Hepatotoxicity And Mechanisms Of Tyrosine Kinase Inhibitors In Dasatinib And Gefitinib

Objective:To evaluate hepatotoxicity of small molecular tyrosine kinase inhibitors in Dasatinib and Gefitinib, and investigate the mechanisms of hepatotoxicity both in vivo and in vitro, furthermore, to find effective drugs for liver protection, provide a viable strategy for the reasonable use of tyrosine kinase inhibitors in the clinic.Methods:Serum hepatic enzyme activity of ALT, AST and LDH were detected, after Sprague-Dawley rats were treated with Dasatinib through intragastric. Liver samples were homogenized to detect GSH, SOD and MDA, sections of liver fixed were stained with hematoxylin and eosin for histopathological analysis. All above were used as an in vivo model system to evaluate Dasatinib-induced liver injury. Rat primary hepatocytes, normal human liver cell lines L02 and Chang liver were applied to investigate mechanism of Dasatinib-induced hepatotoxicity in vitro. The combination of Dasatinib and GSH was analyzed by UV scanning. MTT assay was used to calculate IC50 values of rat primary hepatocytes, normal human liver cell lines L02 and Chang liver after varying concentrations of Dasatinib treatment for 48 h. Apoptosis was evaluated by DAPI staining, DNA gel electrophoresis assay and PI staining for flow cytometry. AO staining was used in detection of autophagy. H2DCFDA stain and flow cytometry were used to test the content of ROS, JC-1 stain and flow cytometry were used to test the change of mitochondrial membrane potential (ΔΨm). Expression and activation of apoptosis, autophagy and oxidative stress related proteins were measured by western blotting. Real-Time PCR was used to analyze the expression level of mRNA in HO-1 and NQ01. siRNA gene silencing technology was used to knock down Atg 5, JNK and p38, in order to evaluate the relationship between autophagy and apoptosis, in addition, the relationship between MAPK pathway and autophagy or apoptosis. Model of human tumor xenografted A549 in nude mice was applied to evaluate antitumor activity of Gefitinib in vivo, meanwhile, detection in activity of ALT, AST and LDH, sections of histopathological analysis were used to evaluate hepatotoxicity of Gefitinib in vivo. IC50 values of Gefitinib in L02 and Chang liver were meansured by MTT assay. Flow cytometry were used to test the content of ROS, autophagy was analyzed by AO stain. Atg 5 was knocked down to evaluate the relationship between autophagy and apoptosis. Western blotting was used in detection of expression and activation of apoptosis, autophagy and oxidative stress related proteins.Results: 1. In vivo, body weights of Dasatinib-treated rats decreased. Serum hepatic enzyme activity of ALT, AST and LDH elevated significantly after Dasatinib treatment.. In addition, GSH, SOD in liver decreased, while MDA increased. And slices of liver for analysis showed diffuse ballooning degeneration of hepatocytes with lobular disarray.2. In vitro, Dasatinib exhibited obvious cytotoxicity in rat primary hepatocytes, L02 and Chang liver cell lines after treatment for 48 h, and IC50 value were 21.1μM, 12.5μM and 15.5μM, respectively.3. DAPI staining exhibited clear chromatin condensation, fragment and apoptotic bodies after Dasatinib treatment, DNA gel electrophoresis assay indicated Dasatinib induced DNA 180-200 bp fragments, resulted in a typical ladder pattern. Westen blotting demonstrated caspase-3 activation and PARP cleavage. All these data revealed that Dasatinib-induced hepatotoxicity was through the process of apoptosis.4. Incubation of GSH and Dasatinib for 5h at 37℃, UV absorption sequential scanning from 200 nm to 260 nm demonstrated both absorption peak of GSH and Dasatinib shifted, which revealed that Dasatinib could form GSH-covalent adducts.5. H2DCFDA staining revealed, Dasatinib increased the ROS level, decreased the content of GSH, induced oxidative stress, activated MAPK pathway, resulted in phosphorylation of p38 kinase, JNK and ERK, and the phosphorylation level increased to a maximum value and then decreased with a further increase in the duration of treatment. NAC, a typical antioxidant, could attenuate Dasatinib-induced oxidative stress. Meanwhile, downstream of oxidative stress, Nrf2 as an antioxidant transcription factor was detached to lead to nuclear translocation, and increased expression level of antioxidative gene HMOX-1and NQO1.6. JC-1 staining showed, Dasatinib could reduce the mitochondrial membrane potential, mitochondrial swelling assay indicated, Dasatinib 30μM caused mitochondrial permeability transition open, release of cytochrome c and the activation of caspase-dependent apoptosis pathway, which results in apoptosis.7. Dasatinib not only induced oxidative stress, but also induced ER stress, the key proteins such as p-e1F2α, p-PERK, ATF4 and CHOP related to ER stress pathway had corresponding change. ER stress could promated the activation of caspase, and lead to apoptosis.8. After further study in normal human liver cell lines L02 and Chang liver, we found autophagy accompanied oxidative stress-induced apoptosis, moreover autophagy played a role in protection of Dasatinib-induced injury. Pretreatment with 3-MA or knock down of Atg 5 would attenuate autophagy and aggravate apoptosis.9. In Dasatinib-induced activation of MAPK pathway, JNK was related to apoptosis, while p38 was related to autophagy. Dasatinib-induced autophagy remained, and apoptosis increased after silence of JNK, however, Dasatinib-induced autophagy attenuated, and apoptosis increased after silence of p38.10. NAC could attenuate Dasatinib-induced ROS level in L02 and Chang liver, reduce the expression of LC3, cleaved caspase-3å'ŒPARP, attenuate autophagy and apoptosis, and protect hepatocytes against Dasatinib-induced hepatotoxicity.11. Gefitinib 200 mg/kg could effectively inhibit human tumor xenografted A549 in nude mice, but pharmacological effect leaded to liver injury at the same time, accompanied by ALT, AST and LDH elevations, and histopathological analysis change.12. After Gefitinib treatment in L02 and Chang liver, IC50 value were 24.1μM and 31.9μM, respectively. Gefitinib could increase ROS level in L02 and Chang liver, activated MAPK pathway, phosphorylation level of JNK, p38 and ERK experienced increased trend.13. Gefitinib induced intensive autophagy in L02 and Chang liver in low concentration, and induced apoptosis in high concentration, the relationship between autophagy and apoptosis was in succession, the autophagy was also protective.Conclusion:Dasatinib and Gefitinib as tyrosine kinase inhibitors revealed obvious hepatotoxicity in vivo and in vitro. Both of them can consume GSH in hepatocytes, increase ROS level, cause oxidative stress, activate MAPK pathway, injury function of mitochondria, finally lead to apoptosis. Meanwhile, Dasatinib and Gefitinib can induce protective autophagy.