Studies On The Effects And Mechanisms Of P38Mediated Autophagy In Dasatinib Induced Hepatotoxicity

Objectives:Dasatinib is the anticancer drug which is clinically used for the treatment of imatinib-resistant chronic myeloid leukemia (CML), it is the second generation of small molecule tyrosine kinase inhibitor. Meanwhile, Dasatinib is in non-small cell clinical phase Ⅱ study of lung cancer. However, hepatotoxicity induced by Dasatinib is one of the serious adverse reactions, which greatly limits the clinical application of Dasatinib, further study of the molecular mechanisms of Dasatinib-induced hepatotoxicity and interferential targets has far-reaching significance. Our previous study found that high dose Dasatinib can inhibit tumor growth, at the same time cause autophagy in liver. Therefore, this study aims to study the role of autophagy in Dasatinib-induced hepatotoxicity, then further explore the mechanism of the autophagy, and looking for small molecule inhibitor which protects Dasatinib-induced hepatotoxicity, provide experimental evidence for protection of Dasatinib-induced hepatotoxicity, expand the breadth and depth of its clinical applications.Methods:Using nude mice xenografted tumor model, ICR mice model, the rat primary hepatocytes and immortalized human hepatocytes Chang Liver, confirmed when Dasatinib-induced hepatotoxicity in clinical treatment doses, it could activate autophagy at the same time, and to explore its effect on Dasatinib-induced hepatotoxicity.(1) ICR mice were given Dasatinib with a dose in clinical treatment of CML for30days, Liver injury was assessed by hematoxylin and eosin (H&E) staining of liver sections and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) activities.(2) A549xenografted nude mice were given Dasatinib with a dose in phase Ⅱ clinical treatment of solid tumor for30days. The anti-tumor efficacy of Dasatinib was assessed by tumor weight, tumor volumn (TV), relative tumor volumn (RTV).(3) Liver injury of nude mice was assessed by serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) activities and hematoxylin and eosin (H&E) staining of liver sections when Dasatinib had an inhibition to tumor.(4) Autophagy was detected by immune transmission electron microscopy (TEM) and Western Blot.(5) ICR mice were treated with supplemental nutrients (10ml/kg), dasatinib (50mg/kg) or both for30days, Food intakes were calculated daily. Origin of autophagy was explored by Western Blot, blood biochemical index and hematoxylin&eosin (HE) staining of liver sections.(6) Rat primary hepatocytes were treated with Dasatinbi by different concentration and time, the occuration of autophagy was studied by AO, MDC stain and Western Blot in Dasatinib-induced hepatotoxicity, as a supplement, autophagy was verified in Chang Liver.(7) ICR mice were treated with dasatinib (50mg/kg/day) or vehicle control for4weeks, and4of16mice of each group were sacrificed every week. Dasatinib-induced the liver injury and autophagy sequence was analyzed by blood biochemical index, HE stain and Western Blot.(8) Hepatocytes were treated with dasatinib and3-MA, Pi-positive cells were quantified by FACS, Total cell lysates were subjected to western blot by anti-LC3, c-PARP, c-caspase-3bodies.(9) Autophagy related protein ATG-5was silenced by small molecular RNA interference technology in primary hepatocytes, then was treated with Dasatinib for24h, PI-positive cells were quantified by FACS, Total cell lysates were subjected to western blot by anti-LC3, c-PARP, c-caspase-3bodies.(10) ICR mice were randomly divided into4groups (n=6mice) and treated with3-MA, dasatinib or both for30days, average body weight and mortality rates were compared, autophagy was observed by TEM, meanwhile, the effect on Dasatinib-induced hepatotoxicity was studied by ALT, AST, LDH and HE stain when autophagy was inhibited.Using rat primary hepatocytes and immortalized human hepatocytes Chang Liver, explored the regulation mechanism of autophagy in Dasatinib-mediated hepatotoxicity, furthermore looked for small molecular inhibitor of targets for intervention.(1) The rat primary hepatocytes and immortalized human hepatocytes Chang Liver were treated by Dasatinib for different time, reactive oxygen species(ROS) was detected.