Mechanisms Of MAPK Pathway And ROS Involved In The Apoptosis Of HL-60 Cells Induced By Resveratrol

【Backgroud and Objectives】Background：Resveratrol,3,4,5-trihydroxy-trans-stilbene,a natural phytalexin was found athigh levels in grapes，polygonum cuspidatum and hellebores. There are two isoforms ofresveratrol, cis-resveratrol and tran-resveratrol which is more stable than cis-resveratrol and hasmore biological effects. It has been reported that RSV has a variety of biological effects e.g.anti-inammatory, anti-oxidative, anti-antibacterial action, anti-tumor and regulating lipid.Specially, the anti-tumor effect of RSV is very important and it takes many researchers attentionthese years. It has been proved that RSV can induce many different cells apoptosis, but themechanisms of apoptosis are not always the same in different cells. We are sure that RSV caninduce the apoptosis of HL-60, but the mechanism is not clear. Therefore the objective of thisresearch is to investigate the mechanism.Objective: 1. To confirm caspase-3 pathway is one of the pathway of HL-60 apoptosis inducedby RSV.2. To confirm whether ROS will be produced in HL-60 cell after treatement withRSV and whether MMP will be change. And we also want to know themechanism of these changes.3. To investigate the effects of PI3K/Akt pathway, ERK pathway and JNK pathwayon the apoptosis of HL-60 cells.【Methods】HL-60 cells were treated with 0.01%DMSO as control and different concentrations of RSVfor 6h, 12h and 24, then MTT assay was performed to detect the cell viability, the caspase-3activity assay kit was used to detect the change of caspase-3 activity and flow cytometry wasperformed to exam the change of MMP and ROS. And the SOD assay kit and NBT method wereperformed to exam the activity of SOD. Then the anti-cancer effect of RSV was attested after theapplication of RSV as single agenta and the combination with RSV and Ac-DEVD-CHO, acaspase-3 inhibitor. MTT was also used to detect the change of the cell viability, and the change of morphological was observed by microscope or fluorescence microscope after stained by DAPI.Then to investigate the effect of ROS, the HL-60 was treated with the combination of RSV andNAC （a ROS inhibitor） or the combination of RSV and SOD, and then the cell viability wasmeasured by MTT. Finally, the level of p-ERK, p-JNK, p-p38 and p-Akt were detected bywestern blotting. Then the changes of cell viability rate were measured by MTT after HL-60 wastreated with RSV as a single agent and the two agents combination of RSV and U0126 or JNKinhibitor or Wortmannin or Metformin.【Results】1. RSV can induce the apoptsis of HL-60 in a time- and concentration-dependent manner.And NAC, SOD, U0126, JNK inhibitor and metformin has no significant effects on theapoptosis of HL-60 induced by RSV.2. The production of superoxide anion of HL-60 was raise after treated with resveratrol butthe H₂O₂ was decreased. And the level of mitochondrial membrane potential was reducedin a time-and concentration-manner after treated with different does of RSV for differenttime.3. The activity of SOD has no significant change after treated with RSV.4. RSV can activate caspase-3 in concentration-dependent manner.5. RSV can down regulate p-ERK and p-JNK and the p-p38 has no change. p-Akt was upregulated after treated with 0～25μM, then when the concentration of RSV is higher, thelevel of p-AKt is begin to down.【Conclusions】1. Caspase-3 pathway is one of the pathways of HL-60 apoptosis induced by RSV.2. RSV can lead to the reduction of the MMP and the production of, and increase theproductiong of superoxide anion. Down regulation of MMP may be one of themechanisms of apoptosis of HL-60 induced by RSV. And H2O2 is one byproduct ofapoptosis.3. The reason of decrease of H2O2 is not the activation of SOD.4. Whether down-regulation of p-JNK and p-ERK play the key role in the apoptosis is need toinvestigate.