Effects And Mechanisms Of Pae On Human Esophageal Cancer Eca-109 Cell Apoptosis In Vitro And In Vivo

Esophageal cancer(EC)is the sixth most common cancer worldwide.It is estimated that there are about 30,000 new cases of esophageal cancer are diagnosed annually in the world,and approximately half of which occurs in China.Esophageal cancer has two maj or histological types:adenocarcinoma(AC) and squamous cell carcinoma(SCC); the latter is the major type in China.Despite advances in surgical techniques and treatment,the prognosis of esophageal cancer remains poor,with very few long-term survivors.There is a need for novel strategies to improve current therapy.It is imperative to develop more effective and low-toxic chemopreventive and chemotherapy agents for EC.Over the past few years,an increased evidence has been obtained showing that upregulation of cyclooxygenase-2(COX-2) expression could be responsible for chronic infl ammation-related cancer promotion and expression of cyclooxygenase-2(COX-2) is involved in the chronic inflammationrelated development of EC.Chemoprevention of cancers by selective COX-2 blockers has attracted great attention in recent years.The preclinical and clinical studies with the use of coxibs to inhibit carcinogenesis in gastrointestinal tract were very encouraging.Survivin is a new member of the family of proteins,which inhibit apoptosis (inhibitor of apoptosis proteins- IAPs).Expression of Survivin was found in esophageal cancer colorectal cancer,bladder cancer,non-small cell lung cancer,and breast cancer.There is some recent data indicating the correlation of poor prognosis and worse response to chemotherapy in patients with EC expressing survivin.Paeonol(Pae),a major active component extracted from the herb Pycnostelma paniculatum(Bunge) K.S.,and the root of the plant Paeonia Suffruticosa Andrew, possesses extensive pharmacological activities such as sedation,hypnosis,antipyresis, analgesic,antioxidation,antiinflammation,and immunoregulation.In our previous study, the antineoplastic activity of Pae has been demonstrated.The present study was designed to investigate the mechanism of apoptosis on human esophageal cancer Eca-109 cell induced by Pae by detecting changes of expressions of COX-2 and Survivin.1.Effect of Pae on the proliferation of human esophageal cancer Eca-109 cell in vitro and in vivo1.1 Effect of Pae on the proliferation of Eea-109 cell in vitroThe antiproliferative effect of Pae on Eca-109 cell line in vitro was measured by MTT assay.The results suggested that Pae at concentrations of 7.81-250mg·L^-1,had inhibitory effect on the proliferation of Eca-109 cell.The higher the concentration of Pae was,the stronger the cytotoxic effect reached,which suggested obvious dose-dependent manner of Pae.The r values of dose-effect curves for single-agent Pae on Eca-109 cell was 0.908.The IC50 values were 98.68 mg·L^-1.And the inhibitory effects were augmented with the prolongation of culture time(24h,48h,to 72h),which had time-dependence 1.2 Inhibition of tumor growth by Pae in BALB/c nude miceThe growth of implanted tumor was markedly inhibited with Pae groups(50,100,200 mg·kg^-1)and CDDP group(5 mg·kg(-1)),the inhibitory rates being 23.54%,27.91%,34.46%,58.71%respectively(P＜0.05 or P＜0.01).The higher the dose of Pae was,the stronger the antitumor effect reached,which suggested the dose-dependent manner of Pae.The r value of dose-effect reached curves for single-agent on Pae Eca-109 cell line was 0.974.P＜0.05.2.Induced apoptosis of human esophageal cancer Eca-109 cell by Pae in vitro and in vivo2.1 Pae induce apoptosis of human esophageal carcinoma Eca-109 in vitro.Terminal-deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL) technique was performed to detect apoptosis The apoptosis index(AI) was analyze the percent of apoptosis cells.The Eca-109 cell treated with Pae showed the typically apoptotic change after 24h.After 24h,48h and 72h,the apoptosis index were (5.2±0.9)%,(14.7±1.8)%and(18.3±2.4)%respectively(P＜0.01),which had tim e-dependence.A Flow Cytometry(FCM) assay was performed to analyze apoptotic rate of Eca-109 cell treated with Pae for 24h,48h and 72h.The sub-G₁ peak appeared before G₁ phase,which represents apoptotie cell population,was observed clearly treated with Pae The apoptotic rate were augmented with the prolongation of culture time(24h,48h and 72h),which had time-dependence.2.2 Pae induce apoptosis of human esophageal carcinoma Eca-109 in vivoIt was found that the apoptotic cells which stained yellow distributed scatterly or diffusely by TUNEL techniques in vivo.The apoptosis indexes of Pae groups were (11.02±2.58)%,(19.80±2.77)%,(24.48±4.35)%and(27.13±4.39)%respectively,which were significantly higher than(4.81±0.83)%in control group.3.Cell cycle perturbation caused by PaeMcycle software was used to analyze the kinetic changes of cell cycle distribution. We also found that Pae can induced cell cycle arrest at the S phase,along with the decrease in the population of G₀/G₁ phase cells,in the human esophageal cancer Eca-109 cell.4.The effect of Pae on expressions of cyclooxygenase-2(COX-2) and Snrvivin of human esophageal cancer Eca-109 cell in vitro and in vivo Immuochemical S-P methods were used to measure the expression of the protein including COX-2 and Survivin.Metamorph biological image software was performed to analyze the results.The results suggested that Pae could down-regulate the expression of COX-2 and Survivin in vitro and in vivo5.ConclusionPae had inhibitory effect on the proliferation,which may be related with the apoptosis-inducing and cell cycle arrest.Pae induce apoptosis of human esophageal carcinoma Eca-109 in vitro and in vivo,the mechanism of that may be related with down-regulation of COX-2 and Suvivin.