Mechanism Of Genetoxic Antineoplastic Drugs Regulating Autophagy And Apoptosis In Tumor Cells

Cancer is one of the most important human diseases.There are>20 million new cases and>9.6 million deaths worldwide each year.Cancer incidence rate in pets also increases 2.5%annually.Cancer poses a serious threat to human and animal health.Veterinarians in China mainly focus on the prevention and control of infectious diseases of livestock as well as virusinduced oncogenesis in poultry.Great attention has been paid in recent years on several common malignancies such as breast cancer and lymphoma in pets.Human and animal tumors share many common characteristics in ongogenesis,progression,and treatment.Animal tumor models have been widely used to study the pathogenesis and therapy.Some anticancer drugs clinically used for treat human tumors are adopted to treat pet tumors.Autophagy is a highly conserved catabolic process in eukaryotic cells that is tigthly regulated by ULK1,which phosphorylates Vps34 and induces the formation of autophagosomes.After fusion with lysosomes to form autophagolysosomes,proteolytic enzymes in lysosomes degrade damaged organelles and misfolded proteins etc.,to produce amino acids to maintain intracellular energy and nutrient homeostasis.mTOR and AMPK are two key kinases that regulate autophagy.The former phosphorylates ULK1 S757 to inhibit its activity and autophagy;the latter phosphorylates and activate ULK1 at multiple sites such as S555 and S777 to induce autophagy.TAK1 is a serine/threonice kinase activated by multiple receptors.Recent studies have shown that monosodium urate（MSU）binds the phosphorylation loop of TAK1 and increases its enzymatic activity.TAK1 can phosphorylate and activate multiple kinases such as JNK and AMPK.TAK1 promotes autophagy by activating downstream AMPK and JNK.Autophagy plays a crucial role in carcinogenesis,tumor growth and progression,drug resistance against chemotherapeutic drugs,angiogenesis inhibitors,genotoxic antitumor drugs such as 5-Fluoro-2-Deoxyuridine（5-FU）or gemcitabine（Gem）.While it is well documented that all these anticancer drugs can induce autophagy in tumor cells and in animal tumor models,the underlying molecular mechanisms remain incompletely understood.Xanthine oxidoreductase（XOR）,a rate-limiting enzyme of purine metabolism,plays an important role in uric acid production and generation of reactive oxygen species（ROS）.Both products have multiple roles in promoting tumorigenesis,inhibiting tumor growth,and regulating immune function in different stages of tumorigenesis.Therefore,XOR functions to suppress or promote tumorigenesis or progression.The molecular mechanisms underlying this remains incompletely understood.Our prior work showed that Gem and 5-FU caused DNA damage and cell cycle arrest in cancer cells,leading to uric acid accumulation and the activation of TAK1 and its downstream MAP pathway to induce the expression of NKG2D ligands.This thesis focuses on the mechanism of XOR-uric acid in genotoxic drug-induced autophagy and apoptosis.1.Molecular mechanism of Gem-induced autophagy in tumor cells:We investigated Gem-induced autophagy in a human cervical cancer cell line（HeLa）and a colon cancer cell line（HT-29）.We found that:1)Gem induced TAK1,JNK,AMPK,ULK1S555 phosphoiylation,LC3 lipidation,and p62 degradation.Chloroquine（CQ）and bafilomycin Al（Baf）reduced the number of autolysosomes in Gem-treated cells;2)Compound C（CC）,an AMPK inhibitor,blocked Gem-induced AMPK and ULK1S555 phosphorylation.5Z-7oxozeaenol（5Z）,a TAK1 inhibitor,and TAK1 siRNA inhibited Gem-induced JNK,AMPK,ULK1S555,p65 and p38 phosphorylation.5Z,CC,and SP-600125,a JNK inhibitor,inhibited Gem-induced LC3 lipidation and p62 degradation,and blocked Gem increased in the number of autophagosomes and autolysosomes.2.The role of XOR and uric acid in genotoxic drugs-induced autophagy:We further investigated the role of XOR in the induction of autophagy by genotoxic drugs and examined whether the XOR product uric acid could induce autophagy.The results showed that allopurinol（AP）,a XOR inhibitor,or XOR-miRNA inhibited Gem-induced TAK1,AMPK and ULK1S555 phosphorylation,and blocked LC3 lipidation;overexpression of XOR enhanced Gem-induced TAK1,JNK,AMPK and ULK1S555 phosphorylation and LC3 lipidation.ATM/Chk1/2 siRNA blocked Gem-induced LC3 lipidation and p62 degradation.Monosodium urate（MSU）induced TAK1,JNK,AMPK,ULK1S5555,p65 and p38 phosphorylation,LC3 lipidation and p62 degradation.5Z or TAK1 siRNA inhibited MSU-induced JNK,AMPK,ULK1S555,p65 and p38 phosphorylation.The number of autophagosomes and autolysosomes in 5Z and MSU co-treated cells was significantly lower than that of cells treated with MSU.3.Effects of Gem-induced autophagy on apoptosis:Finally,we explored whether Gem-induced autophagy affects apoptosis.Gem induced cleavage of Caspase8,Caspase3,and PARP.CQ.Beclin-1 siRNA,5Z,TAK1 siRNA and AP enhanced Gem-induced apoptosis.Overexpression of XOR inhibited Gem-induced apoptosis.CQ or 5Z and Gem co-treatment have a significant inhibitory effect on cell proliferation compared with CQ or 5Z alone.In summary,our study shows that XOR catalyzes excess amounts of purine in genotoxic drug-treated cancer cells to produce uric acid,which activates TAK1 and its downstream kinases,JNK and AMPK,to induce autophagy.Inhibition of autophagy enhances Geminduced apoptosis and inhibits cell proliferation.Our study improves our understanding on the molecular mechanisms of genotoxic drug-induced autophagy.