Studies On The Antitumor Differentiation Therapy Of Tetrandrine

Cancer is a disease caused by the abnormal proliferation and differentiation of cells governed by tumorigenic factors induced gene mutation.As we increasingly know about meaning of the abnormal cell differentiation in development and therapy of malignant tumors,the differentiation therapy of tumor become one of the hotspots in oncology research.Inducing differentiation of tumor cell generally apply to leukemia therapy,which refers to the morphology and biochemistry marks of tumor cell are differentiate forword to the normal cells after induced by the differentiation inducer.The latest differentiation therapy strategy of tumor can also be used for targeting promote cell differentiation of cancer stem cells to lose stem cell properties and differentiate into common tumor cells with epithelial phenotype,which can overcome the strong drug-resistance,high invasive and recurrent rate caused by cancer stem cells in the process of cancer treatment.Traditional chemotherapy is focus on killing tumor cells and may have side effects to normal cells at the same time.By contrast,differentiation therapy has the advantages of low toxicity and can gently facilitate differentiation of tumor cells until death.Among non-solid tumors(leukemia),acute megakaryoblastic leukemia(AMKL)is a rare and heterogeneous sub-type of acute myeloid leukemia due to megakaryocytopoiesis blocked at a particular stage of differentiation and proliferate abnormally in the development process.Clinical treatment of AMKL is still a big challenge because of lack of effective drugs.The feature of leukemia cells from AMKL patients is malignant proliferation and refuse to go terminal differentiation into mature megakaryocyte.Therefore,promoting AMKL cells to megakaryocyte differentiation can be used as an effective therapeutic strategy.The researchers hope to develop novel drugs to induce AMKL cells to megakaryocyte terminal differentiation and exit the malignant proliferation.Among solid tumors,breast cancer is a malignant tumor with highest incidence of women and seriously threaten the women's health and life safety all over the world.Although the clinical treatment of breast cancer is relatively mature,the metastasis and recurrence rate of breast cancer has been maintained at a high level.Researchers have isolated a kind of rare cell subgroup from breast tumor tissue cells which have infinite ability to self-renewal,multi-directional differentiation potential and high tumorigenicity called breast cancer stem cells(breast cancer stem cells,BCSCs).These cells are thought to be closely related to recurrence,invasion and metastasis and radiation and chemotherapy resistance of breast cancer.So,the researchers committed to developing new treatment strategies for breast cancer which can selectively or target to kill BCSCs or eliminate the properties of stem cells and differentiate into common cancer cells.Thus,we can overcome the strong drug resistance,high invasive and recurrent of BCSCs in the process of cancer treatment.Increasing studies have shown that Chinese medicine or Chinese medicine effective component has not only positive therapeutic effect on tumor cells,but also make advantages in relieving the side effects of chemotherapy,enhanced the effect of chemotherapy and improve the body's immune that can improve patients' quality of life.Tetrandrine.a natural product,is a bisbenzylisoquinoline alkaloid that originally isolated from the roots of the Chinese herb Stephania tetrandra S Moore.Recent years,many studies suggested that tetrandrine has pharmacological potential in cancer therapy with multiple bioactivities.This paper preliminary studied the differentiation therapeutic effect of tetrandrine in anti-AMKL and anti-breast cancer.After dose and time gradient treatment with tetrandrine,we found that high-dose of tetrandrine induced cell apoptosis in leukemic cells,low-dose of tetrandrine inhibited cell proliferation,induced autophagy and promote megakaryocyte differentiation.When use the autophagy inhibitor 3-MA or knockout the autophagy related gene ATG7 to inhibit the tetrandrine induced autophagy,we found the megakaryocyte differentiation effect of tetrandrine attenuated or eliminated,that demonstrated autophagy plays a critical role in tetrandrine-induced megakaryocytic differentiation.Further the underlying mechanism study showed that the intracellular ROS levels were significantly increased in the tetrandrine-treated leukemic cells.This ROS accumulation can be effectively eliminated by the antioxidants N-acetyl cysteine(NAC)and we found tetrandrine induced autophagy and megakaryocyte differentiation also reduced after NAC treatment that suggest ROS involved in autophagy and megakaryocyte differentiation in response to tetrandrine.Additionally,tetrandrine activate Notchl signaling which were inhibited by DAPT or shRNA against the Notch 1 gene,resulting in the inhibition of tetrandrine-induced autophagy and differentiation.We also found the p-Akt levels were by tetrandrine in the leukemia cells at the early time points.The inhibition of Akt by the PI3K/Akt inhibitor wortmannin or overexpression of constitutively active form of Akt can suppress or enhance tetrandrine-induced autophagy and differentiation respectively.These results demonstrated tetrandrine-induced autophagy and differentiation is dependent on Notchl and Akt signaling.Further study demonstrated that tetrandrine functioned through ROS/Notchl/Akt signaling cascade pathway.Notably,tetrandrine did not affect cell proliferation and failed to promote megakaryocyte differentiation in normal mouse fetal liver cells although it induced autophagy,suggesting a specific effect of tetrandrine in malignant megakaryobalsts.Consistent with our in vitro findings,tetrandrine also showed potent anti-leukemia effect in K562 subcutaneous tumor xenograft models in athymic nude mice and in 6133 AMKL mouse model.Remarkably,the tetrandrine treatment significantly prolongs the survival of the mice with AMKLIn addition,we found that low-dose(2 ?M)tetrandrine without kill breast cancer cells directly can reduce breast cancer stem cells and inhibit cell migration,suggest tetrandrine may facilitate BCSCs lose their stem cell properties and differentiate into common cancer cells.As Low level of ROS can stabilize cancer stem cells,we found that the tetrandrine can downregulate the expression of superoxide dismutasel(SOD1)and hydrogen peroxide enzyme(Catalase),also activate ROS in breast cancer cells.Further use ROS scavenger NAC or exogenous use SOD1 or Catalase to eliminate the accumulation of ROS can rescue the EMT and BCSCs inhibition by tetrandrine.As a recognized calcium ions(Caa+)antagonist,we also found that tetrandrine can downregulate the expression of mitochondrial calcium uniporter(MCU).After treated with the Ca2+ agonist Ionomycin or overexpress MCU,can also eliminate the EMT and BCSCs inhibition by tetrandrine.The results show that ROS activation and mitochondrial Ca2+ signal reduction both involved in regulation of tetrandrine inhibit EMT and BCSCs.Taken together,this thesis is focus on studying the antitumor differentiation therapy of tetrandrine,and we found that the anti-AMKL and anti-breast cancer effects of tetrandrine by induction of megakaryocyte differentiation and targeting BCSCs.We also make a further preliminary research on the underlying mechanism.All these results suggest that tetrandrine may be a potential differentiation-inducing compound for antitumor therapy.Tetrandrine may be a novel strategy to cure AMKL and breast cancer clinically.