| Surgery,chemotherapy,and radiation therapy are considered to be the main methods of traditional treatment for malignant tumors.Cholangiocarcinoma and hepatocellular carcinoma are the two most common primary tumors in the hepatobiliary system,but they are not sensitive to chemical drug treatment.In order to treat the two most common primary tumors in the hepatobiliary system,some new anti-tumor drugs are being screened.Since the development of anti-tumor drugs is a time-consuming and expensive task,people hope to use existing drugs to study their new pharmacological effects and improve the therapeutic effects of cholangiocarcinoma.Chloroquine(CQ)is widely used clinically for its anti-malarial effects.At the same time,chloroquine,as a classical autophagy inhibitor,blocks the binding of autophagosomes to lysosomes by changing the acidic environment of lysosomes,so that a large amount of proteins to be degraded accumulate in cells.The inhibition of autophagy by chloroquine resulted in the accumulation of a large number of damaged proteins in the cytosol and induced endoplasmic reticulum stress(ERS),which ultimately led to cell death.To date,studies have shown that chloroquine can induce TRAIL-mediated apoptosis in glioma cells.Our experiments found that cisplatin can increase the ERS of Hela cells and increase the level of autophagy;and blocking autophagy can significantly increase the chemosensitivity of HeLa cells to cisplatin.It is suggested that by studying the mechanism of chloroquine against cholangiocarcinoma,new pharmacological effects may be found,which may provide new targets for the treatment of cholangiocarcinoma.ERS can induce autophagy in a variety of ways.Autophagy can help reduce endoplasmic reticulum stress and promote cell survival.Unobstructed autophagy is an effective way to reduce ERS-induced protein accumulation,and the coordination of the two ways ensures cell survival.Recent studies have found that autophagy not only eliminates misfolded proteins,but also participates in the regulation of cell survival and death,playing a "molecular central platform" role.It can also trigger apoptosis under severe or long-term stress conditions,indicating a more complex cross-talk between the two,thus better understanding the key nodes of ERS and autophagy to promote survival or pro-apoptotic signal transition,through treatment Sexual intervention in the target molecules of the Unfold protein response(UPR)pathway to regulate autophagy and reduce endoplasmic reticulum stress is expected to provide a new and effective strategy for the treatment of various diseases.DNA damage-inducing transcript 3(COP)is considered to be one of the most important molecules in mediating ER stress-induced apoptosis.On the one hand,activated CHOP plays a pro-apoptotic role by up-regulating the Bcl-2 family pro-apoptotic protein Bax;on the other hand,CHOP promotes the transcriptional activation of TRAIL receptor 2(DR5)and the Caspas-8-dependent exogenous apoptotic pathway.Inhibition of CHOP expression protects cells from apoptosis induced by ER stress injury;it has also been found that CHOP-deficient mice are more tolerant to the endoplasmic reticulum stress-inducing agent,Tunicamycin.This suggests that CHOP plays a key role in targeting endoplasmic reticulum stress-induced apoptosis.Further studies have confirmed that in the ERS-apoptotic signaling pathway,there is an autophagy-dependent death-inducing signaling complex,which is called iDISC(Intracellular death-inducing signaling complex),and iDISC is closely related to the autophagy substrate P62 and initiate apoptosis by activation of caspase8.CHOP may be the core molecule that links multiple apoptosis pathways induced by endoplasmic reticulum stress.Therefore,in this study,cholangiocarcinoma cells were used as research objects,and chloroquine was used as an experimental tool to further elucidate the role of autophagy in the treatment of cholangiocarcinoma by studying chloroquine-regulated endoplasmic reticulum-autophagy network in tumor cells.In order to use chlorquine for new drugs,it provides a new experimental basis for the treatment of cholangiocarcinoma.Methods:1.Observe the effect of CQ on the apoptosis of cholangiocarcinoma cell line QBC939.The cells were treated with different concentrations of CQ for 24 h,and the survival rate of the cells was detected by MTT assay.The cells were treated with 50 μM CQ for 12 h and 24 h,and the morphology of the nuclei was detected by Hochest 33258 staining to further reflect the apoptosis.The proportion of apoptosis was measured by flow cytometry.2.Detect the effect of CQ on apoptosis of QBC939 cells.The expression of Caspase-3,Caspase-8,PARP,BAX and BAK proteins was detected by immunoblotting in QBC939 cells at 50 hM for 6h,12 h and 24 h,and the expression of proteins was further determined by quantitative analysis..3.Detection of inhibition of autophagy by CQ and regulation of autophagy-induced apoptosis pathway.The autophagy marker protein LC3 and the autophagy substrate P62 were detected by immunoblotting to determine the effect of CQ on the autophagy process.Autophagy-induced protein expression and activity changes were detected by immunoblotting,co-immunoprecipitation and Caspase-8 activity assay.4.To examine the effects of CQ on transcription and translation of endoplasmic reticulum stress-related genes.84 endoplasmic reticulum stress-related