| ObjectiveSorafenib is the most widely used first-line drug in the treatment of advanced hepatocellular carcinoma(HCC).Unfortunately,the resistance to sorafenib in advanced HCC often limit the therapeutic effects.Increasing evidence indicated that artesunate may be a prospective candidate drug in HCC.However,whether artesunate could reverse the resistance of HCC to sorafenib remains unknown.In this study,we integrated the technology of "chemical biological probe fishing target","transcriptome expression data mining","biomolecular network topology calculation"and "clinics-animal-cell"-based multi-dimensional experimental validations.The aim of this study was to evaluate the reversal effects and to investigate the underlying molecular mechanisms of artesunate on drug resistance of HCC to sorafenib.Methods1.The "direct targets of artesunate against HCC-differentially expressed genes between sensitive and resistant liver tissues to sorafenib-anti-HCC targets of sorafenib" interaction network construction and analysis(1)We first identified the target profile of sorafenib-resistant cell intervened by artesunate using artesunate chemical probe and large-scale chemical proteomics experiments.(2)On the basis of GEO database(GSE143235,2020),transcriptomic expression data from sensitive or resistant tumor tissues after long-term treatment with sorafenib were collected.Differentially expressed genes between sensitive and resistant tumor tissues were screened as sorafenib resistance-related genes.(3)Anti-HCC targets of sorafenib were extracted from high-quality reference and database by Python.(4)The interaction network of "artesunate direct target-sorafenib resistance-related gene-sorafenib target" were constructed based on the links among genes.The key network targets of artesunate reversing resistance of HCC to sorafenib were analyzed by calculating network topology feature and functional mining.2.Clinical samples were used to investigate the associations of Actin Filament Associated Proteinl Like 2(AFAP1L2),one of direct targets of artesunate reversing sorafenib resistance in HCC,with malignant progression and poor prognosis in patients with HCC(1)A total of 99 HCC tissue samples and 18 non-cancerous liver tissue samples from 100 patients with HCC were collected from the Fifth Medical Center of Chinese people’s Liberation Army General Hospital.(2)The expression of AFAP1L2 in HCC and non-cancerous liver tissues was detected by RT-qPCR,and the above results were compared and analyzed between the two groups.(3)Kaplan-Meier survival analysis and Log-rank test were carried out to evaluate the correlation between the expression level of AFAP1L2 and the prognosis of HCC patients using the transcriptome expression data of 364 HCC clinical samples collected from Kaplan-Meier-plotter online database.3.The efficacy and characteristics of artesunate in reversing the drug resistance of HCC to sorafenib were evaluated using sorafenib-resistant cell line and animal model(1)Sorafenib was used to induce HepG2 cells continuously for more than 9 months to establish sorafenib-resistant cell model-HepG2R based on the "drug gradient concentration increasing method".The successful construction of drug-resistant cells was detected by cytotoxicity,cell proliferation,apoptosis and Western blot.The success of the drug-resistant cell model was detected by cytotoxicity,cell proliferation,apoptosis and Western blot.In addition,3.5μM sorafenib and 25 μM artesunate were used to interfere with HepG2R cells,and cytotoxicity,cell proliferation and apoptosis were carried out to evaluate the reversal effect of artesunate on drug resistance of HCC cells to sorafenib.(2)The HepG2 cell line stably knocked down or over-expressed AFAP1L2 was established.by lentivirus expression vector,and 3.5μM sorafenib was given to interfere with it.Then the experiments of cytotoxicity,cell proliferation and apoptosis were carried out to investigate the changes of drug resistance of HepG2 cells to sorafenib after knockdown or enforced expression of AFAP1L2.Furthermore,the above cell lines were treated with 25 μM artesunate,and the same detection experiments were carried out to investigate whether artesunate reversing the drug resistance of HCC cells to sorafenib depends on the inhibition of AFAP1L2.