Betulinic Acid Induces ROS-dependent Cell Death By Inhibiting The NF-?B Pathway In Human Multiple Myeloma

Objective: Multiple myeloma(MM)is the second most prevalent hematological malignancy.Despite enormous progress in treatment and diagnosis,many patients do not respond to current therapies and ultimately succumb to the disease.Betulinic acid(BA),as a natural plant product,showed its antitumor bioactivities in many other solid malignant tumors,but few investigations on hematological malignancies.The purposes of this research were to confirmed its cytotoxic and tumor inhibitory effects on MM cells,and more importantly,to investigate deeply its potential molecular targets and mechanisms.Methods:MM cells were treated with various concentrations of BA for different hours and cell viability was determined using CCK-8 assay.The proliferation of MM cells were observed under microscope by Ed U incorporation assays.Nuclei morphological changes were observed by Hoechst 33342 staining and cells apoptosis rate was measured by Annexin V/PI double staining.The cell cycle,mitochondrial membrane potential(MMP),intracellular reactive oxygen species(ROS)were determined by fluorescent probes PI,JC-1,DCFH-DA,respectively.Proteins related to cell cycle,mitochondrial apoptotic pathway and NF-?B system were evaluated by western blot.The expression level of NF-?B p65 and its intracellular localization were detected by immunofluorescent staining.Nude mouse xenograft model in vivo was established and the isolated tumor tissues were used to analyze the expression level of NF-?B p65 and Ki-67 by immunohistochemistry(IHC),and the protein NF-?B p65 by western blot.Results: Our data showed that BA had a cytotoxic effect on cell viability and inhibited the proliferation of MM cells in dose-and time dependent manner.Flow cytometry results showed BA promoted cell apoptosis and induced cell cycle arrest at the S phase in MM cells.JC-1 results indicated the damaged mitochondria.Further studies had demonstrated BA treatment induced the overwhelming intracellular reactive oxygen species(ROS)in U266 cells and activated the mitochondrial apoptosis pathway.When the ROS scavenger N-acetyl-L-cysteine(NAC)used,the BA-induced MM cells apoptosis partly reversed.Moreover,our western blot results showed that BA significantly reduced the levels of NF-?B pathway in a time-and dose-dependent manner.In addition,we proved NF-?B pathway had regulatory effects on celluar ROS as well as cell viability,by up-regulating and down-regulating NF-?B activity with TNF-? and BAY 11-7082 respectively.In agreement,BA also suppressed the tumor growth of MM cells in vivo via inhibition of the NF-?B pathway.Conclusions: Collectively,we found for the first time that BA exerted its cytotoxic effect and tumor inhibition role of MM cells both in vitro and in vivo.The main conclusions are as follow:1.The potential mechanisms mainly include mitochondrial-apoptosis induction,cell cycle blockade,MMP disruption,intracellular ROS accumulation and NF-?B signaling inhibition.2.By blocking the NF-?B pathway that breaks redox homeostasis,BA,as a potent NF-?B inhibitor,boosted accumulation of ROS and then the cell death.3.BA also inhibits the growth of MM xenograft tumors in vivo4.These promising findings provided new insight into the mechanisms of antitumor effects of BA on MM.Therefore,BA is perhaps a prospective new therapeutic alternative for clinical application in the near future.