume was calculated using the formula (V=a*b2/2). At the end of week 5, all mice were sacrificed by cervical dislocation. Tumors were dissected from the body and the tumor growth curve and inhibition rate were also calculated. All samples for Western blot were frozen immediately in liquid nitrogen and stored at -80℃for analysis. Paraffin embedded tissues were processed and the morphological alterations of tumors were examined by light microscope following HE staining. The cell cycle and apoptosis of tumor single cell suspension were measured by FCM analysis Expression of PPAR-γ, PTEN, Skp2 and P27^kip1 in tumors was detected by Western blot.Results:1 Immunohistochemical analysis1.1 PPAR-γ, PTEN, Akt, pAkt, Skp2 and P27^kip1 expression in HCCThe positive rates of PPAR-γ,Akt,pAkt and Skp2 expression in HCC were significantly higher than those in normal liver tissue groups (P<0.05), while PTEN and P27^kip1 expression in HCC were significantly lower than those in normal liver tissue groups (P<0.05). Positive PPAR-γexpression was correlated with tumor size, cancer-embolus in portal vein and TNM stage. The positive rates of PPAR-γexpression in the cases with cancer-embolus in portal vein, larger tumor size (>5cm) and advanced TNM stages were much higher than those of the control (P<0.05). Low PTEN expression was correlated with tumor size, cancer-embolus in portal vein, invasion or metastasis and TNM stage. The rates of low PTEN expression in the cases with cancer-embolus in portal vein, larger tumor size (>5cm), invasion or metastasis and advanced TNM stages were much higher than those of the control (P<0.05). High Akt/pAkt expression was respectively correlated with tumor size, invasion or metastasis and TNM stage. The rates of high Akt/pAkt expression in the cases with invasion or metastasis, larger tumor size (>5cm) and advanced TNM stages were much higher than those of the control respectively (P<0.05). High Skp2 expression was correlated with tumor size, cancer-embolus in portal vein and TNM stage. The rates of high Skp2 expression in the cases with cancer-embolus in portal vein, larger tumor size (>5cm) and advanced TNM stages were much higher than those of the control (P<0.05). Positive P27^kip1 expression was correlated with tumor size, tumor amount and TNM stage. The positive rates of P27^kip1 expression in the cases with single tumor, larger tumor size (>5cm) and advanced TNM stages were much lower than those of the control (P<0.05).1.2 Survival analysis.Patients with low PTEN expression, high Akt expression, high pAkt expression and high Skp2 expression had a significantly worse patients'survival time than those with converse expression respectively (P<0.05, log-rank test).No significant difference was observed in patients'survival time between the two groups distinguished with PPAR-γ/P27^kip1 expression respectively (P>0.05, log-rank test).1.3 The relationship between expression of PPAR-γ, PTEN, Akt, pAkt, Skp2 and P27^kip1According to the analysis of Spearman rank correlation, PTEN expression in HCC was negatively correlated with the expression of Akt, pAkt and Skp2 respectively, while positively correlated with the expression of P27^kip1. Skp2 expression in HCC was positively correlated with the expression of pAkt, while negatively correlated with the expression of P27^kip1. No significant correlation was observed in HCC between PPAR-γand PTEN,Skp2,pAkt and P27^kip1 expression.2 The effects of Wortmannin on HepG2 and possible mechanism.MTT results showed that Wortmannin significantly inhibited the proliferation of HepG2 cells, with a time- and dose-dependent manner, the maximum inhibitory rate was (74.93±3.25)%; FCM results showed that Wortmannin induced the apoptosis of HepG2 cells; The ratio of cells in G0/G1 phase was significantly increased (P<0.05), while that in S phase was significantly decreased (P<0.05), a significant dose- and time-depended response correlation could be also found. It was showed that wortmannin could induce an arrest of cell cycle in G0/G1 phase. Moreover, with the increasing concentration of wortmannin and prolonging of treatment time, Skp2 expression at both protein and mRNA level was shown