Effects And Mechanisms Of Pae On Apoptosis In Human Hepatoma Cell Lines

Background Hepatocellular carcinoma (HCC) is the third most common cancer in China where the incidence is 30.3/100 000. Over the past twenty years, the mortality was increased 41.7% which constituted 42% of all death number in the world. In recent years, the incidence of HCC is increasing in several developed countries, as a result of increased prevalence of HCV infection. The optimal treatment of HCC is hepatic resection. However, more than 80% of patients present with advanced or unresectable disease, and for those patients who do undergo resection, the recurrence rates can be as high as 50% at 2 years. Unfortunately, chemotherapy which is a significant way in the treatement of cancer has not play a key role for HCC patients.Systemic chemotherapy is an important means of hepatocellular carcinoma,especially no surgical indications of advanced hepatocellular carcinoma,Although various chemotherapeutic drugs have been used for the treatment of hepatocellular carcinoma, they are generally not effcetive either when used alone or in combination therapy with other drugs.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 HCC.Chinese herb medicine with the high performance and low venenosus characteristic has attentioned extensively in the prevention and cure of cancer. 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 in vivo and in vitro. Furthermore, we also demonstrated that the Pae could enhanced the antiproliferative effect of cisplatin on human hepatocarcinoma cell lines HepG2, SMMC-7721 and human esophageal carcinoma cell lines Eca-109, SEG-1. The present study was designed to investigate the the possible mechanism of apoptosis in human hepatoma cell lines HepG2,Bel-7402 induced by Pae, in order to develop an effective therapy for HCC.Objective to explore the effect of Pae on apoptosis in HepG2,Bel-7402cell lines and possible mechanismMethods Pae on HepG2,Bel-7402 cell lines was measured by MTT assay.The apoptosis rate was deteceted by flow cytometry and the morphological change of apoptosis was observed by TUNEL. The expression of COX-2,cIAP-1,XIAP,Survivin were assayed by western-blot and immunohistochemical staining.Results1. Effect of Pae on the proliferation of human hepatoma cell lines HepG2,Bel-74021.1 Effect of Pae on the proliferation of human hepatoma cell lines HepG2Investigated the effect of Pae on the proliferation of HepG2 by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra-zolium bromide(MTT) assay. The results suggested that Pae at concentrations of 7.81-62.5mg/L, had the inhibitory effect on the proliferation of HepG2 cells which was dose- and time-dependence.1.2 Effect of Pae on the proliferation of human hepatoma cell lines Bel-7402Investigated the effect of Pae on the proliferation of Bel-7402 by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra-zolium bromide(MTT) assay. The results suggested that Pae at concentrations of 7.81-62.5mg/L, had the inhibitory effect on the proliferation of Bel-7402 cells which was dose- and time-dependence.2. The apoptotic effect of Pae on human hepatoma cell lines HepG2,Bel-7402 2.1 The apoptotic assay by TUNELMorphological evidence of apoptosis was assayed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL).The typically apoptotic changes include chromatin condensation and deformed and fragmented nuclei. The results suggested that the proliferation was prosperous in control group.However, the cell density were decreased significantly and the the number of apoptotic cells increased obviously in treated groups.2.2 The apoptotic detection by FCMA Flow Cytometry (FCM) assay was performed to analyze apoptotic rate. The sub-G1 peak, which appeared before the G0/G1 phase that represents apoptotic cell population, was observed clearly in the HepG2,Bel-7402 cell lines treated with Pae. The apoptotic peak was dramatically increased when the cells were exposed to Pae.The apoptotic rate of Pae at concentrations of 7.81-62.5mg/L on HepG2 is (2.3±1.8)%,(15.6±3.2)%,(27.6±3.9)%(33.2±2.7)%,(45.3±1.7)% respectively;The apoptotic rate of Pae at concentrations of 7.81-62.5mg/L on Bel-7402 is (1.8±1.2)%,(12.5±3.1)%,(25.1±4.2)%,(37.5±3.7)%,(50.3±4.5)%respectively.3.Possible mechanism of Pae on the inducing apoptosis effect on human hepatoma cell lines HepG2,Bel-7402The expression of COX-2 Survivin XIAP cIAP-1were assayed by Western-Blot and Immunocytochemical analysis. The results showed that COX-2,Survivin,XIAP,cIAP-1 were down-regulated after Pae treated.ConclusionPae at some concentration had inhibitory effect and induce apoptosis on HepG2,Bel-7402 cell line, which may be associated with the decreased expression of COX-2,Survivin,XIAP,cIAP-1.