Pterostilbene Has Anti-tumor Activity In T-cell Leukemia/lymphoma By Suppressing The ERK1/2

ObjectivePterostilbene is a natural 3,5-dimethoxy analog of trans-resveratrol that has been reported to have anti-tumor,anti-oxidant and anti-inflammatory effects.T-cell leukemia/lymphoma is one of the more aggressive yet uncommon non-Hodgkin lymphomas.The aim of this study was to confirm the effect of pterostilbene in T-cell leukemia/lymphoma.Methods(1)Jurkat and Hut-78 cells(2×10~5cells/mL)were treated with various concentrations of pterostilbene for 24 or 48 h.PBMCs were treated with of pterostilbene for 48 h.The cell proliferation was detected by cell counting kit-8(CCK-8)and a microplate reader.(2)Jurkat and Hut-78 cells were treated with various concentrations of pterostilbene for24 h.The cell cycle was detected by PI and a flow cytometer.(3)Jurkat and Hut-78 cells were treated with various concentrations of pterostilbene for24 h and 48 h,peripheral blood mononuclear cells(PBMCs)were treated with different doses of pterostilbene for 48 h.The cell apoptosis was detected by Annexin V/PI kit and a flow cytometer.(4)Jurkat and Hut-78 cells were treated with various concentrations of pterostilbene for48 h.The generation of reactive oxygen species(ROS)and mitochondrial membrane potential(MMP)were detected by ROS kit and JC-1 kit,respectly,by a flow cytometer.(5)Jurkat and Hut-78 cells were treated with various concentrations of pterostilbene for 48h.The protein levels of caspase,cell cycle and extracellular regulated protein kinases(ERK)1/2 signaling pathways were detected by western blot.Results(1)Jurkat and Hut-78 cells(2×10~5cells/mL)were treated with different concentrations(0,10,15,20,25,30,35 or 40μM)of pterostilbene for 24 or 48 h,the calculated IC50(50%cell growth inhibitory concentration)values were 17.83μM(Jurkat)and 22.74μM(Hut-78)at 48h.PBMCs were treated with(0,10,20,40,80μM)of pterostilbene for 48 h.Pterostilbene inhibited the growth of Jurkat and Hut-78 cells in a dose-and time-dependent manner.In addition,pterostilbene has no toxicity in PBMCs,suggesting that pterostilbene is a safe compound in our this study.(2)Pterostilbene(0,5,10,20μM)treatments for 24 h induced a S-phase arrest in Jurkat and Hut-78 cells.Cells were treated with pterostilbene(0,5 or 10μM)for 24 h to test protein expression levels by western blot.Cdc25A,CDK2 and cyclinA2 protein levels were dramatically decreased in the pterostilbene-treated group compared with the control group.(3)The results showed that pterostilbene treatment for 24 h or 48 h markedly induced apoptosis of Jurkat and Hut-78 cells in a dose-and time-dependent manner.Moreover,the result showed that pterostilbene treatments(20,40,80μM)have no toxicity in PBMCs,further suggesting that pterostilbene is a safe agent for treatment of T-cell leukemia/lymphoma.Our results demonstrated that pterostilbene treatment induced the cleavage of caspase 3,PARP,caspase 9 and caspase 8 proteins.(4)We detected the effect of pterostilbene treatment for 48 h on MMP using the JC-1MMP Detection Kit.The result showed that MMP was greatly decreased in pterostilbene-treated cells compared with the control group.To detect ROS levels in the DNA damage response,we evaluated ROS generation in Jurkat and Hut-78 cells treated with pterostilbene(10μM)for 48 h by flow cytometry.The results showed that the 10μM pterostilbene group had visibly increased ROS levels compared with the control group.(5)Cells treated with pterostilbene showed decreased levels of phospho(active)-ERK1/2,while there was no significant change in total ERK1/2.Conclusion(1)Pterostilbene inhibites T-cell leukemia/lymphoma cells proliferation in a time-and dose-dependent manner.(2)Pterostilbene arrests the cell cycle of T-cell leukemia/lymphoma cells at S phase.(3)Pterostilbene induces T-cell leukemia/lymphoma cells apoptosis in a time-and dose-dependent manner.(4)Pterostilbene induces mitochondrial membrane potential declines and reactive oxygen species generation in T-cell leukemia/lymphomas cells.(5)Pterostilbene decreases the level of phospho-ERK1/2.