(3)Mitophagy inhibitor,chloroquine,and artesunate were used to treat drug-resistant cell line HepG2R at the same time.The experiments of cytotoxicity,cell proliferation and apoptosis were carried out to investigate the reversal effect of artesunate on drug resistance of HCC cells to sorafenib,and to verify whether artesunate reversing sorafenib resistance depends on the activation of mitophagy in the opposite direction.(4)The orthotopic xenograft(PDOX)animal model resistant to sorafenib was established based on the patient-derived orthotopic xenograft animal model by oral administration of sorafenib for 3 weeks.The success of drug resistance animal model was evaluated by Spectrum In Vivo Imaging System,MicroPET/CT,Kaplan-Meier survival analysis,H&E staining,liver brain index,general photos of the liver and so on.Furthermore,30 mg/kg/2 days sorafenib and low dose of artesunate(30 mg/kg/2 day)or high dose of artesunate(60 mg/kg/2 day)were used to interfere with sorafenib resistant animals.The effects of artesunate on reversing the drug resistance of animals resistant to sorafenib were also investigated by Spectrum In Vivo Imaging System and MicroPET/CT.(5)The same drug grouping,dosage and detection methods were used to investigate whether artesunate can enhance the inhibitory effect of sorafenib on tumor in drugsensitive animals based on the established orthotopic xenograft animal model sensitive to sorafenib.4.The target and mechanism of artesunate in reversing the drug resistance of HCC to sorafenib were verified using sorafenib-resistant cell line and animal model(1)The regulation mode and activity trend of AFAP1L2/SRC(SRC Proto-Oncogene,Non-Receptor Tyrosine Kinase)/FUNDC1(FUN14 Domain Containing 1)axis and mitophagy in HCC cells resistant to sorafenib were detected using Western blot,immunofluorescence double staining and co-immunoprecipitation based on the established sorafenib resistant cell model HepG2R.In addition,HepG2R was treated with 3.5 μM sorafenib and 25 μM artesunate.To determine the target and mechanism of artesunate in reversing the drug resistance of HCC to sorafenib,Western blot,immunofluorescence double staining,Pulldown-wb,cellular thermal shift assay(CETSA),surface plasmon resonance(SPR)and other experiments were used to detect the regulation of artesunate on AFAP1L2/SRC/FUNDC1 axis and mitophagy in drug-resistant cells.(2)HepG2 cell lines with low or over-expression of AFAP1L2 were treated with 3.5μM sorafenib.Then Western blot and immunofluorescence double staining were used to detect the changes of AFAP1L2/SRC/FUNDC1 axis and mitophagy in HepG2 cells after AFAP1L2 knockdown or overexpression.Furthermore,the above cell lines were treated with 25 μM artesunate.Western blot and immunofluorescence double staining were also used to detect the regulatory effect of artesunate on AFAP1L2/SRC/FUNDC1 axis and mitophagy,and to verify whether artesunate reversing sorafenib resistance depended on the regulation of AFAP1L2.(3)Western blot,immunohistochemistry and transmission electron microscope were used to detect the regulation mode and activity trend of AFAP1L2/SRC/FUNDC1 axis and mitophagy based on the established orthotopic xenograft animal model resistant to sorafenib.Furthermore,sorafenib of 30 mg/kg/2 day and low dose artesunate(30 mg/kg/2 day)or high dose artesunate(60 mg/kg/2 day)were used to interfere with sorafenib resistant animals.The regulation of artesunate on AFAP1L2/SRC/FUNDC1 axis and mitophagy was also detected by the above detection method,and the target and mechanism of artesunate reversing sorafenib resistance were identified.(4)In order to clarify the target and mechanism of artesunate enhancing the inhibitory effect of sorafenib on HCC,the same administration groups,doses and detection methods were used to detect the regulation mode and activity trend of AFAP1L2/SRC/FUNDC1 axis and mitophagy in orthotopic xenograft animal model sensitive to sorafenib,and the changes of these signal axes and mitophagy in HCC drug-sensitive animals treated with sorafenib combined with artesunate.Results1.Artesunate may intervene sorafenib resistance though targeting AFAP1L2/SRC/FUNDC1 axis and mitophagy(1)A total of 58 direct target proteins of artesunate against HCC were identified through biological probe fishing experiment,and 141 genes related to sorafenib resistance in HCC were obtained by clinical transcriptome data mining.A total of 53 known target proteins of sorafenib were collected from high-quality literature and database.The ID of the above-mentioned drug target proteins were converted into the Official Gene Symbol of their coding genes,which is convenient for subsequent integrated analysis.(2)After constructing the interaction network of"artesunate anti-HCC targetsorafenib resistance-related gene-sorafenib anti-HCC target",and carrying out network topology feature calculation and function mining,we found that artesunate may interfere with SRC and its downstream mitophagy activation key protein FUNDC1 by regulating its direct target AFAP1L2,and then affect the process of mitophagy.Finally,artesunate play its role in reversing the drug resistance of HCC to sorafenib.2.AFAP1L2 was significantly associated with malignant clinicopathological characteristics and poor prognosis of patients with HCC(1)The results of comparative analysis between groups showed that the gene expression of AFAP1L2 in HCC tissues was significantly higher than that in noncancerous liver tissues.(2)According to Kaplan-Meier survival analysis and Log-rank test analysis,it was found that the overall survival time of patients with high expression of AFAP1L2 was significantly shorter than that of patients with low expression of AFAP1L2,that is,high expression of AFAP1L2 predicted poor prognosis of patients with HCC.3.Artesunate effectively reversed the drug-resistant HCC cells and animals to sorafenib(1)After continuous induction of sorafenib,the IC50 of HCC cells to sorafenib was significantly increased,the ability of cell proliferation was significantly enhanced,the apoptosis rate was significantly decreased,and the expression of multi-drug resistance-related marker genes was significantly increased,indicating the successful construction of sorafenib-resistant cells.Furthermore,3.5 μM sorafenib and 12.5 μM,25 μM and 50 μM artesunate were used to interfere with sorafenib-resistant cells.And it was found that artesunate could inhibit the proliferation of drug-resistant cells,induce apoptosis and reverse the resistance of drug-resistant cells to sorafenib in a dose-dependent manner.(2)After the stable overexpression of AFAP1L2 in HepG2,the IC50 of sorafenib was significantly increased,the ability of cell proliferation was significantly enhanced,and the apoptosis rate was significantly decreased,which was contrary to the characterization of HCC cells with low AFAP1L2,indicating that the high expression of AFAP1L2 can induce the drug resistance of HCC cells to sorafenib.Furthermore,we treated the above cells with artesunate,and found that the effects of artesunate on the proliferation and apoptosis of HepG2 with overexpressing AFAP1L2 were significantly weakened,indicating that artesunate reversing the drug resistance of sorafenib depends on the inhibition of AFAP1L2.(3)Chloroquine,an inhibitor of mitophagy,and artesunate were used to treat drugresistant HCC cells at the same time.It was found that chloroquine significantly reduced the mitophagy activated by artesunate.Chloroquine also reduced the inhibitory effect of artesunate on proliferation and apoptosis of drug-resistant cells,indicating that artesunate reverses drug resistance by activating mitophagy.(4)The liver tumors in the HCC group and sorafenib resistant group continued to grow,while those in the sorafenib sensitive group continued to decrease or remain unchanged.The results of H&E staining showed that there were tumor foci,necrotic areas,vacuolar-like cells and inflammatory infiltration in the liver tissues of the HCC group and the drug resistant group,while those in the drug sensitive group were significantly reduced.The results of liver-brain index showed that the liver-brain index in the drug-sensitive group was significantly lower than that in the HCC group,while the liver-brain index in the drug-resistant group was significantly higher than that in the drug-sensitive group,close to or even larger than that in the HCC group.Results of general photos of the liver also showed that there were a large number of tumors and nodules in the liver tissue of the drug-resistant group,which indicated that the animal model of drug resistance to sorafenib was successfully constructed.