Suppression Of ER-resident Protein RCN1 By HDAC Inhibitors Reverses Drug-resistance Of Lung Cancer And Enhances The Efficiency Of Chemotherapy

IntroductionAlthough the treatment of malignant tumors has made great progress,whatever radiotherapy or chemotherapy,targeted treatment or immunotherapy,multi drug resistance(MDR)would eventually occur,When the tumor cells become resistant to one anti-tumor drug,many mechanisms,such as metabolism and gene mutation,mediated the cells to become resistant to other drugs with completely different structures and mechanisms of action,so they can not play the role of anti-tumor,and the cancer cell become multi drug resistance.Not only multi drug resistance seriously restricts the clinical therapeutic effect,but also affects the quality of patients'life.Therefore,to overcome the drug resistance and reduce the side effects of drugs have always been a crucial issue in the field of tumor treatment To explore the mechanism of drug resistance is the key to finding new therapeutic targets and reversal agents,and also providing ideas for the high efficiently usage of anti-tumor drugs.Lung cancer is a malignant tumor with high incidence rate and high mortality rate worldwide.Radiotherapy,chemotherapy and immunotherapy are the main treatment methods for metastatic lung cancer.Because chemotherapy is still the first-line treatment for lung cancer,and the mechanism of drug resistance is also a mainly point.At present,the strategies to overcome the drug resistance of lung cancer mainly include:1)research for the mechanism of drug resistance,and find new reversal drugs,for example,the high expression of drug pump protein P-gp is one of the drug resistance mechanisms of docetaxel(Doc),while the affinity of cabazitaxel to P-gp is low,which can be a alternative treatment plan,it is used to treat patients with docetaxel resistance;2)choose a new treatment plan,for example,immunotherapy after failure of first-line chemotherapy,which is also a new reversal method;3)choose a combination chemotherapy plan without cross resistance,for example,after failure of first-line chemotherapy with platinum based drugs,docetaxel(Doc)can be used for treatment,and the survival time of lung cancer patients was prolonged with the combination chemotherapy plan.In this study,we found that the expression of reticulocalin-1(RCN1)was high in lung cancer,and the expression of RCN1 in a variety of tumor resistant cells increased significantly,which was closely related to the drug resistance of tumor cells.Therefore,we focus on the role of RCN1 in lung cancer drug resistance,and through the screening of drugs depressing RCN1,we found that HDAC inhibitors can significantly reduce the expression of RCN1,and can significantly increase the sensitivity of drug-resistant cells to chemotherapy drugs,especially adriamycin,reduce their toxicity,and preliminarily explore the mechanism of HDAC inhibitors can significantly reduce the expression of RCN1.RCN1,as an endoplasmic reticulum protein,its physiological and pathological functions are rarely studied,mainly focusing on the expression of tumor.In this paper,we researched the relationship between RCN1 and multidrug resistance of tumor and its influence on chemotherapy drugs,to provide a new basis for the clinical use of drug-resistant lung cancer patients.ResultsPart ?:Endoplasmic reticulum protein RCN1 mediates multidrug resistance of lung cancer1?RCN1 is highly expressed in lung cancer and is related to the survival of cancer cellOur research group has found that RCN1 is highly expressed in a variety of tumor cells,including prostate cancer,lung cancer and so on.The results showed that RCN1 was expressed in immortalized HBE,small cell lung cancer cells(H1688,H446)and non-small cell lung cancer cells(A549,H460,H1299).The expression of RCN1 protein in lung cancer tissue was further analyzed.The results of immunohistochemistry showed that the expression of RCN1 protein in lung cancer was significantly higher than adjacent tissues,indicating that RCN1 may be related to the occurrence of cancer.The results showed that RCN1 expression significantly negatively correlated with the survival of lung cancer patients,which acquired from overall survival analysis of lung cancer patients by TCGA database.The survival rate of A549 and H1688 cells was significantly decreased,while depressing RCN1 protein.Therefore,the