Hadi Bakhtiari1, Ali Gheysarzadeh1, Mustafa Ghanadian2, Mahmoud Aghaei3. 1. Department of Clinical Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran. 2. Department of Pharmacognosy, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran. 3. Department of Clinical Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran. Electronic address: maghaei@pharm.mui.ac.ir.
Abstract
AIM: 15-Hydroxy-8(17),13(E)-labdadiene-19-carboxylic acid (HLCA) isolated from Juniperus foetidissima, has been recently identified as an antiproliferative agent; however, the molecular basis of antiproliferative effects of HLCA remains unknown. To investigate it, the current study has emphasized the hypothesis that HLCA induced cell death is a consequence of intracellular reactive oxygen species (ROS) production followed by cell cycle arrest and apoptosis. MAIN METHODS: Human ovarian OVCAR-3 and Caov-4 cells were treated with various concentrations of HLCA (48 h) and the measurement of intracellular ROS was considered. Then, the potential of HLCA in promoting apoptosis was investigated via flow cytometry, western blot, and caspase activity assay. Also, the inhibitory effect of HLCA on the cell cycle was evaluated using flow cytometry and western blot analysis. KEY FINDINGS: We found intracellular (ROS) accumulation in HLCA-treated cells. Subsequent observation of the increment in pro-apoptotic Bax as well as the decrement in antiapoptotic Bcl2 revealed that the HLCA-induced cytotoxicity may be triggered by the intrinsic pathway of apoptosis. Our subsequent experiments suggested that caspase-9 and -3 were activated and led the cells to apoptosis during the process. Cell cycle disruption at the G1 phase via down-regulation of cyclin D1 and Cyclin-dependent kinase 4 (CDK4) was another proved mechanism by which HLCA exerts its antiproliferative effects on the ovarian cell lines, OVCAR-3 and Caov-4, especially at relatively lower concentrations. SIGNIFICANCE: This is the first study that reveals the apoptotic effects of HLCA, suggesting its therapeutic potential as an effective anti-tumor agent. However, further in vivo studies are required to confirm these effects.
AIM: 15-Hydroxy-8(17),13(E)-labdadiene-19-carboxylic acid (HLCA) isolated from Juniperus foetidissima, has been recently identified as an antiproliferative agent; however, the molecular basis of antiproliferative effects of HLCA remains unknown. To investigate it, the current study has emphasized the hypothesis that HLCA induced cell death is a consequence of intracellular reactive oxygen species (ROS) production followed by cell cycle arrest and apoptosis. MAIN METHODS:Human ovarian OVCAR-3 and Caov-4 cells were treated with various concentrations of HLCA (48 h) and the measurement of intracellular ROS was considered. Then, the potential of HLCA in promoting apoptosis was investigated via flow cytometry, western blot, and caspase activity assay. Also, the inhibitory effect of HLCA on the cell cycle was evaluated using flow cytometry and western blot analysis. KEY FINDINGS: We found intracellular (ROS) accumulation in HLCA-treated cells. Subsequent observation of the increment in pro-apoptotic Bax as well as the decrement in antiapoptotic Bcl2 revealed that the HLCA-induced cytotoxicity may be triggered by the intrinsic pathway of apoptosis. Our subsequent experiments suggested that caspase-9 and -3 were activated and led the cells to apoptosis during the process. Cell cycle disruption at the G1 phase via down-regulation of cyclin D1 and Cyclin-dependent kinase 4 (CDK4) was another proved mechanism by which HLCA exerts its antiproliferative effects on the ovarian cell lines, OVCAR-3 and Caov-4, especially at relatively lower concentrations. SIGNIFICANCE: This is the first study that reveals the apoptotic effects of HLCA, suggesting its therapeutic potential as an effective anti-tumor agent. However, further in vivo studies are required to confirm these effects.