(5)Sorafenib combined with low and high dose artesunate could effectively inhibit the growth of tumor in sorafenib-resistant nude mice and significantly prolong the overall survival time of drug resistant nude mice(p<0.05).The results of pathological observation showed that after the intervention of sorafenib and low and high dose artesunate,the tumor foci of liver tissue was significantly reduced,the necrotic area and vacuolar-like cells were significantly reduced,and the inflammatory infiltration was significantly alleviated.The results of liver-brain index and general photos of the liver showed that the combination of sorafenib and low-and high-dose artesunate may significantly reduce the number of liver tumors and liver-brain ratio in drug-resistant animals(p<0.05).(6)Sorafenib combined with low and high dose artesunate interfered with the animal model of HCC sensitive to sorafenib,the changes of tumor growth status,overall survival time,histopathological changes,tumor number and liver-brain ratio were the same as those of(5),suggesting that artesunate may enhance the inhibitory effect of sorafenib both on drug-resistant and drug-sensitive tissues(p<0.05).4.Artesunate reversed the resistance of drug-resistant HCC cells and animals to sorafenib through regulating AFAP1L2/SRC/FUNDC1 axis-mediated mitophagy(1)The results of Western blot and immunofluorescence double staining showed that AFAP1L2/SRC/FUNDC1 axis was highly expressed in sorafenib-resistant cells,and mitophagy was inhibited.In addition,CO-IP results showed that AFAP1L2 can directly bind to SRC.When drug-resistant cells were treated with sorafenib combined with different doses of artesunate,artesunate could inhibit the AFAP1L2/SRC/FUNDC1 axis in drug-resistant cells in a dose-dependent manner and over-activate the inhibited mitophagy.In addition,Pulldown-wb,CETSA and SPR showed that artesunate could directly inhibit AFAP1L2 protein expression.(2)The experimental results based on the stable knockdown or overexpression of AFAP1L2 in HepG2 cell lines showed that the expression levels of SRC and FUNDC1,the downstream molecules of AFAP1L2,were significantly decreased or increased after AFAP1L2 was knocked down or overexpressed,and the level of mitophagy showed the opposite trend.Furthermore,artesunate was used to interfere with the above cell lines,and it was found that the inhibitory effect of artesunate on AFAP1L2/SRC/FUNDC1 axis and the induction of mitophagy were significantly weakened,which indicated that the regulation of artesunate on SRC/FUNDC1 axis and mitophagy was dependent on AFAP1L2.(3)The experimental results based on sorafenib-resistant animal model showed that the AFAP1L2/SRC/FUNDC1 axis was significantly up-regulated and mitophagy was significantly inhibited in drug-resistant animals,which was consistent with the results based on drug-resistant cell model.Furthermore,artesunate inhibited the expression of AFAP1L2/SRC/FUNDC1 axis in drug-resistant animals in a dose-dependent manner and significantly increased the level of mitophagy in drug-resistant animals treated with sorafenib combined with low-and high-dose artesunate.These findings showed that artesunate may effectively inhibit AFAP1L2/SRC/FUNDC1 axis and over-activate inhibited mitophagy in drug-resistant animals.(4)The experimental results based on sorafenib drug-sensitive animal model showed that artesunate inhibited the expression of AFAP1L2/SRC/FUNDC1 axis in drugsensitive tissues in a dose-dependent manner and significantly increased the level of mitophagy in drug-sensitive animals treated with sorafenib combined with low and high dose artesunate.These findings suggest that artesunate can significantly inhibit the AFAP1L2/SRC/FUNDC1 axis both in drug-resistant animals and drug-sensitive animals and over-activate the inhibited mitophagy.ConclusionOur findings suggest that AFAP1L2/SRC/FUNDC1 axis-mediated mitophagy inhibition may induce the resistance of HCC to sorafenib,and artesunate may activate mitophagy in sorafenib-resistant cells though the AFAP1L2/SRC/FUNDC1 axis,subsequently leading to inducing the apoptosis of HCC cells and reversing the resistance of HCC to sorafenib. |
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