high expression of RCN1 in lung cancer tissues and cells related to cell survival,and lung cancer patients survival.2?The expression of RCN1 in drug-resistant cells of different types of tumor are significantly increased,which suggest that RCN1 related to drug resistance1.The expression of RCN1 increased significantly in many tumor resistant cells,and decreased the expression of RCN1 inhibited the survival of resistant cellsWe used paclitaxel induced multidrug resistance cells H460/Tax and A549/Tax,docetaxel induced multidrug resistance cells PC3/Doc,vincristine induced multidrug resistance cell KB/VCR to analyze the expression of RCN1.The results showed that compared with the corresponding sensitive cell lines,the expression of RCN1 resistant cells was improved,while down-regulation of RCN1 expression,significantly inhibited the survival of drug-resistant cells.2.Effect of RCN1 expression on the efficacy of chemotherapy drugsMTT assay was used to detect the effect of decreased or increased rcnl expression on the efficacy of chemotherapy drugs.We chose docetaxel(Doc),VP 16,5-fluoropyrimidine(5-FU)and gemcitabine(GEM).The results showed that after overexpression of RCN1 in H446 cells,the sensitivity of cells to the above drugs decreased to some extent,indicating that the overexpression of RCN1 may relate to cell drug resistance.We used A549/Tax resistant cells decreased the expression of RCN1,and analyzed the response of drug-resistant cells to drugs.The results showed that the drug-resistant cells decreased the sensitivity of RCN1 to cisplatin(CDDP)and doxorubicin(Doxo);on the contrary,the resistance of A549 cells to drugs slightly increased after overexpression of RCN1.The results showed that the up-regulation of RCN1 expression was involved in the process of drug resistance.Part ?:Mechanism of HDAC inhibitor down regulating RCN1 and its sensitization effect on chemotherapy drugsDue to the up-regulation of RCN1 expression leading to drug resistance of cells,we screened small molecule compounds which down-regulating the expression of RCN1,and explored the possibility of reversing the resistance mediated by RCN1 through drug combination.1?HDAC inhibitors inhibit RCN1 mRNA expression in lung cancer cells1.Screening of small molecule drugs which down-regulate RCN1We selected drugs for lung cancer and other commonly used drugs or inhibitors for preliminary screening,including:1)DNA damage drugs,cisplatin(CDDP),doxorubicin(Doxo);2)microtubule targeted drugs docetaxel(Doc);3)epidermal growth factor receptor(EGFR)targeted drugs,gefitinib(Gefi);4)HDAC inhibitors(HDACi),trichostatin A(TSA),vorinostat(suberoylanilide hydroxamic acid,SAHA).The results showed that RCN1 showed two bands(total RCN1 with smaller molecular weight and glycosylated RCN1 with larger molecular weight).Gefitinib and docetaxel showed an up-regulation trend of RCN1,while TSA and SAHA,inhibitors of HDAC,significantly reduced total RCN1.We used A549 and A549/Tax cells to further determine the concentration dependence of TSA and SAHA on the inhibition of RCN1.Therefore,HDAC inhibitors can significantly down regulate the expression of RCN1,which may be one of the mechanisms of HDAC inhibitors reversing drug resistance.2.Mechanism of HDAC inhibitor down regulating RCN1 expressionBecause TSA and SAHA,inhibitors of HADC,which can regulate the expression of target genes at the transcription and protein levels through various mechanisms,we explored the mechanism of HDAC inhibitors down-regulating the expression of RCN1 from protein translation,degradation and transcription.(1)HDAC inhibitors did not affect the translation of RCN1.We first analyzed the half-life of RCN1 in A549/Tax.When we added actinomycin CHX,the protein synthesis inhibitor,the half-life of RCN1 was about 12h,but in the presence of HDAC inhibitors,the half-life of RCN1 did not change significantly,indicating that HDAC inhibitors did not affect the protein synthesis of RCN1.(2)Autophagy induced by HDAC inhibitors does not affect the expression of RCN1 Because TSA and SAHA can inhibit protein level through autophagy mechanism,we first analyzed whether SAHA can reduce RCN1 by inducing autophagy and promoting RCN1 degradation.The results showed that SAHA can significantly induce LC3II,decrease p62 and NBR1 protein level,indicating that SAHA activate autophagy.When HCQ is used to block autophagy,it does not reverse inhibition of RCN1.In the same time autophage induced by starvation and BOR neither decrease RCN1 significantly.Above all,SAHA can not influence translation and degration of RCN1 protein,we need to research more.