Study On The Role Of GCS In Regulating Verapamil In Reversing Multi-drug Resistance To Chemotherapy In Gastric Cancer

Background:Gastric carcinoma is one of the most common digestive malignant tumors in the world,originating from gastric mucosal epithelium.According to the statistics of 2021,there are nearly 1.08 million new gastric cancer cases in the world,accounting for 5.6%of all new gastric cancer cases.There are about 760,000 cases of gastric cancer death every year,making it the fourth leading cause of cancer death worldwide.According to the statistics in 2020,the new incidence rate and mortality rate of gastric cancer in China are the third highest.Early gastric cancer patients often have no obvious clinical symptoms or atypical symptoms,with concealment,no specificity,can be hidden,can also exist for a long time.This is also closely related to the imbalance in the distribution of medical and health resources in China and the low cancer screening rate.At this time,patients with gastric cancer regrettably missed the best opportunity for radical surgery.At this time,they lost the best opportunity for surgical treatment,most advanced gastric cancer patients are treated by surgery combined with perioperative chemotherapy,radiotherapy and biological targeted therapy.At the same time,because of the insensitivity of gastric cancer to radiation and the side effects of radiotherapy,the use of radiotherapy is limited.Therefore,medical chemotherapy has become one of the main treatment for advanced gastric cancer,in order to prolong the survival period and improve patient quality of life.For patients with early gastric cancer,even if they have the opportunity to undergo radical surgery,at least 80%of patients will have local recurrence or distant metastasis within 2 to 3 years after surgery,and the 5-year survival rate is less than 30%.The preferred treatment for advanced gastric cancer is internal medicine chemotherapy.Studies showed that multiple drug resistance(MDR)is the main cause of chemotherapy failure.Tumor multi drug resistance refers to the phenomenon that after a drug acts on tumor cells and produces drug resistance,tumor cells also produce cross resistance to other chemotherapy drugs with different molecular structures and mechanisms.The multi drug resistance of tumor cells can be divided into natural resistance and acquired resistance.The drug resistance of tumor involves many mechanisms,such as the decrease of intracellular drug concentration,the change of drug target molecules,the metabolic detoxification,the imbalance of DNA damage repair function and so on.The resistance of gastric cancer cells to chemotherapeutic drugs caused by various reasons is an extremely difficult problem in multidisciplinary treatment of gastric cancer,which seriously restricts the survival of cancer patients.Therefore,overcoming multi drug resistance of tumor cells and improving the efficacy of chemotherapeutic drugs have become an urgent problem for cancer treatment.Further in-depth study of the molecular mechanism of drug resistance of gastric cancer cells and search for drugs and methods that can be used in clinical practice are important points for the study of gastric cancer treatment.With the continuous invention and development of a variety of methods for clinical treatment methods,especially for the treatment of cancer is also increasingly abundant.In addition to simple surgery,chemotherapy,radiotherapy,new interventional therapy makes tumor patients venefit in the entire treatment.Target artery infusion chemotherapy is the application of puncture,intubation technology,under the guidance of X-ray fluoroscopy angiography,the catheter will be accurately and selectively inserted into the related blood supply target artery of the tumor focus,through DSA to analyze and diagnose the location,quantity and shape of the focus,and then the high concentration of chemotherapy drugs will be accurately and directly infused into the focus through the catheter,which has strong lethality and high curative effect The distribution of drugs in unrelated tissues and organs is less,and the systemic side effects are very low.Since the 1880s,foreign doctors have tried to use target artery infusion drugs to treat esophageal cancer,and found that they can significantly improve the lethal effect of