Studies On Molecular Mechanisms Of Tetrandrine-induced Differentiation In Leukemia

Cancer is a serious disease that threatens human health.It is estimated that 14.1 million people develop into cancer by the World Health Organization statistics in 2014.And the death rate is extremely high,especially in the developing countries.Leukemia is a high incidence,a great deal of malignant clonal diseases of hematopoietic stem cells.The pathogenic factors mainly include virus infection,chemical factor,radiation induction,chromosome aberration and hereditary factors.At present,the main means of treatment of leukemia chemotherapy,targeted therapy,immunotherapy,stem cell transplantation and so on.Nowadays clinically used for the treatment of leukemia chemotherapeutic drugs mainly to promote cell differentiation.However,with the increase of medication time and dosage,leukemia cells are also more likely to develop resistance to chemotherapeutic drugs,therefore,it is very important for finding new chemotherapeutic drugs.Many traditional Chinese medicine was found to have very significant anti-tumor.Tetrandrine is a bisbenzylisoquinoline alkaloid isolated from the roots of Stephaniae tetrandrae.Many studies have shown that tetrandrine has a good anti-tumor effect.This study mainly focused on the anti-leukamia effect of tetrandrine and its molecular mechanism in vitro and in vivo.Leukemia chemotherapy is mainly to induce cell differentiation.And many studies have shown that induced differentiation is associated with autophagy.Our study found:2 ?M tetrandrine can induce K562?THP-1 and AML patient leukemia cells differentiation.Wright-Giemsa staining?NBT reduction assay?flow cytometry of cell surface CD 14 and CD11b expression and Western blot detection of CD 14 protein have proved the occurrence of cell differentiation.On the other hand,tetrandrine remarkably induced leukemia cells autophagy in dose-and time-dependent manner.Western blot analysis LC3 protein levels?acridine orange staining detection autophagic vacuoles and the formation of puncta with the GFP-LC3 fusion protein prove that tetrandrine can induce autophagy in leukemia cell.Dual fluorescence of ptf-LC3 and LC3-II transformation demonstrated the generation of autophagic flux in cells after tetrandrine treatment.A common specific inhibitor of autophagic/lysosomal protein degradation,3-methyladenine,significantly inhibited the occurrence of cell autophagy and thus inhibited cell differentiation.And knockdown of autophagy-related gene atg7 also blocked the occurrence of tetrandrine-induced cell differentiation.All experiments demonstrate that tetrandrine-induced cell differentiation is associated with autophagy.Reaction oxygen species(ROS)was crucial signaling molecules in oxidative stress response.Some reports suggested that ROS may be involved in drug-induced cell differentiation.Studies on molecular mechanisms of tetrandrine-induced differentiation in leukemia,the reaults suggested that tetrandrine can promot intracellular ROS accumulation,which was an important event in tetrandrine induced autophagy and differentiation,Then NAC and Tiron can rescue tetrandrine-induced cell differentiation and autophagy.The c-myc was an important gene involved in the fate of cells.Some reports indicated that c-MYC protein can regulate drug-induced differentiation of leukemia cells.In this study we found that 2?M tetrandrine was able to reduce c-MYC protein expression and c-MYC mRNA level was also inhibited.Then we overexpression c-MYC in leukemia cells found that tetrandrine-induced cell differentiation was inhibited.Moreover,a small molecular c-MYC inhibitor 10058-F4 enhanced tetrandrine-induced differentiation of leukemia.In addition,we also found that c-MYC protein changes was an early event of tetrandrine-induced cell autophagy.To determine the effect of tetrandrine on healthy hematopoietic cells.Mice embryos were taken after 12.5 days of conception,then we isolated normal hematopoietic cells from mouse embryos.We found that 2 ?M tetrandrine has no cytotoxicity effects in the hematopoietic cells by MTS and apoptosis detection.However,the detection of autophagy found that 2?M tetrandrine can promote autophagy in hematopoietic cells.At the same time,we also found same results with human cord blood cells.In this study,to investigate the induced differentiation effect of tetrandrine in vivo,we established subcutaneous tumor xenograft models in athymic nude mice using the vector and overexpression c-MYC THP-1 cells.By comparing the size and the weight of tumor,we found that tetrandrine also has a good effect of anti-tumor in animals.Notably,we found that 25 mg/kg and 50 mg/kg tetrandrine treatment were well tolerated by all mice,as the animal did not display weight loss.Immunohistochemistry results showed that tetrandrine effectively promoted CD 14 expression and decreased c-MYC in THP-1 vector,but caused weak influences in overexpression c-MYC tumors.Detection the MDA levels in tumors using the MDA kit can indicate that tetrandrine also promotes the accumulation of ROS in animals.Western blot or immunohistochemistry analysis LC3 and western blot analysis c-MYC protein levels were also consistent with in vitro experiments.In summary,we found that ROS generation and c-MYC suppression play important role in tetrandrine-induced autophagy and differentiation.Therefore,tetrandrine can induce leukemia cell differentiation and autophagy and tetrandrine may be a promising agent for the treatment of leukemia.