(3)HDAC inhibitors inhibit the expression of RCN1Because HDAC inhibitors do not affect the protein level of RCN1,and the main role of HDAC inhibitors is to regulate histone acetylation and affect gene transcription.So we analyzed the effect of HDAC inhibitor on RCN1 mRNA.The results showed that SAHA significantly reduced the transcription of RCN1 in different cells in a concentration dependent manner,indicating that SAHA inhibited the gene expression of RCN1.We further analyzed the promoter of RCN1 and found that there were multiple binding sites of transcription factors,including SP1,Myc,TFAP4,C-JUN,GATA2,NF-?B,etc.After SAHA treatment,we found that SAHA could significantly inhibit Myc,TFAP4 and p65(NF-?B subunit).Overexpression of TFAP4 and p65 can reverse the inhibition of RCN1 with SAHA in varying degrees,indicating that SAHA inhibit the transcription of RCN1 mRNA by inhibiting the expression of transcription factors p65 and TFAP4.2?HDAC inhibitors down regulate RCN1 and increase the sensitivity of drug-resistant cells to doxorubicin1.Screening of HDAC inhibitor combination drugsBecause the high expression of RCN1 mediates the drug resistance of tumor,and HDAC inhibitor can down regulate the expression of RCN1,does HDAC inhibitor reverse the drug resistance of tumor by combining chemotherapy drugs?The results of CI curve showed that HDAC inhibitor combined with doxorubicin,gemcitabine and cisplatin had a obviously inhibitory effect on cell A549,while HDAC inhibitor combined with doxorubicin and gemcitabine had a good inhibitory effect on resistant cell A549/Tax,especially doxorubicin.2.Animal experiments showed that HDAC inhibitor combined with doxorubicin could reverse drug resistance to a certain extentThe antitumor effect of SAHA combined with doxorubicin was analyzed.The results showed that SAHA inhibited the tumor tissue of A549 and A549/Tax cells,and that doxorubicin had better antitumor effect on sensitive cells than drug-resistant cells,while SAHA combined with doxorubicin had significant antitumor effect on drug-resistant cells In addition,Ki67 positive staining cells were significantly reduced and proliferation was inhibited.3.HDAC inhibitor combined with doxorubicin significantly reduced the expression of RCN1,p65 and TFAP4Doxorubicin can promote the expression of RCN1 in both sensitive and drug-resistant cell groups,while SAHA and its combination can down regulate the expression of RCN1,and also reduce the expression of transcription factors p65 and TFAP4,indicating that inhibition of RCN1 with SAHA is involved in the process of reversing drug resistance.Conclusion1.RCN1 is highly expressed in tumor tissue,while RCN1 is related to cell survival,and patient survival.2.The expression of RCN1 in drug-resistant tumor cells increased significantly,which was involved in the drug resistance of tumor cells.Down regulation of RCN1 can increase the sensitivity of cells to drugs.3.HD AC inhibitors can inhibit the expression of RCN1 by inhibiting the expression of p65 and TFAP4.4.The combination of HD AC inhibitor SAHA and doxorubicin can reverse drug resistance to a certain extent,and the down-regulation effect of SAHA on RCN1 may be one of the mechanisms of reversing drug resistance.Innovation and deficiency1.Innovation:(1)The high expression of RCN1 is involved in the drug resistance of tumor,while the down regulation of RCN1 increase the sensitivity of cells to drugs.RCN1 may be the target of drug resistance.(2)HDAC inhibitors down regulate the expression of RCN1 and increase the sensitivity of drug-resistant cells to doxorubicin.The combination of the two drugs can significantly inhibit the proliferation of drug-resistant cells and the growth of tumor tissue.It provides a new method to overcome multidrug resistance.2.Deficiencies(1)The mechanism of multidrug resistance mediated by RCN1 has not been explored.Preliminary data suggest that RCN1 may relate to the promotion of DNA damage repair,which needs further study.(2)The mechanism of inhibiting RCN1 of SAHA is relatively weak,which needs to be further explored.(3)The protein degradation pathway of RCN1 has not been elucidated and needs to be analyzed carefully.