Troglitazone Inhibits Growth Of Liver Cancer Cells And Its Functional Mechanism

BACKGROUD &OBJECTIVE :Primay hepatic cancer (PHC) is now the most common solid tumor in the world and the third leading cause of cancer-related death. More than 500 000 new cases are currently diagnosed yearly, with an age-adjusted worldwide incidence of 5.5–14.9 per 100 000 population and its incidence is increasing worldwide because of the dissemination of hepatitis B and C virus infection. Unfortunately, PHC carries a dismal prognosis due to rapid growth rate and nonspecific clinical presentation during the early stages of the disease .Surgical resection is considered to be the curative treatment of patients with PHC. However, only 10-20% of PHC are found to be resectable at the time of diagnosis. Liver transplantation, which offers the potential to both resect the entire potentially tumor-bearing liver and eliminate the cirrhosis, holds great theoreticalappeal in treating this disease.However, transplantation is also not widely available,because of the limited supply of donor organs and great ecnomy burden.In addition ,recurrence of PHC is frequent even after curative surgical treatment. Systemic chemotherapy has never been shown to prolong survival in patients with PHC .Thus, The development of effective therapeutic agents is urgently needed for the control of this disease.Peroxisome proliferator-activated receptors (PPARs) belong to the ligand-dependent nuclear receptor superfamily which includes receptors for steroid, retinoid, and thyroid hormones. Three isoforms have been identified to date, namely, PPARa, PPARγ, and PPARc. PPARγis one of the most intensively studied PPAR isoforms, with diverse roles regulating lipid homeostasis, cellular differentiation, proliferation and the immune response. PPAR-γexerts its effects by forming heterodimers with the 9-cis-retinoid X receptor after ligand activation. Once activated by a ligand,this heterodimer binds to a specific DNA sequence designated peroxisome proliferator response element (PPRE) located in the promoter region of PPAR t?arget genes and regulates their transcription . Ligands for PPARγinclude the natural prostaglandin 15-deoxy-12,14-prostaglandin J2 (15d-PGJ2),polyunsaturated fatty acids, a variety of nonsteroidal antiinflammatory drugs and a class of oral antidiabetic agents, the thiazolidinediones (TZDs). PPARγwas initially recognized as a key regulator of adipogenic differentiation ,glucose homeostasis, and inflammation. Recent studies have demonstrated that PPARγwas highly expressed in certain human cancers and its ligands exhibit antineoplastic effects on tumor cells by affecting differentiation,inducing growth arrest and apoptosis,in different cancer types.It is therefore suggested that PPARγligands as a promising new therapeutic alternative in the treatment of cancer.The purpose of this study was to further clarify the molecular mechanism responsible for the anti-PHC effects of troglitazone in order to evaluate the potential therapeutic use of these drugs in PHC.METHODS:1. Cells growth was assessed by the 3-(4-,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.2. Cell cycle was analyzed by flow cytometry.3. TUNEL (terminal deoxynucleotidyl transferase-mediated nick end labeling of DNA fragmentation sites) was used to study apoptosis of cultured cells.4. Morphological changes were examined under phase-contrast microscopy.5. Differentiation marker albumin was determined by bromocresol green dye-binding method. Dedifferentiation marker AFP was measured by chemiluminescence immunosorbent assay.6. Immunocytochemistry was performed assessing the subcellular location ofβ-catenin and survivin, expression of actived-caspase-3 and E-cadherin.7. Expression of Cyclin D1,c-Myc,survivin and Bax were detected by Western blotting.8. Statistical analysis: Data were expressed as mean±SD. Statistical correlation of data was checked for significance by the ANOVA and paired Student's t test. P<0.05 was considered significant.RESULTS:1. Effect of troglitazone on growth of HepG2 cellsHepG2 cells were incubated with varying concentrations(5,10,20,40,80,and 100μmol/L) of troglitazone for 120 h. Cells growth was determined by an MTT assay. The percentage of cells growth were 96.8%±1.22%,53.4%±1.2%,42.3%±1.2%,31.4%±1%,13.6%±0.8% and 9.6%±0.7%,respectively.Troglitazone significantly inhibited the growth of HepG2 cells in a dose-dependent manner. 