The Mechanism Of Ursodeoxycholic Acid For The Inhibition Of Hepatocellular Carcinoma

Background and ObjectiveLiver cancer represents 6%and 9%of the global cancer incidence and Imortality burden,respectively.With an estimated 746 000 deaths iin 2012.liver cancer is the second most common cause of death from cancer worldwide.Liver cancer is the fifth most common cancer in men(554 000 new cases,8%of the total)and the ninth most common in women(228 000 cases,3%of the total).Almost three quarters of the new cases occur in areas with low and medium human development;more than half of the global incidence and mortality is in China.Given the high fatality of liver cancer(overall mortality-to-incidence ratio,0.95),the geographical pattherns and trends for mortality are very similar to those observed for incidence.The most important etiological factors for hepatocarcinogenesis remain viral infections,usually involving HBV or HCR,and toxi injury,typically initiated by ingestion of aflatoxin or consumption of alcohol.HCC is most commonly associated with chronic liver disease.Cirrhosis is not,moreover,required for development of HCC.Evidence from experimental animal models and human studies suggested that UDCA exerted chemopreventive activity against colorectal carcinogenesis.Sung-Chul Lim et al.proved that UDCA-induced apoptosis is mediated by death receptor 5(DR5)expression,which is regulated by the raft formation/ROS production/PKC? activation pathway and DR5 localization into lipid rafts in gastric cancer cells.And they proved UDCA significantly decreased tumor growth in gastric cancer xenograft mice."Autophagy" is broadly defined as a mechanism by which intracellular and extracellular substrates are delivered to lysosomes for degradation.This process is required for the maintenance of cellular homeostasis,generation of amino acids for sustained viability during periods of starvation,and enhanced protection against pathogens.On the basis of the delivery route and cargo specificity,three different types of autophagy have been distinguished—macroautophagy,microautophagy,and chaper-one-mediated autophagy.It is not a surprise,therefore,that autophagy has a fundamental role in cancer and that perturbations in autophagy can contribute to malignant disease.Autophagy worked in various aspects of tumor suppression including the response of cells to nutrient and hypoxic stress,the control of programmed cell death,and the connection to tumor-associated immune responses.Indeed,oncogene activation can inhibit autophagy,in part through a mechanism similar to one used for inhibition of apoptosis.Thus,enhanced autophagy correlates with activation of anti-tumor immunity,and its downregulation may allow malignant growths to avoid immune surveillance.The microtubule-associated protein 1A/1B light chain 3(LC3)puncta indicated the cancer with the upregulated of autophagy.Lim SC et al.thought that UDCA reduced ATG5 levels notably,an essential component of autophagy,in the SNU601/WT(gastric cancer celline).DNA is the template for the basic processes of replication and transcription,making the maintenance of genetic stability critical for viability.Even before the discovery of the double helix in 1953,it was known that exogenous agents that we are exposed to in our everyday lives,such as X-rays,ultraviolet light,and various chemicals,can cause genetic changes that can promote cancer.It has been estimated that each human cell is subject to approximately 70,000 lesions per day.The majority of lesions(75%)are single-strand DNA breaks,which can arise from oxidative damage during metabolism or base hydrolysis.ssDNA breaks can also be converted to DNA double-strand breaks,which although much less frequent,are more dangerous.Gmma-H2AX is one of the most widely used marker to measure DNA damage.In our research,we study that if UDCA could inhibit HCC by autophagy or DNA damage repair.Part ?The mechanism of UDCA for the inhibition of HCC in vitroObjectives1.To evaluate if UDCA could inhibit the growth of HCC in vitro.2.To examine the expression of y-H2AX,LC3B and P53 of HCC cell lines with UDCA.Methods1.A CCK-8 assay is based on the measurement of activity of a marker associated with the number of viable cell.Our results showed different decreases in OD(450 nm)values in cells treated with UDCA(0.6,0.8,0.9,1.0,1.1,1.2 mmol/L)for 24 h,48 h and 72 h.2.The migration of HCC cells was assessed using