The Effect Of Rosiglitazone And Troglitazone On Human Laryngeal Cancer Hep-2 Cell

Laryngeal cancer is otorhinolaryngology primary malignant tumor in the epithelium, accounting for 11%-22% of otorhinolaryngology malignant tumors. Although in recent decades the treatment of laryngeal cancer has made considerable progress, but the survival rate and improvement in the quality of life of patients still need to improve. With the new chemotherapy drugs emerging, the treatment of laryngeal cancer is also transit to the comprehensive treatment consisted of surgery, radiotherapy and chemotherapy, so the research and exploration of laryngeal more and more effective chemotherapy with far-reaching significance.Peroxisome proliferator-activated receptor (peroxisome poliferator-activited receptor, PPAR) is a nuclear transcription factor with multiple biological effects involved in a variety of physiological responses of the body, especially in the fat storage and metabolism play an important unparalleled role. The current studies have shown that PPAR is divided into three subtypes (α,δ,γ), in which PPARy subtypes and tumor most closely, so the study on it functions in the tumor development also is the most extensive and in-depth. a lot of reports have shown that PPARy expression in various tumor tissues, when applied with PPARy agonists will result in reducing the malignant biological behavior of tumor cells such as inhibition of tumor cell proliferation and of invasion, angiogenesis and induction of apoptosis etc. Therefore in recent years PPARy as a target with the rapid progress in drug research, which has become is one of hot research and development of antitumor drugs.PPARy agonists can be divided into natural agonists and synthetic agonists two categories. Synthetic PPARy agonists thiazolidinedione compounds, (TZDs) is most representative, which have the applyzed clinical treatment of diabetes. In recent years, a large number of studies have shown that TZDs can effectively inhibit a variety of tumor cell proliferation, inhibit tumor angiogenesis, block cell cycle progresses, induce apoptosis. Moreover, TZDs as a diabetes treatment drug, because of its good curative effect, less side-effects widely used in clinical practice for many years, so TZDs inhibit the proliferation of the tumor is much more meaningful. Studies have shown that the inhibitory mechanism of TZDs is very complicated, and even some academics have suggested that TZDs anti-tumor effect were through activate PPARy and independent PPARy ways, but the anti-tumor effect of TZDs is needless to be questioned.In ordor to study the impact of TZDs on the laryngeal Hep-2 cells, TZDs on behalf of drug rosiglitazone (RGZ) and troglitazone (TRG) first in vitro on the Hep-2 cells, using MTT, flow cytometry, Real-time PCR, ELISA, western, etc, molecular biology techniques in vitro to observed the impact of TZDs on Hep-2 cell proliferation, apoptosis and hypoxia-induced HIF-la and VEGF and to explore the impact mechanism; Then, based on the observation of our vitro experiments, we observed the impact of the RGZ and TRG on human laryngeal cancer Hep-2 cells nude mice transplanted tumor and explored it mechanism, and the toxicity of RGZ and TRG in this process. For take PPARy as a target for prevention and treatment of laryngeal cancer provide a theoretical basis and experimental evidence, for TZDs development, clinical application provide valuable experimental evidence. This study is divided into the following three parts:The first chapter:the impact of RGZ and TRG on human laryngeal cancer Hep-2 cell proliferation, apoptosis and its mechanismMethods1. The proliferative activity of the Hep-2 cells treated with RGZ or TRG at 12.5,25,50,100μmol/L for 12,24,36,48,60 or 72h was respectively analyzed by MTT assay.2. The apoptosis rate and cell cycle distribution of the Hep-2 cells treated with 50μmol/L RGZ or TRG for 0,24,48 or 72h were respectively analyzed by FCM.3. The expression of COX-2 and p65 mRNA and protein, VEGF mRNA in the Hep-2 cells treated with 50μmol/L RGZ or TRG for 0,24,48 or 72h were respectively analyzed by Real-time PCR and western.4. The VEGF protein levels in Hep-2 cell culture supernatant after treated with 25,50,100μmol/L RGZ or TRG for 24h were detected by ELISA.Results1.The proliferative activity of the cells treated with RGZ or TRG was inhibited obviously. The inhibitory rate of cell growth in dose-dependent and time-dependent manner (P<0.05)2. FCM results showed that after treated with 50μmol/L RGZ or TRG for 0h, 24,48,72h, the proportion of the cells in G0/G1 stage increased in a time dependent manner(P<0.01); that cells in S stage decreased (P<0.01); that cells in G2/M stage also decreased(P>0.05). the apoptosis rate of the cells increased gradually with the prolonging action time of RGZ or TRG, There were significant differences among different time groups(P<0.01).3. Real-time PCR and western results showed that the expression of COX-2,p65 and VEGF mRNA and protein in Hep-2 cells decreased in a time dependent manner after treat with 50μmol/L RGZ or TRG for Oh,24,48,72h, There were significant differences among different time groups(P<0.01).4. The ELISA results showed that the VEGF protein levels in Hep-2 cell culture supernatant decreased in a dose dependent manner after treated with 25,50,100μmol/L RGZ or TRG for 24h. There were significant differences among different time groups(P<0.01).The second chapter:the RGZ and TRG inhibit hypoxia induced HIF-1αand VEGF Expression in Hep-2 cellsMethods1. The proliferative