2. Low doses of troglitazone induced differentiation of HepG2 cells.2.1 Cell Cycle AnalysisAfter exposure of HepG2 cells to 10μmol/L of troglitazone for 120 h, Flow cytometry analysis demonstrating troglitazone caused increase in the G0/G1 phase(67.6±0.5% vs.56.3±1.5%, t=12.02,P < 0.01 ) and decrease in the S phase ( 20.6±0.5%vs. 25±1.0%,t=6.5,P<0.01)as compared to control,indicating that troglitazone induced G0/G1 phase arrest of the cell cycle.2.2 Effect of troglitazone on differentiation markerUntreated HepG2 cells were all negative for E-cadherin expression. After exposure of HepG2 cells to 10μmol/L of troglitazone for 120 h, about 80% cells showed E-cadherin expression ,as demonstrated by immunocytochemistry. Simultaneously,a reduction of AFP level[850.3±37.8(ug/L) vs.471.3±61.1(ug/L),t=9.13,P < 0.01 ) and a dramatic increase of albumin level[0.53±0.05(g/L) vs.0.3±0.1%(g/L), t=3.5,P=0.025 ) were observed in 10μmol/L of troglitazone treated cells.2.3 Effect of troglitazone on ?-catenin subcellular localization and expression of c-Myc and cyclin D1After exposure of HepG2 cells to 10μmol/L of troglitazone for 120 h, ?-catenin translocated from nucleus into the cytoplasma. These effects were accompanied by reduced expression of c-Myc and cyclin D1, which are two of of ?-catenin target genes involved in the control of cell proliferation and differentiation.3 High doses of troglitazone induced differentiation of HepG2 cells.3.1 Morphological changes were examined under phase-contrast microscopy.Troglitazone induced dramatic morphologic changes of cell death on HepG2 cells. As a untreated control, cells were flat and elongate. In contrast, after treated with 20μmol/L and 30μmol/L troglitazone for 24 h,partial cells became rounded in shapes. Further, the cell membrane shrunk with a condensed cytoplasm,subsequently lost contact with neighboring cells, and finally floated into medium.3.2 Cell Cycle AnalysisWhen analyzed by flow cytometry, PI-stained apoptotic cells appeared as a sub-G0/G1 peak, which represents apoptotic cells with subdiploid DNA staining. In the untreated control cells, none of cells were found in sub-G0/G1 peak, but after 20μmol/L and 30μmol/L troglitazone for 24 h, 25.5±1.1% and 43.9±1.7% cells were found in sub- G0/G1 peak,respectively.3.3 TUNELCells were treated with 20μmol/L and 30μmol/L troglitazone for 24 h, they exhibited typical morphological changes of apoptosis including cytoplasmic and nuclear shrinkage, chromatin condensation and fragmentation.Troglitazone significantly increased the number of TUNEL positive cells in a concentration-dependent manner, TUNEL positive cells reached 22.4±1.2% at the troglitazone concentration of 20μmol/L ,and 38.6±0.8% at the troglitazone concentration of 30μmol/L.3.4 Effect of troglitazone on actived caspase-3 expressionAfter treated with 20μmol/L and 30μmol/L troglitazone for 24 h. The percentage of actived caspase-3 positive cells was 21.2±1.4% and 35.8±2.4%,respectively. None of cells in the untreated control cells were positive .The actived caspase-3 showed intense cytoplasmic and nuclear staining.3.5 Effect of troglitazone on subcellular localization of anti-apoptotic factor survivinIn the untreated control cells, survivin immunostaining is predominantly in the nucleus. After treated with 20μmol/L and 30μmol/L troglitazone for 24 h, survivin incompletely translocated from nucleus into the cytoplasma. Simultaneously, the expression intensity was reduced.3.6 Western blottingAfter treated with 20μmol/L and 30μmol/L troglitazone for 24 h ,the expression of anti-apoptotic factor survivin was downregulated by troglitazone, while pro-apoptotic proteins Bax had no change.CONCLUSION:1. Troglitazone significantly inhibited the growth of HepG2 cells in a dose-dependent manner. 2. Different doses of troglitazone had different molecular mechanism on inhibiting hepatic cancer cells proliferation.3. Low doses(10μmol/L) of troglitazone induced differentiation of HepG2 cells.The underdlying mechanism maybe correlated with regulating ?-catenin signaling.4. High doses(20 and 30μmol/L) of troglitazone induced apoptosis of HepG2 cells. These effects involve modulation on either the localization or expression of survivin,and activation of caspase-3.