Effects Of Recombinant Adenovirus Harboring Melittin Gene, Under The Control Of Transcription Regulatory Element Of A-fetoprotein(rAFP), On Hepatocarcinoma Cellline HepG2 And Its Related Mechanism

IntroductionHuman hepatocellular carcinoma(HCC), with 500,000-1,000,000 new cases and causing approximately 1,000,000 deaths annually, is the fifith most common malignancy tumor in the world. Looking for better treatment of liver cancer treatment has become a major difficulty .As the potential remedy to HCC, the gene therapy has attracted extensive attention and been the hot research topic recently.Melittin can inhibit the growth of tumor in vitro. However, the severe adverse effect of hemolysis greatly limits the clinical application of melittin. To avoid this adverse effect,we assume that melittin gene transfection mediated by recombinant adenovirus,under the control of transcription regulatory element of a-fetoprotein(rAFP),could specifically inhibit proliferation of AFP-positive cells, because hepatocarcinoma cells are very insensitive to adenovirus. Based on this idea,fitstly we successfully construct recombinant adenoviruses carrying melittin gene, under the control of AFP(α-fetoprotein) transcription regulatory element (rAFP),then experiments in both vitro and vivo indicated it could indeed specifically inhibit proliferation of AFP-positive cells. Therefore, the aim of this study is to investigate the attacking mode and molecular mechanism of Ad-rAFP-Mel after it was transduced into HepG2 cells, and provide the theoretical basis for its clinical application, as well as extensive researches on Chinese medicine.Objectives1. To study the effects of melittin on the proliferation of HepG2 cells in vitro, and observe the correspondent morphological changes.2. To explore the related mechanism of death of HCC HepG2 cells caused by Ad-rAFP-Mel.3. To provide experiment evidences for the clinical application of traditional Chinese toxicant in treatment of HCC with gene recombination technologies.Methods1. Phase-contrast microscope, HE stain Hoechst 33258 stain and transmission electron microscope were used to observe the apoptosis and necrosis caused by Ad-rAFP-Mel,and morphological changes of the HepG2 cells and their organelles.2. Apoptosis induced by Ad-rAFP-Mel was detected by FCM using the apoptosis kit including dying with PI and Annexin V-FITC. 3. Rhodamine 123 and PI dyes was used to check the changes of mitochondrial membrane potential of the Ad-rAFP-Mel infected HCC cells determined by FCM, while Fura-2 dye was used to detect the intracellular calcium ion concentration determined by FCM too, both of them demonstrated the state of biomenbrane.4. Western blotting was used to analyze the protein express of caspase family which is essential in cells for apoptosis.Results1. Morphological observation showed that the number of viable cell declined with the increasing of time and multiplicity of infection. Both apoptosis and nectosis cells were found by HE stain ,then the apoptosis of cells was assured through Hoechst33258 stain.2. Treatment of HepG2 cells with Ad-rAFP-Mel at a certain concentration (MOI=100, or above) could induce remarkable apoptosis in a time and concentration dependent manner.3. The mitochondrial membrane potential began to decline from the 12th hour after the transfection, and it declined gradually within 72 hours with the increasing of time and multiplicity of infection,there were significant differences of MMP when all Ad-rAFP-Mel transfection groups were compared with control group and Ad-rAFP group (P<0.05).4.The intracellular calcium concentration began to increase from the 8th hour after the treatment, and it increased gradually with the increasing of time and multiplicity of infection, there were significant differences when all Ad-rAFP-Mel transfection groups were compared with control group and Ad-rAFP group (P<0.05).5. Western blot analysis revealed that Ad-rAFP-Mel transfection induced activation of caspase 9 and 3 8hrs after the Ad-rAFP-Mel transfection, and an upward trend had been seen within 72hrs, but showed no effect on activation of caspase 12.Conclusions1. Ad-rAFP-Mel could inhibit the growth of HepG2 cells,the character of cell death including both nectosis and apoptosis.2. Ad-rAFP-Mel could lead to the reduce of mitochondrial membrane potential and increase of intracellular calcium concentration., this phenomena may be explained through the changes of biomembrane structure caused by it.3. Ad-rAFP-Mel could up-regulate the protein level of caspase family and induce the apoptosis of HepG2 cells, hence, which we presumed were correlated.