decreasing (P<0.05), while there were no difference detected in the PPAR-γ, PTEN and Akt expression by Western blot and RT-PCR. However, western blot analysis indicated that treatment with wortmannin resulted in an increased expression of P27^kip1 at protein level, but a decreased expression of pAkt and Skp2.3 The effects of ROZ and GW9662 on HepG2 and possible mechanismMTT results showed that ROZ can significantly inhibit the proliferation of HepG2 cells, with a time- and dose-dependent; FCM results showed that ROZ induced the apoptosis of HepG2 cells; The ratio of cells in G0/G1 phase was significantly increased (P<0.05), while that in S phase was significantly decreased (P<0.05), a significant dose- and time-depended response correlation could be found. It was showed that ROZ could induce an arrest of cell cycle in G0/G1 phase. We also found PPAR-γand PTEN mRNA expression were increased gradually and Skp2 mRNA expression was decreased in HepG2 cells upon different concentrations and different time of ROZ treatment (P<0.05). Though P27^kip1 mRNA expression was no significant difference upon ROZ treatment, but the expression of P27^kip1 protein was significantly increased; Moreover, Western blot analysis indicated that ROZ increased PPAR-γand PTEN expression and decreased pAkt and Skp2 protein expression (P<0.05). The ROZ-induced anti-cancer effect was significantly reduced when the cells were treated with both ROZ and PPAR-γantagonist GW9662.4 Effects of ROZ on tumor growth in vivoSubcutaneous tumor growth was first palpable 5 days after injection of HepG2. At day 36, there was a significant difference in tumor volume between ROZ treated mice and control mice (0.82±0.12cm3 v 1.39±0.22cm3; p<0.05). Hence the difference between the two groups was more pronounced at week 6 (1.24±0.26cm3 v 2.59±0.79cm3; p<0.05). The volume inhibition rate was 52.13% and the weight inhibition rate was 65.63% when study finished. None of the study animals died during the study. The histopathologic alterations in ROZ treated group mice showed lots of necrotic tumor cells, cells arranging loosely, fibrous hyperplasia, and lymphocyte infiltration in local tumor tissues; Compared with the control, apoptosis rate in ROZ treated group was significantly higher (19.42±2.70)%, and the ratio of cells in G0/G1 phase was significantly increased, while that in S phase cells was decreased significantly (P<0.05); pAkt and Skp2 protein expression in the ROZ treated group was significantly lower than that in control group, but P27^kip1 protein expression was significantly increased (P<0.05). PPAR-γand PTEN protein expression were higher in the ROZ treated group, but this change did not reach statistical significance.Conclusion:1 PPAR-γ, Akt, pAkt and Skp2 expression were increased in hepatocellular carcinoma while PTEN and P27^kip1 expression were decreased. Higher expression of PPAR-γ, Akt, pAkt, and Skp2, and lower expression of PTEN and P27^kip1 play an important role in the development, invasion and metastasis of HCC. PTEN, Akt, pAkt, and Skp2 might serve as the key prognostic factors in HCC.2 Wortmannin could inhibit HepG2 cells proliferation; induce apoptosis and G0/G1 phase arrest. Blocking PI3K/Akt signaling in HepG2 cells resulted in decreased Skp2 protein expression and increased P27^kip1, which suggesting that Skp2 might be involved in the PI3K/Akt-dependent regulation of P27^kip1.3 ROZ could inhibit HepG2 cells proliferation; induce apoptosis and G0/G1 phase arrest. ROZ could up-regulate the expression of PTEN, subsequent induce the inhibition of the PI3K/Akt signaling, decrease Skp2 and increase P27^kip1 expression. The ROZ-induced anti-cancer effect was significantly inhibited by PPAR-γantagonist GW9662. It is suggested that the anti-cancer effects of ROZ in HepG2 cells might be mediated by PPAR-γ-PTEN-PI3K/Akt-Skp2- P27^kip1 signaling pathway.4 ROZ could significantly inhibit the growth of human hepatocellular carcinoma xenografts in nude mice without obvious side effects. As showing the same effects and mechanisms as in vitro, in vivo studies supported the possibility that PPAR-γagonists might become a new clinical treatment for the cancer strategy.