Pterostilbene Induced Apoptosis Of Multiple Myeloma Cells Through AMPK Activation

Part Ⅰ : Pterostilbene induced the apoptosis of multiple myeloma cells via AMPK activation.Objective: To investigate the pro-apoptosis effect of pterostilbene treatment on myeloma cells and explore the underlying mechanism.Methods: MM cells were cultured in the different concentrations of pterostilbene and compared with the control group in vitro.After 48 h,CCK8 was performed to explored cell proliferation and the IC50 value was detected.The relatively sensitive RPMI8226 and ARH77 cells were used for subsequent experiments.The cell apoptosis was tested by flow cytometry.The protein expression of caspase family and PARP was detected by Western blotting.Western blotting was also used to examine the expression of AMPK and p AMPK.Co-treatment with PTE and compound C,then apoptosis was evaluated through flow cytometry,and western blotting was also used to the expression of p AMPK,PARP.After PTE treatment,the protein levels of FASN,ACC and p ACC were performed,the protein expression levels of FASN was detected after co-treatment with compound C.CCK8 was performed to explored cell proliferation inhibition after orlistat treatment.Palmitic acid was supplemented in PTE or orlistat treatment group,then detect apoptosis using flow cytometry.The protein levels of m TOR,p4 EBP,4EBP,p70S60 K and p-p70S60 K were tested too.The protein expression levels of FASN was detected after co-treatment with compound C.Results: After intervention by PTE,the proliferation of myeloma cells decreased significantly in dose dependent manners.The number of apoptosis were markedly induced with increasing doses of PTE accompanied by the cleaved activation of caspase3,caspase9 and PARP.PRMI8226 and ARH77 cells were intervened with different concentrations of PTE.After PTE treatment,the protein expression level of p AMPK was increased,corelating with the degree of PTE concentration.After cotreatment with compound C,PTE’s ability to induce apoptosis was partially reduced,the expression of p AMPK and PARP were partially reduced,too.After PTE treatment,the FASN protein level was decreased and ACC was phosphorylated.After cotreatment with compound C,FASN expression were partially reversed.Cell proliferation was decreased after orlistat treatment.After supplementing palmitic acid in PTE or orlistat treatment group,MM cell apoptosis was partly restored.The expression level of pm TOR decreased as well as the expression levels of p4 EBP and p-p70S60 K.After cotreatment with compound C,4EBP expression were partially reversed.Conclusion: Pterostilbene can inhibit the proliferation and induce apoptosis of multiple myeloma cells in vitro.PTE could induce AMPK phosphorylation in myeloma cells,and simultaneously interfere with the activity or expression of downstream target protein.Activation of AMPK is directly related to the level of myeloma cell apoptosis.Part Ⅱ: Role of autophagy in apoptosis of multiple myeloma cells induced by AMPK activation in pterostilbeneObjective: To investigate whether pterostilbene induced autophagy via the AMPK signaling pathway and clarify the role of autophagy in the apoptotic inducting of Pterostilbene.Methods: The production of autophagosomes of PRMI8226 cells after PTE treatment was examined by TEM.MDC was used as a specific autophagolysosome marker to analyze the autophagic process.Western blotting was used to texted the protein levels of ATG5,Beclin1 and LC3.Western blotting was performed to test the protein levels of LC3 with PTE alone or cotreatment with CC.Annexin V / PI flow cytometry assay was performed to test apoptosis with PTE alone or cotreatment with 3MA.Results: TEM and MDC fluorescence showed that PTE induced autophagy in PRMI8226 cells,with increased accumulation of autophagy markers,such as LC3-II,Beclin1 and ATG5.The protein levels of LC3Ⅱwas reduced after co-treated with CC.The apoptotic effect of PTE was further enhanced after blocking the autophagic flux by 3MA.Conclusion: Pterostilbene could induce autophagy through the AMPK signaling pathway.Autophagy acts as a protective role in PTE induced apoptosis.PTE combined with autophagy inhibitor could further induce myeloma cell apoptosis.PartⅢ: Exploring the safety and effectiveness of pterostilbene in inducing multiple myeloma cell apoptosis in vivoObjective: To explore the effectiveness and safety of pterostilbene intervention in inducing MM cell apoptosis in vivo.Methods: The NOD / SCID mice were applied to establish PRMI8226 subcutaneous transplantation tumor model.The mice were intraperitoneally injected with PTE.Observing the growth of tumor bearing mice and compared with DMSO control group.Tunel experiment and immunohistochemistry of cleaved caspase3 were performed to detect the apoptosis in mice.The protein expression levels of p AMPK,pm TOR and FASN in the subcutaneous tumor tissue was detected by immunohistochemical staining.Pharmacological toxicity assessment and HE staining were tested for side effects.Results: Xenograft tumors receiving pterostilbene treatment demonstrated a reduced tumor volume compared with xenograft tumors receiving DMSO.Tumor necrosis was observed through Tunel assay and immunohistochemistry of cleaved caspase3.The expression level of p AMPK in the subcutaneous transplantation tumors of PTE-treated mice was much higher than the control group,and the levels of the downstream target proteins FASN and m TOR were decreased.The proliferation of myeloma cells decreased significantly.Compared with the control group,tumor bearing mice performed no significant changes in body weight,food and water intake,or activity.No obvious toxic effects were found in the heart,liver or kidney.Conclusion: Pterostilbene can also inhibit tumor growth in vivo with no significant side effects.