title: Berberine and S allyl cysteine mediated amelioration of DEN+CCl4 induced hepatocarcinoma.
abstract: BACKGROUND
  Diethylnitrosamine (DEN) and carbon tetrachloride (CCl4) have been used as initiator and promoter respectively to establish an animal model for investigating molecular events appear to be involved in development of liver cancer. Use of herbal medicine in therapeutics to avoid the recurrence of hepatocarcinoma has already generated considerable interest among oncologists. In this context studies involving S-allyl-cysteine (SAC) and berberine have come up with promising results. Here we have determined the individual effect of SAC and berberine on the biomolecules associated with DEN+CCl4 induced hepatocarcinoma. Effective therapeutic value of combined treatment has also been estimated.
  
  
  METHODS
  ROS accumulation was analyzed by FACS following DCFDA incubation. Bcl2-Bax and HDAC1-pMdm2 interaction were demonstrated by co-immunoprecipitation. Immunosorbent assay was performed to analyze PP2A and caspase3 activities. MMP was determined cytofluorimetrically by investigating JC-1 fluorescence. AnnexinV binding was demonstrated by labeling the cells with AnV-FITC followed by flow cytometry.
  
  
  RESULTS
  CytochromeP4502E1 mediated bioactivation of DEN+CCl4 induced Akt dependent pMdm2-HDAC1 interaction that led to p53 deacetylation, probable cause of its degradation. In parallel, oxidative stress dependent Nrf2-HO1 activation increased Bcl2 expression which in turn stimulated cell proliferation. SAC in combination with berberine inhibited Akt mediated cell proliferation. Activation of PP2A as well as inhibition of JNK resulted in induction of apoptosis after 30 days of treatment. Extension of combined treatment reverted tissue physiology towards control. Co-treated group displayed normal tissue structure.
  
  
  CONCLUSIONS
  SAC and berberine mediated HDAC1/Akt inhibition implicates the efficacy of combined treatment in the amelioration of DEN+CCl4 induced hepatocarcinoma.
authors.affiliation: Molecular Biology and Tissue Culture Laboratory, Post Graduate Department of Zoology, Vidyasagar College, Kolkata-700006, India.
authors.forename: Dipanwita
authors.initials: D
authors.lastname: Sengupta
authors.forename: Kaustav Dutta
authors.initials: KD
authors.lastname: Chowdhury
authors.forename: Avik
authors.initials: A
authors.lastname: Sarkar
authors.forename: Soumosish
authors.initials: S
authors.lastname: Paul
authors.forename: Gobinda Chandra
authors.initials: GC
authors.lastname: Sadhukhan
mesh_terms: D000477:Alkylating Agents / Q000633:toxicity; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001599:Berberine / Q000494:pharmacology*; D015153:Blotting, Western; D002251:Carbon Tetrachloride / Q000633:toxicity*; D006528:Carcinoma, Hepatocellular / Q000139:chemically induced / Q000378:metabolism / Q000517:prevention & control*; D053148:Caspase 3 / Q000378:metabolism; D002478:Cells, Cultured; D003545:Cysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D004052:Diethylnitrosamine / Q000633:toxicity*; D005434:Flow Cytometry; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D056284:Histone Deacetylase 1 / Q000378:metabolism; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D008113:Liver Neoplasms / Q000139:chemically induced / Q000378:metabolism / Q000517:prevention & control*; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D018384:Oxidative Stress / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D000477:Alkylating Agents; D000970:Antineoplastic Agents; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D017382:Reactive Oxygen Species; D001599:Berberine; D004052:Diethylnitrosamine; C065299:S-allylcysteine; D045304:Cytochromes c; D002251:Carbon Tetrachloride; D019392:Cytochrome P-450 CYP2E1; D053148:Caspase 3; D056284:Histone Deacetylase 1; D003545:Cysteine
keywords: 2′,7′-Dichlorofluorescein diacetate, TCA, tri choloro-acetic acid; 5,5-dithiobis (2 nitrobenzoic acid); B; BSA; Berberine; CYP2E1; Carbon tetrachloride; DCFDA; DTNB; Di ethyl nitrosamine; FACS; FITC; GR; HDAC1; HO1; JNK; MMP; NADPH; NBT/BCIP; NaCN; Nrf2; Oxidative stress; PCNA; POT; PP2A; PT; ROS; S; S allyl cysteine; S+B; SAC treated group; SOD; berberine treated group; bovine serum albumin; c-Jun N terminal kinase; co-treated group; cytochromeP4502E1; fluorescence activated cell sorter; fluorochrome iso-thiocyanate; glutathione reductase; hemoxygenase1; histone deacetylase type1; mitochondrial membrane potential; nicotinamide adenine dinucleotide 2′-phosphate reduced; nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate; nuclear factor erythroid 2 related factor 2; p53 acetylation; pMdm2; phosphorylated Mdm2; post treated group; proliferating cell nuclear antigen; protein phosphatease 2A; reactive oxygen species; sodium cyanide; superoxide dismutase; treatment with combination of SAC and berberine;NS, Non significant