the 24-well polycarbonate membrane cell migration assay kit according to the manufacturer's instructions.Four views were examined per Transwell plate and the cells/view were counted at 200×magnification.Each experiment was performed in triplicate.3.The expression of y-H2AX was assessed using qRT-PCR and Western Blot according to the laboratory instructions and the manufacturer's instructions.4.The expression of LC3B and p53 was assessed using qRT-PCR and Western Blot according to the laboratory instructions and the manufacturer's instructions.Results1.A significant difference was observed between the control groups and the experimental groups of 7721 cells that were treated with different dosages UDCA at 24 h(Student's t-test,P<0.05).A statistically significant difference was also observed between the experimental groups and the control groups of 7721 cells 48 h/72 h after UDCA treatment(Student's t-test,P<0.05).The viability of HepG2 and Hep3B2.1-7 cells was significantly reduced after treatment with UDCA compared with cells that were not treated with UDCA(Student's t-test,P<0.05).The viability of L02 was not significantly reduced after treatment with UDCA 24h(Student's t-test,P>0.05).2.UDCA could inhibit the migration of 7721(Student's t-test,P<0.05),HepG2 cells(Student's t-test,P<0.05)and Hep3B2.1-7 cells(Student's t-test,P<0.05).And this inhibition was intensified as the dosage increased.3.UDCA inhibits the level of ?-H2AX in vitro.Additionally,these differences in expression were statistically significant(Student's t-test,P<0.05).4.UDCA improves the level of LC3B and p53 in vitro.Additionally,these differences in expression were statistically significant(Student's t-test,P<0.05).Conclusions1.UDCA could reduce the viability of HCC cells,and the inhibition enhanced as the dosage of UDCA increased.2.UDCA could inhibit the migration of HCC cells,and the inhibition enhanced as the dosage of UDCA increased.3.UDCA could inhibit the level of ?-H2AX of HCC cells,and the inhibition enhanced as the dosage of UDCA increased.4.UDCA improves the level of LC3B and p53 of HCC cells,and the expression of LC3B/p53 increased as the dosage of UDCA increased.5.UDCA could inhibit HCC by autophagy.6.UDCA could inhibit HCC by DNA damage repair of DSBs.Part ?The mechanism of UDCA for the inhibition of HCC in vivoObjectives1.To evaluate if UDCA could inhibit the growth of HCC in vivo.2.To examine the expression of y-H2AX,LC3B and P53 of HCC xenograft nude mice with UDCA.MethodsApproximately 2×106 cells(resuspended in 100 ?l of DMEM)were injected subcutaneously into the posterior axillary of BALB/c(nu/nu)nude mice.Three to 4 weeks later,the mice were assigned to different treatment groups.The mice were used in experiments 7 to 8 weeks after the inoculation of the tumor cells.UDCA was dissolved in the drinking water of the mice.There were three groups:the control group,the low dose UDCA group(90mg/kg/d)and high dose UDCA group(270 mg/kg/d).Results1.7-8 weeks after transplantation,tumor growth in nude mice that were treated with UDCA was significantly slower than that in control mice that were not treated with UDCA(Student's t-test.P<0.05).2.Hematoxylin-eosin staining of tumors from nude mice showed that the percentage of cells that underwent cell death increased as the dosages of UDCA increased3.IHC staining and WB of sections from paraffin-embedded xenograft tumors showed an increase in LC3B expression in tumors from mice treated with UDCA.Additionally,these differences in expression were statistically significant(Student's t-test,P<0.05).4.IHC staining and WB of sections from paraffin-embedded xenograft tumors showed an decrease in y-H2AX expression in tumors from mice treated with UDCA.Additionally,these differences in expression were statistically significant(Student's t-test,P<0.05).Conclusions1.UDCA could inhibit the growth of HCC xenograft nude mice,and the inhibition enhanced as the dosage of UDCA increased.2.UDCA could promote cell death of HCC xenograft nude mice,and the promotion enhanced as the dosage of UDCA increased.3.UDCA could inhibit the level of y-H2AX of HCC xenograft nude mic,and the inhibition enhanced as the dosage of UDCA increased.4.UDCA improves the level of LC3B of HCC xenograft nude mice,and the expression of LC3B increased as the dosage of UDCA increased.5.UDCA could inhibit HCC by autophagy.6.UDCA could inhibit HCC by DNA damage repair of DSBs.