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Potential therapeutic role of Tridham in human hepatocellular carcinoma cell line through induction of p53 independent apoptosis.

Jaganathan R, Ravinayagam V, Panchanadham S, Palanivelu S - BMC Complement Altern Med (2013)

Bottom Line: Chromatin condensation, DNA fragmentation and apoptotic bodies, which are structural changes characteristic of apoptosis, were found following TD treatment of Huh7 cells.The p53 gene expression was found to be unaltered in TD treated cells.These results suggest that TD induces apoptosis of Huh7 cells through activation of Bax and triggered caspase cascade, independent of p53 function.

View Article: PubMed Central - HTML - PubMed

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.

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.

Conclusion: 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.

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Time and dose dependent effect of TD on antiproliferative (based on time kinetics) in Huh7 cells by Tryphan blue exclusion assay. Values are expressed as mean ± SD for three independent experiments.
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Figure 2: Time and dose dependent effect of TD on antiproliferative (based on time kinetics) in Huh7 cells by Tryphan blue exclusion assay. Values are expressed as mean ± SD for three independent experiments.

Mentions: Viability of Huh7 cells treated with TD was assessed by trypan blue assay which showed a more effective action after 48 h treatment when compared to 24 and 72 h treatment (Figure 2). Huh7 cells when treated with TD, showed cell death at the end of 48 h. In the present study, the aqueous extract of TD induced cytotoxicity to Huh7 cells in a dose and time dependent manner. The results indicate that exposure of Huh7 cells to TD triggers the pathway of apoptosis leading to decrease in viability of Huh7 cells.


Potential therapeutic role of Tridham in human hepatocellular carcinoma cell line through induction of p53 independent apoptosis.

Jaganathan R, Ravinayagam V, Panchanadham S, Palanivelu S - BMC Complement Altern Med (2013)

Time and dose dependent effect of TD on antiproliferative (based on time kinetics) in Huh7 cells by Tryphan blue exclusion assay. Values are expressed as mean ± SD for three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222730&req=5

Figure 2: Time and dose dependent effect of TD on antiproliferative (based on time kinetics) in Huh7 cells by Tryphan blue exclusion assay. Values are expressed as mean ± SD for three independent experiments.
Mentions: Viability of Huh7 cells treated with TD was assessed by trypan blue assay which showed a more effective action after 48 h treatment when compared to 24 and 72 h treatment (Figure 2). Huh7 cells when treated with TD, showed cell death at the end of 48 h. In the present study, the aqueous extract of TD induced cytotoxicity to Huh7 cells in a dose and time dependent manner. The results indicate that exposure of Huh7 cells to TD triggers the pathway of apoptosis leading to decrease in viability of Huh7 cells.

Bottom Line: Chromatin condensation, DNA fragmentation and apoptotic bodies, which are structural changes characteristic of apoptosis, were found following TD treatment of Huh7 cells.The p53 gene expression was found to be unaltered in TD treated cells.These results suggest that TD induces apoptosis of Huh7 cells through activation of Bax and triggered caspase cascade, independent of p53 function.

View Article: PubMed Central - HTML - PubMed

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.

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus