Shikonin analogue (SA) 93/637 induces apoptosis by activation of caspase-3 in U937 cells.
Beta,beta-dimethyl acryl shikonin is an extract from the root of plant Arnebia nobilis which has been shown to possess anti-cancer activity. However, its toxicity limited further development of shikonin as a therapeutic agent. Subsequently, several analogues of beta,beta-dimethyl acryl shikonin were synthesized. One of these analogues, shikonin 93/637 was found to be significantly less toxic compared to shikonin. This study is aimed to determine the cell cycle associated differences in the susceptibility of U937 cells to apoptosis induced by shikonin analogue 93/637 (SA). Lower concentrations of SA (approximately 100 nM) showed no significant changes in cell growth. However, higher concentrations (approximately 500 nM) resulted in growth inhibition of U937 cells after 48 h of treatment with SA as measured by MTT assay. Flow cytometric analysis showed that SA treatment resulted in blocking of cell cycle progression in G1 phase. Decreased expression of Cyclin D, CDK 4 and PCNA was observed with SA treatment corroborating the G1 block. DNA gel electrophoresis showed an oligonucleotide ladder pattern, a distinct characteristic of DNA fragmentation associated with programmed cell death. Ribonuclease protection assay revealed inhibition of bcl2 expression at transcriptional level. SA treatment also resulted in induction of caspase-3 activity. The results suggest the involvement of bcl2 and Caspase-3 in SA induced apoptosis of human U937 cells.
Thangapazham
Rajesh L
RL
Center for Combat Casualty and Life Sustainment Research, Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA.
Singh
Anoop K
AK
Seth
Pankaj
P
Misra
Namita
N
Mathad
Vijayavitthal T
VT
Raj
Kanwal
K
Maheshwari
Radha K
RK
D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism*; D002470:Cell Survival; D002934:Cinnamates / Q000494:pharmacology*; D053938:DNA Fragmentation; D004789:Enzyme Activation; D005434:Flow Cytometry; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D009285:Naphthoquinones / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D009369:Neoplasms / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D014158:Transcription, Genetic; D020298:U937 Cells
C524845:5,8-dihydroxy-2-(1'-cinnamoyloxy-4'-methylpent-3'-enyl)-1,4-naphthoquinone; D002934:Cinnamates; D009285:Naphthoquinones; D013778:Tetrazolium Salts; D013844:Thiazoles; C016101:shikonin; D012260:Ribonucleases; D053148:Caspase 3; C022616:thiazolyl blue