Paeonol Enhances The Antiproliferative Effect Of Doxorubicin On HepG2 Cell Line And The Possible Mechanism

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,chemotheray which is a significant way in the treatement of cancer has not play a key role for HCC patients.Doxorubicin(Dox) is perhaps the most widely used agent in the treatment of HCC.However,the response rate of Dox is only 4%-10.5%and the dose-limiting toxicity constrained essentially the clinical application furtherly.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 growth-inhibitory effect of Pae combined with Dox in human hepatoma cell lines HepG₂ and the possible mechanism,in order to develop an effective combination therapy for HCC.1 Effect of Pae combined with DOX on the proliferation of human hepatoma cell line HepG2Investigated the effect of Pae,Dox and Pae combined with Dox on the proliferation of HepG2 respectively by 3-[4,5-dimethylthiazol-2-y1]-2, 5-diphenyltetra-zolium bromide(MTT) assay.The results suggested that Pae at concentrations of 7.81-250mg/L,had the inhibitory effect on the proliferation of HepG2 cells which was dose- and time-dependence.The r values of dose-effect curves for single-agent Pae on HepG2 cell was 0.9612.The IC₅₀ values was 64.30 mg/L.Dox with the concentration of 7.81-250 mg/L also had the analogical effect on the HepG2 ceils which was dose- and time-dependence.The r values of dose-effect curves for single-agent Pae on HepG2 cell was 0.9231.The IC₅₀ values was 0.56 mg/L.Furthermore,various concentrations of Pae(15.63,31.25,62.5mg/L) combining with different concentrations of Dox(7.81-250mg/L) showed synergistically inhibitory effect on the proliferation of the HepG2 cell line.2.The apoptotic effect of Pae combined with Dox on HepG2 cells2.1 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,especially in the combination group. 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 cell line treated with Pae or Dox alone or both.The apoptotic peak was dramatically increased when the cells were exposed to Pae combined with Dox which indicated that Pae had synergistic effect with Dox on inducing apoptosis of HepG2.2.3 Cell cycle perturbation of Pae combined with Dox Mcycle software was used to analyze the kinetic changes of cell cycle distribution.We found that administed groups showed the cell were accumulated at the S phase and decreased at the G0/G1,G2/M.That were significant in combined group which indicated that the Pae enhanced the cell cycle arrest effect of Dox on HepG2 at the S phase.3.Possible mechanism of Pae combined with Dox on the inducing apoptosis effect on HepG2The expression of Bcl-2 and Bax were assayed by Westem-Blot and COX-2, Survivin,Caspase-3 were measured by Immunocytochemical analysis.The results showed that Bel-2,Bax,COX-2,Survivin and Caspase-3 were expressed in different extent.But the Bax and Caspase-3 were up-regulated,the Bcl-2,COX-2 and Survivin were down-regulated after Pae or/and Dox treated,especially in combined group. 4 ConclusionPae at some concentration could enhance the antiproliferation and inducing apoptosis effect of Dox on HepG2 cell line,which may be associated with the cell cycle perturbation and the increased expression of Bax and Caspase-3 and the decreased expression of Bcl-2,COX-2 and Survivin.