(2) The rat primary hepatocytes and immortalized human hepatocytes Chang Liver were treated by Dasatinib for different time, JNK, ERK, p38and their phosphorylation of MAPK were detected by Western Blot.(3) JNK, ERK was silenced by small molecular RNA interference technology in primary hepatocytes, then were treated with Dasatinib for24h, PI-positive cells were quantified by FACS, Total cell lysates were subjected to western blot by anti-LC3, c-PARP, c-caspase-3bodies.(4) p38was silenced by small molecular RNA interference technology in primary hepatocytes, then were treated with Dasatinib of different concentration for24h, PI-positive cells were quantified by FACS, Total cell lysates were subjected to western blot by anti-LC3, c-PARP, c-caspase-3bodies.(5) Autophagy was detected by AO, MDC stain to assess the effect of p38agonist ISO on Dasatinib.(6) PI-positive cells were quantified by FACS to assess the effect of p38agonist ISO on Dasatinib.(7) Autophagy and apoptosis protein LC3, c-PARP, c-caspase-3and p-p38were analyzed by Western Blot to assess the effect of ISO on Dasatinib in rat primary hepatocytes and immortalized human hepatocytes Chang Liver Using ICR mice to study the protective effect of ISO on Dasatinib-induced hepatotoxicity in vivo. Meanwhile human non-small cell lung cancer A549, prostate cancer PC3, breast cancer MDA-MB-231and A549xenografted nude mice model were adopted to study the antitumor effect of ISO on Dasatinib.(1) ICR mice were randomly divided into4groups (n=6mice) and treated with ISO (8ng/kg), dasatinib (50mg/kg) or both for30days, average body weight, blood biochemical index, HE staining of liver sections were detected to assess the effect of ISO on Dasatinib-induced hepatotoxicity. Total liver lysates were analyzed by western blot using antibodies against c-PARP and c-caspase-3to study the effect of ISO on Dasatinib.(2) Total liver lysates were analyzed by western blot using antibodies against LC3and p-p38to study the effect of ISO on Dasatinib.(3) The effect of ISO on Dasatinib-induced autophagy was observed with TEM.(4) A549, PC3and MDA-MB-231cells were treated with protective concentration of ISO, dasatinib or both for24hours, apoptosis rate was determined by FACS following PI staining to assess the antitumor effect of ISO on Dasatinib in vitro.(5) Nude mice transplanted with A549human xenografts were randomly divided into4groups and treated with ISO, dasatinib or both for30days. Tumor weight, tumor volumn, relative tumor volumn were detected to assess the antitumor effect of ISO on Dasatinib.Results:1) Autophagy was activated after clinical dose Dasatinib treatment and induced hepatotoxicity, inhibition of autophagy could enhance Dasatinib-induced hepatotoxicityWe treated ICR mice treated with a clinical dose of dasatinib for30days. In agreement with clinical reports, dasatinib stimulated liver dysfunction in vivo. Tumor growth with50mg/kg dasatinib treatment for30days was significantly inhibited compared to control group, the inhibitory rate was55.9%. However, the body weight of dasatinib-treated animals was decreased. Meanwhile, the serum hepatic enzyme activity of ALT, AST and LDH were2-3fold than control. Furthermore, treated mice exhibited histopathological changes in the liver, these results characterized the occurrence of dasatinib-induced liver damage when there was anti-tumor effect. We employed TEM to detect the presence of autophagic vacuoles, dasatinib increased endogenous conversion of LC3-I to LC3-II levels in liver tissues. In addition, we compared the food intake between the Dasatinib treated and non-treated groups and found no significant difference in ICR mice for30days, we added supplemental nutrients to the dasatinib group and that the dasatinib-induced LC3-Ⅰ to LC3-Ⅱ levels and the serum hepatic enzyme activity, supplemental nutrients could not changeover pathological injury induced by Dasatinib, which suggested that autophagy was directly activated by dasatinib, but not starvation. Treatment with dasatinib induced the accumulation of AO and MDC in the cytoplasmic vacuoles in a time-and concentration-dependent manner. The conversion of the LC3(LC3-Ⅰ to LC3-Ⅱ) and dramatic degradation of p62was also observed after dasatinib treatment in primary cultured rat hepatocyte in a time-and concentration-dependent manner, indicating dasatinib induces autophagic flux in tissues and cells. The serum and liver tissues of ICR mice at different