Goniothalamin-induced oxidative stress, DNA damage and apoptosis via caspase-2 independent and Bcl-2 independent pathways in Jurkat T-cells.
Goniothalamin (GTN) isolated from Goniothalamus sp. has been demonstrated to induce apoptosis in a variety of cancer cell lines including Jurkat T leukemia cells. However, the mechanism of GTN-induced apoptosis upstream of mitochondria is still poorly defined. In this study, GTN caused a decrease in GSH with an elevation of reactive oxygen species as early as 30 min and DNA damage as assessed by Comet assay. Analysis using topoisomerase II processing of supercoiled pBR 322 DNA showed that GTN caused DNA damage via a topoisomerase II-independent pathway suggesting that cellular oxidative stress may contribute to genotoxicity. A 12-fold increase of caspase-2 activity was observed in GTN-treated Jurkat cells after 4h treatment and this was confirmed using Western blotting. Although the caspase-2 inhibitor Z-VDVAD-FMK inhibited the proteolytic activity of caspase-2, apoptosis ensued confirming that caspase-2 activity was not crucial for GTN-induced apoptosis. However, GTN-induced apoptosis was completely abrogated by N-acetylcysteine further confirming the role of oxidative stress. Since cytochrome c release was observed as early as 1h without any appreciable change in Bcl-2 protein expression, we further investigated whether overexpression of Bcl-2 confers resistance in GTN-induced cytotoxicity. Using a panel of Jurkat Bcl-2 transfectants, GTN cytotoxicity was not abrogated in these cells. In conclusion, GTN induces DNA damage and oxidative stress resulting in apoptosis which is independent of both caspase-2 and Bcl-2.
Inayat-Hussain
S H
SH
Faculty of Allied Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia. salmaan@medic.ukm.my
Chan
K M
KM
Rajab
N F
NF
Din
L B
LB
Chow
S C
SC
Kizilors
A
A
Farzaneh
F
F
Williams
G T
GT
D000972:Antineoplastic Agents, Phytogenic / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053143:Caspase 2 / Q000378:metabolism / Q000502:physiology*; D061945:Caspase Inhibitors; D020552:Comet Assay; D045304:Cytochromes c / Q000378:metabolism; D004249:DNA Damage; D004250:DNA Topoisomerases, Type II / Q000737:chemistry; D004791:Enzyme Inhibitors; D005434:Flow Cytometry; D005978:Glutathione / Q000378:metabolism; D054335:Goniothalamus / Q000737:chemistry; D006801:Humans; D019169:Jurkat Cells; D009842:Oligopeptides / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism / Q000502:physiology*; D011753:Pyrones / Q000633:toxicity*; D017382:Reactive Oxygen Species; D015398:Signal Transduction / Q000187:drug effects; D013778:Tetrazolium Salts; D013844:Thiazoles
D000972:Antineoplastic Agents, Phytogenic; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D009842:Oligopeptides; D019253:Proto-Oncogene Proteins c-bcl-2; D011753:Pyrones; D017382:Reactive Oxygen Species; D013778:Tetrazolium Salts; D013844:Thiazoles; C427308:benzoylcarbonyl-valyl-aspartyl-valyl-alanyl-aspartyl-fluoromethyl ketone; C103280:goniothalamin; D045304:Cytochromes c; D053143:Caspase 2; D004250:DNA Topoisomerases, Type II; C022616:thiazolyl blue; D005978:Glutathione