chemotherapy drugs on tumor tissues,and the toxic and side effects are significantly less than that of systemic meridional chemotherapy.Verapamil(VER)is a widely used clinical calcium channel blocker in heart disease,which has the effect of reversing MDR in tumor cells.It has been thought that anti-tumor drug resistance is mainly achieved through suppression of P-glycoprotein(P-gp)pathway.However,recently,a growing number of researchers have found that it increases the cells' susceptibility to drugs through nonclassical pathways.Experimental studies show that VER can significantly improve the sensitivity of various tumor cells to chemical drugs.In-vitro study reported that 6.0-10.0 ?mol/L VER concentration,higher than the safe vein concentration(1.0-2.0?moL/L),can effectively reverse tumor drug resistance.Exceeding the safe vein concentration of VER causes sinus bradycardia,atrioventricular block,and other serious toxic and side effects,thus restricting its wide application in clinics as a reversal agent of tumor multi drug resistance.Previous animal experiments have shown that the concentration of verapamil in tissue is 50-100 times higher than that in peripheral blood,which can successfully reverse multi-drug resistance of tumors without causing adverse cardiovascular reactions.In our study,the target artery perfusion with verapamil combined with chemotherapy can significantly improved the efficacy of advanced gastric cancer,with an overall effective rate of up to 75%,but there were still some poor efficacy.Therefore,it is urgent to find the key genes of VER to reverse the drug resistance of tumor cells and enhance its efficacy.Most cytotoxic agents kill tumor cells by inducing apoptosis.The tolerance of tumor cells to apoptosis is one of the important mechanisms of MDR.Apoptosis related factors become the targets of drug resistance and can mediate MDR together with other pathways.Autophagy has dual effects on the drug resistance of tumor cells.On the one hand,apoptosis tolerance is an important mechanism of tumor drug resistance.Autophagy can promote tumor drug resistance by antagonizing apoptosis induced by chemotherapy drugs;on the other hand,autophagy may be a way of death of apoptosis tolerant tumor cells.Studies have shown that autophagy and apoptosis are closely related.Firstly,there is a cross between the two signaling pathways.For example,the interaction between Bcl-2 family members and autophagy protein Beclinl through BH3 domain prevents Beclinl from participating in the assembly of pre autophagosome and the binding of Bcl-2 and apoptosis protein,thus promoting apoptosis and inhibiting autophagy.In addition,ATG5 translocated from cytoplasm to mitochondria after calpain cleavage,and combined with Bcl-x to promote the release of cytochromec and promote apoptosis.Glucosylcemmide Synthase(GCS)is the key enzyme in the glycosylation of ceramide,and the increase of its activity can lead to the generation of tumor acquired multi-drug resistance.GCS can catalytic uridine diphosphate glucose sugar base combined with ceramide,make the ceramide into no cytotoxicity of glucose amide(GlcCer),GlcCer is decorated a series of glycosyl transferase,synthetic higher forms of sheath glycolipids,the sheath of sugar fat cells not only maintain the normal structure and function,is also involved in cell proliferation,differentiation,and can be against the apoptosis induced by drugs.Previous studies of our group showed that verapamil reversed the multi-drug resistance of some tumor cells.Several studies have suggested that verapamil competitiveness combines with P-gp to achieve reverse effects by acting as a competitive substrate for the drug resistant cell efflux pump and by increasing intracellular chemotherapeutic drug accumulation.In this study,molecular markers that can effectively predict individual differences in VER's ability to reverse chemotherapy resistance are selected to provide a clinical efficacy prediction method and theoretical basis for the combined use of TACE+VER,which is of great significance for the improvement of verapamil individualized treatment.In our previous study,real-time PCR and Western blotting showed that the expression of Bcl-2 in verapamil combined with doxorubicin group was low.The specific mechanism of verapamil combined with chemotherapeutic drugs to enhance