time suggested that dasatinib-induced liver dysfunction began at3weeks, and autophagy occurred at4weeks. After exposed to dasatinib and/or the specific inhibition of autophagosome formation with3-MA for24hours, we found that apoptosis induction by dasatinib was markedly enhanced in the presence of3-MA, as determined by PI staining, accompanied with the increase of c-PARP and c-caspase-3. The apoptosis rates and apoptosis protein of hepatocyte cells significantly increased (p<0.01) in the Atg5knockdown group compared to negative control group;3-MA treatment aggravated dasatinib-induced body weight loss and alse further increased serum ALT, AST and LDH levels. In particular,2out of6mice died after treated with3-MA and dasatinib. By H&E staining, centrilobular necrosis was evident in mice livers after dasatinib treatment, which was further exacerbated by3-MA treatment, these data indicated that induction of autophagy protected against dasatnib-induced liver injury in vivo.2) p38-MAPK was required for Dasatinib-induced autophagy, small molecular p38agonist isoproterenol hydrochloride(ISO) could protect the hepatotoxicity of Dasatinib via activated p38meditated autophagy.Hepatocytes treated with12.5μM dasatinib for various times (0-7h) were evaluated, and, compared to untreated cells, the ROS level in dasatinib-treated hepatocytes increased significantly, and to the maximum at7h. Dasatinib treatment induced a sharp increase in the phosphorylation of p38, JNK and ERK through Western Blot. Knockdown of JNK and ERK expression also reduced the expression of the apoptotic proteins cleaved caspase-3and cleaved PARP, which were induced by dasatinib treatment, although knockdown had no effect on the conversion of LC3. The inhibition of p38activity by siRNA sitmulated dasatinib-induced hepatocyte cell death, and dramatically blocked LC3-II induction by dasatinib.10nM ISO could enhance acidic vesicular organelles induced by Dasatinib with AO and MDC stain. The apoptosis rate of primary cell decreased from48.0%to26.0%FACS following PI staining, and Chang liver cell decreased from48.0%to26.0%FACS following PI staining. Western blot analysis revealed a significant increase in the amount of LC3-II after dasatinib plus the ISO compared to dasatinib treatment alone, which was accompanied by decreased caspase-3and PARP cleavage. These results suggest p38activation is an essential step in the stimulation of autophagy and enhancement of p38activity, may protect against dasatinib-induced liver damage in vitro.3) ISO mitigated dasatinib-induced liver dysfunction in mice without altering its anticancer activity in vivo and vitro.The results of ICR mice in vivo suggested, compared with the control and ISO group, ISO (8ng/kg) could upregulated the dasatitinib-induced body weight loss, and interestingly, this drug also reduced dasatinib-mediated increase ALT, AST and LDH levels; In addition, centrilobular necrosis widespread apoptotic cells and moderate multifocal inflammation were evident in the livers of mice treated with dasatinib by H&E staining, whereas this type of liver damage was virtually absent in ISO-treated mice. Furthermore, ISO decreased the level of activated caspase-3compared to treatment of dasatinib treatment alone. TEM and western bloting were performed to confirm that ISO enhanced dasatinib-induced autohagy in mice liver. In three antitumor cell lines A549, PC3, MDA-MB-231, the proportion of apoptotic cells after10μM dasatinib treatment did not decrease with lOnM ISO. Meanwhile, administration of ISO at a dose of8ng/kg each day for30days produced no significant difference in mean tumor weight or RTV compared to the50mg/kg dasatinib-alone group. These results indicated that ISO has potential clinical application for mitigating the liver injury resulting from dasatinib treatment without influencing its anticancer activity.Conclusions:This study reveals Dasatinib-induced hepatotoxicity along with its anti-tumor efficiency, and protective autophagy via p38signaling can resist Dasatinib-stimulated hepatotoxicity, which provides a novel mechanism for the hepatotoxicity induced by Dasatinib. Importantly, ISO attenuated dasatinib-induced liver failure without influencing its anticancer activity, which provides novel, feasible for managing the clinic applications of Dasatinib and protect its hepatotoxicity.