Inhibition of mitochondrial cytochrome c release and suppression of caspases by gamma-tocotrienol prevent apoptosis and delay aging in stress-induced premature senescence of skin fibroblasts.

Suzana Makpol,Wan Zurinah Wan Ngah,Norhazira Abdul Rahim,Chua Kien Hui

doi:10.1155/2012/785743

Abstract

In this study, we determined the molecular mechanism of γ-tocotrienol (GTT) in preventing cellular aging by focusing on its anti-apoptotic effect in stress-induced premature senescence (SIPS) model of human diploid fibroblasts (HDFs). Results obtained showed that SIPS exhibited senescent-phenotypic characteristic, increased expression of senescence-associated β-galactosidase (SA β-gal) and promoted G0/G1 cell cycle arrest accompanied by shortening of telomere length with decreased telomerase activity. Both SIPS and senescent HDFs shared similar apoptotic changes such as increased Annexin V-FITC positive cells, increased cytochrome c release and increased activation of caspase-9 and caspase-3 (P < 0.05). GTT treatment resulted in a significant reduction of Annexin V-FITC positive cells, inhibited cytochrome c release and decreased activation of caspase-9 and caspase-3 (P < 0.05). Gene expression analysis showed that GTT treatment down regulated BAX mRNA, up-regulated BCL2A1 mRNA and decreased the ratio of Bax/Bcl-2 protein expression (P < 0.05) in SIPS. These findings suggested that GTT inhibits apoptosis by modulating the upstream apoptosis cascade, causing the inhibition of cytochrome c release from the mitochondria with concomitant suppression of caspase-9 and caspase-3 activation. In conclusion, GTT delays cellular senescence of human diploid fibroblasts through the inhibition of intrinsic mitochondria-mediated pathway which involved the regulation of pro- and anti-apoptotic genes and proteins.

Highlights

Replicative senescence is the final in vitro state reached by all primary cells in culture and is characterized by decreased total cell numbers and loss of proliferation capacity, in which they remain alive and metabolically active [1]
Control young human diploid fibroblasts (HDFs) displayed the normal spindle shape characteristic of fibroblast cells, while prolonged exposure to 20 μM H2O2 resulted in HDFs exhibiting senescence features such as cells were enlarged and flattened with increased size of nucleus
The present study explored the molecular mechanism of GTT in inhibiting apoptosis and delaying cellular aging in H2O2-induced premature senescence (SIPS) of HDFs

Summary

Introduction

Replicative senescence is the final in vitro state reached by all primary cells in culture and is characterized by decreased total cell numbers and loss of proliferation capacity, in which they remain alive and metabolically active [1]. The term stress-induced premature senescence (SIPS) was introduced to represent an irreversible growth arrest of proliferative cells induced by exogenous oxidative agents. The imbalance between cellular oxidants and antioxidants leads to oxidative damage to cellular macromolecules contributing to the decline in cellular functions and progressive organism aging [3]. Vitamin E in particular alpha-tocopherol protects biological membrane from oxidation by acting as a radical chain-breaking molecule. Other biological functions of vitamin E that are unrelated to its antioxidant properties include its roles in cellular signaling, gene expression, immune response, and apoptosis are considered to be of importance [4]

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