title: Potential therapeutic role of Tridham in human hepatocellular carcinoma cell line through induction of p53 independent apoptosis.
abstract: BACKGROUND
  Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths reported worldwide. The incidence is higher in Asia and Africa, where there is greater endemic prevalence of hepatitis B and C. The devastating outcome of cancer can be minimized only by the use of potent therapeutic agents. Tridham (TD) has been acknowledged since olden days for its wide spectrum of biological properties and was used by traditional practitioners of Siddha and other indigenous systems of medicine. The present study aims at investigating the mechanistic action of TD by assessing the antiproliferative and pro-apoptotic effects on human hepatocellular carcinoma cell line (Huh7).
  
  
  METHODS
  Cell viability and apoptosis assay using MTT analysis and trypan blue staining, DAPI staining, DNA fragmentation, cell cycle analysis, mitochondrial membrane potential, real-time reverse transcription-polymerase chain reaction, western blotting and immunofluorescence staining were determined in Huh7 cells.
  
  
  RESULTS
  Viability studies of TD treated Huh7 cells showed an inhibition in cell growth in time and dose dependent manner. Chromatin condensation, DNA fragmentation and apoptotic bodies, which are structural changes characteristic of apoptosis, were found following TD treatment of Huh7 cells. DAPI staining and agarose gel electrophoresis confirmed the induction of apoptosis by TD. Cell cycle analysis of Huh7 cells treated with TD exhibited a marked accumulation of cells in the sub-G1 phase of the cell cycle in a dose dependent manner. Immunofluorescent staining for Ki-67 showed a higher level of expression in untreated cells as compared to TD treated cells. We observed a significant loss in the mitochondrial membrane potential and the release of cytochrome c into the cytosol in TD treated cells. Down regulation of Bcl-2, up regulation of Bax and Bad as well as activation of caspases-3 and 9 were also observed. The p53 gene expression was found to be unaltered in TD treated cells.
  
  
  CONCLUSIONS
  These results suggest that TD induces apoptosis of Huh7 cells through activation of Bax and triggered caspase cascade, independent of p53 function. This study throws light on the mechanistic action of TD in triggering apoptosis in Huh 7 cells.
authors.forename: Ravindran
authors.initials: R
authors.lastname: Jaganathan
authors.forename: Vijaya
authors.initials: V
authors.lastname: Ravinayagam
authors.forename: Sachdanandam
authors.initials: S
authors.lastname: Panchanadham
authors.affiliation: Department of Pathology, Dr, ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai, Tamilnadu 600113, India. pshanthi9@yahoo.co.in.
authors.forename: Shanthi
authors.initials: S
authors.lastname: Palanivelu
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D045304:Cytochromes c / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism*; D008515:Medicine, Ayurvedic; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D010936:Plant Extracts / Q000737:chemistry / Q000494:pharmacology*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
chemical_list: D010936:Plant Extracts; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D045304:Cytochromes c