activity of the Hep-2 cells treated with RGZ and TRG at 12.5,25,50,100μmol/L for 12,24,36,48,60,72h under hypoxia was analyzed respectively by MTT assay.2. The level of hypoxia induced VEGF protein in the supernatants of cultured Hep-2 cells and VEGF mRNA treated with 50μmol/L RGZ or TRG at 25,50,100μmol/L were respectively analyzed by ELISA and Real-time PCR.3 The expression of HIF-1αprotein and mRNA in the Hep-2 cells treated with RGZ or TRG at 25,50,100μmol/L under hypoxia respectively were analyzed by western and Real-time PCR.4. The expression of HIF-1α, p-AKT/AKT, p-ERK1/2/ERK1/2, p-STAT3/STAT3 protein in the Hep-2 cells treated with RGZ or TRG at 25,50,100μmol/L under hypoxia were respectively analyzed by western.5. The impact of RGZ or TRG on invasion of Hep-2 cells under normoxia and hypoxia were detected by transwell.6. The impact of RGZ or TRG on MMP-2 activity in Hep-2 cells under normoxia and hypoxia were detected by zymography.Results1. MTT results showed that the proliferative activity of the cells treated with RGZ or TRG was inhibited under hypoxia, this effect even more obviously than that under normoxia. The inhibitory rate of cell growth in dose and time dependent manner(P<0.01).2. Real-time PCR and ELISA results showed that the level of secretive VEGF protein in the supernatants of cultured Hep-2 cells and mRNA in cells decreased in a time dependent manner after treated with RGZ or TRG at 25,50,100μmol/L, There were significant differences among different groups(P<0.01). while HIF-la mRNA have not obvious changes between different time groups (P<0.01).3. Western results showed that the expression of HIF-1α,p-AKT,p-ERK1/2 and p-STAT3 protein in Hep-2 cells decreased in a time dependent manner after treated with RGZ or TRG at 25,50,100μmol/L under hypoxia, There were significant differences among different groups(P<0.01); while AKT,ERK1/2 and STAT3 protein have not obvious changes among different groups (P<0.01).4. transwell invasive studies results showed that RGZ or TRG can inhibit Hep-2 cells invasion, There were significant differences among different groups(P<0.01).5. Zymography results showed that RGZ or TRG can inhibit Hep-2 cell MMP-2 activity under normoxia and hypoxia. There were significant differences among different time groups(P<0.01).The third chapter:the impact of RGZ and the TRG on human laryngeal cancer Hep-2 cell nude mice xenograftsMethods1. After establishment of human laryngeal carcinoma xenografts in nude mice were randomly divided into control group, RGZ experimental group and TRG experimental group, n=6, the treatment group for each 3d to the local injection of RGZ and TRG on tumor tissue at 50μmoL/L; control group was injected with 0.5% DMSO. Were treated 4w.2. Nude mice were observed every day for diet, activity and other physiological conditions. Each tumor size and nude body mass were measured every week, drown tumor growth curve. when the mice were killed at the end of treatment after 4W treatment, stripped and weighted tumor, calculated the quality of tumor inhibition rate. the changes of brain, heart, liver, lung, spleen and kidney and other important organs was observed under optical microscope after HE staining.3. The expression of CD34 antigen of in each nude mice xenografts group was detected by immunohistochemistry method, and calculated staining results for MVD values.4. The expression of COX-2,p65,VEGF and HIF-1αmRNA and protein in each nude mice xenografts group was detected by Real-time PCR and western.Results 1. Compared with the control group, RGZ and TRG inhibited tumor growth, the control group and experimental group of nude mice tumor volume, tumor mass were significantly difference, there were statistically significant difference among three groups (P<0.05).2. No significant adverse effects in nude mice was found during the course of treatment. There were no significant organic changes of heart, liver, lung, spleen, kidneys and other important organs in each group at the end of treatment.3. Compared with the control group, experimental group MVD in tumor tissue was significantly reduced, the difference of MVD values among the three groups were statistically significant (P<0.01).4. Real-time and western PCR results showed that VEGF,COX-2 and NF-κB mRNA and protein and HIF-1αprotein expression was significantly lower in the RGZ and TRG experimental group tumor xenografts in nude mice than that in the control group, the difference between the three groups was statistically significant(P<0.05).Conclusion1. The RGZ and the TRG inhibited Hep-2 cells proliferation in a concentration and time-dependent manner; block Hep-2 cells in the G0/G1 phase and induce apoptosis, its mechanism were related to inhibition of COX-2 and p65 protein and mRNA and VEGF mRNA expression, and reduced VEGF protein secretion.2. The RGZ and the TRG inhibited Hep-2 cells proliferation, inhibited VEGF expression in a dose-dependent manner under hypoxia, inhibited HIF-la protein expression in a dose-dependent manner under hypoxia. Its mechanism were related to inhibiting AKT, ERK1/2 and STAT3 protein phosphorylation and promoting the degradation of HIF-1αprotein through the proteasome pathway, inhibited the invasion of Hep-2 cells under normoxia and hypoxia, its mechanism were relate to reduced activity of the MMP-2.3. The RGZ and the TRG can inhibit laryngeal carcinoma xenografts growth, reduce the value of tumor MVD, down-regulateCOX-2, p65, HIF-1αand VEGF expression in Hep-2 cell xenografts in nude mice. the body had no significant toxic side effects.