genes were screened by PCR Array gene chip technology,and the genes with significant changes at the transcriptional level were verified by immunoblotting,and the changes in protein expression were determined by quantitative analysis.5.To examine the effect of CQ on the expression of apoptosis-related genes induced by endoplasmic reticulum stress.The expression of apoptosis-related genes in the downstream of endoplasmic reticulum stress core factor CHOP was quantitatively analyzed by qPCR technique to determine the expression changes of these apoptotic genes.6.Detection of endoplasmic reticulum stress,autophagy and apoptosis-related protein expression in tumor tissues of patients with cholangiocarcinoma.Tumor tissue samples from patients with cholangiocarcinoma were stained by immunohistochemistry,and the expression of endoplasmic reticulum stress and autophagy-related proteins in different tissue samples were observed.Results:1.After CQ was applied to QBC939 cells,the cell survival rate decreased with the increase of concentration;under the condition of 50 μM drug,the cells showed typical apoptosis characteristics of nuclear fragmentation and staining with time;Furtherly,the results of flow cytometry showed that the percentage of apoptosis increased with time.2.CQ induced the expression of apoptosis-related proteins Caspase-3,Caspase-8 and c-PARP in QBC939 cells,and also expressed the endoplasmic reticulum stress-related proteins GRP78 and CHOP,and the Bcl2 family pro-apoptotic protein.The expression levels of BAX and BAK also gradually increased,indicating that CQ can induce apoptosis through various pathways.3.The addition of CQ resulted in the accumulation of a large amount of P62 in the cells,and the expression level of LC3 was significantly increased,and the increasing trend of both was time-dependent,indicating that CQ has a significant inhibitory effect on autophagy.4.The results of PCR Array gene chip showed that the transcription level of most endoplasmic reticulum stress-related genes in QBC939 cells treated with chloroquine changed,and the expression levels of CHOP,p-ERK,IRE1α,GADD34 and GRP78 increased significantly.The change of CHOP was the most significant,which was nearly 10 times higher.The results of further immunoblotting were consistent with the results of PCR Array.This indicates that CQ can induce ERS in QBC939 cells.5.The prolongation of CQ incubation time,the gene expression of DCR1,DCR2,DCR3,DCR4,DCR5 and TNFR2 increased significantly.However,there was no significant change in gene expression between the two death receptors,Trail and Fas.This suggests that chloroquine may initiate death receptor pathway-dependent apoptosis by inducing CHOP,a key molecule of endoplasmic reticulum stress.6.GRP78 and P62 are highly expressed in tumor tissues of patients with cholangiocarcinoma.This suggests that tumor cells are at a relatively high level of endoplasmic reticulum stress and autophagy,and this relatively high steady state is a critical homeostasis between adaptive response and pro-apoptosis.Conclusions:1.CQ can induce the expression of BAX and BAK,Caspase-3,Caspase-8 and c-PARP in Bcl2 family of cholangiocarcinoma cells,and also express high levels of endoplasmic reticulum stress-related proteins GRP78 and CHOP.This suggests that inhibition of autophagy may induce apoptosis in cholangiocarcinoma cells through multiple pathways or interactions between these pathways.2.Combined with PCR-aray high-throughput experiments and qPCR results,chloroquine may initiate death receptor-dependent apoptosis by inducing CHOP,a key molecule of endoplasmic reticulum stress.In addition to mediating apoptosis in the endoplasmic reticulum pathway,CHOP activation may also indirectly promote transcription of cell death-related receptor DCR,providing a molecular basis for apoptosis in the death receptor pathway.3.CQ inhibition of autophagy leads to the accumulation of P62,LC-II and other proteins,thereby enhancing the binding of P62 and Caspase-8,promoting the activation of Caspase-8,and inducing apoptosis.We speculate that the proteins accumulated by autophagy inhibitition may provide a platform for Caspase-8 self-activition.4.GRP78 and P62 are highly expressed in tumor tissues of patients with cholangiocarcinoma.This suggests that cholangiocarcinoma is at a relatively high level of endoplasmic reticulum stress and autophagy,and clinical patient tissue specimens suggest that targeting the endoplasmic reticulum/autophagy pathway may be a new therapeutic target.In summary,QBC939 cells under the stimulation of CQ,showed significant autophagy inhibition,which led to a large accumulation of autophagy substrate P62 and its misfolded protein,further aggravated endoplasmic reticulum stress.CQ regulates the endoplasmic reticulum-autophagy network to induce apoptosis through multiple pathways.Staining of specimens from patients with cholangiocarcinoma furtherly confirmed that autophagy levels and endoplasmic reticulum stress levels in tumor cells were maintained at a relatively high critical homeostasis.Breaking this homeostasis by external intervention may lead to apoptosis of tumor cells.Therefore,we promoted apoptosis through the intervention of chloroquine on the endoplasmic reticulum-autophagy network,providing a new entry point for the application of chloroquine in anti-tumor therapy. |
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