the sensitivity of gastric cancer cells to chemotherapeutic drugs by inhibiting Bcl-2 is not clear,so we continue to further study to find P-gp In addition to protein related genes,more genes related to the reversal of drug resistance of verapamil were identified,which provided a theoretical basis for the treatment of gastric cancer with verapamil combined with chemotherapy drugs via target artery infusion.Chapter 1.The correlation between the expression level of P-gp and the reversal of VER's resistance to chemotherapy in gastric carcinoma cellsObjective:To investigate the correlation between hMDR1/P-gp level and the difference of VER resistance in gastric cancer cell lines.Methods:1.The expression levels of hMDR1/P-gp in gastric carcinoma cell lines(SGC-7901,BGC-823 and AGS)were detected by real-time quantitative PCR and western blot;2.The drug sensitivity of gastric carcinoma cell lines to three chemotherapeutic drugs(5-FU,ADM and DDP)was detected by CCK-8;3.The drug sensitivity of gastric carcinoma cell lines to verapamil combined with three chemotherapeutic drugs(5-FU,ADM and DDP)was detected by CCK-8;4.Evaluation of the ability of verapamil to reverse chemotherapy drug resistance in gastric carcinoma cells.Result:1.The expression level of P-gp was significantly different in GC cell lines.Compared with SGC-7901,the expression level of AGS was the highest.2.The IC50 values of DDP in BGC-823 and AGS are significantly higher than those in SGC-7901.The IC50 value of ADM and 5-Fu in BGC-823 is lower than those in the rest of the two cell lines.3.After VER is added,the IC50 values of DDP,ADM,and 5-Fu in the three cell lines(SGC-7901,BGC-823 and AGS)declined to different extents,indicating that VER can increase the sensitivity of the three aforementioned chemotherapeutics to different extents.4.The resistance of SGC-7901 to VER reversal ADM,which has significant difference with BGC-823(Relative IC50=1.66),is the strongest(Relative IC50=6.77).Conclusion:No simple correlation between the expression level of hMDR1/P-gp and VER resistance is found.Chapter 2.Construct gastric cancer drug-resistant cell line and verify its drug-resistant efficiencyObjective:To construct a 5-Fu resistant gastric carcinoma cell line.Methods:Drug concentration gradient increasing method combined with intermittent induction was used to establish the 5-Fu resistant sub-line of gastric carcinoma cell line SGC-7901/5-Fu.Relevant tests were performed monthly,and drug sensitivity was determined by CCK-8 method.Result:After 6 months,SGC-7901/5-Fu cell line was established,and it had cross resistance to ADM,DDP and other anticancer drugs.Conclusion:The SGC-7901/5-Fu cell line has the phenotypic characteristics of drug resistance,and drug resistance can be stable.Chapter 3.Screening,confirmation and mechanism study of differential genes mediated by VER in the reversal of chemotherapy resistance of gastric carcinoma cellsObjective:To screen the differential gene and confirm the differential resistance of VER to the chemotherapeutic drug resistance of gastric carcinoma cells,and to explore the mechanism of its action.Methods:1.Screening of possible differential genes by bioinformatics analysis and literature;2.Real-time quantitative PCR test of expressions of candidate genes in gastric carcinoma cells;3.Western blot test of expression of candidate proteins in gastric carcinoma cells;4.Changes in VER reversal of chemotherapy resistance following GCS upregulation or downregulation.5.Annexin V-FITC/PI double-staining test of gastric carcinoma cell apoptosis.Result:1.We found a total of 8 genes,namely,hMDR1,LRP,GST-?,GCS,TOPO ?,PrPc,MGrl-Ag and CIAPIN1.2.The qRT-PCR showed that the expression of LRP,GCS and TOPO II was different after VER treatment.The GCS gene was the most significant difference(p<0.01).So this research focuses on the possible mechanism of VER resistance reversal from GCS.3.Western blot verified the differences expression of GCS protein after treatment with VER.Combined with qRT-PCR results,we focus on the possible mechanism of VER resistance reversal from GCS.4.In the SGC-7901 and SGC-7901/5-Fu cells,overexpression of the GCS gene(pEGFP-GCS),VER reversal of ADM chemotherapy resistance was significantly enhanced.While downregulation of GCS gene(siR-GCS),VER reversal of ADM chemotherapy resistance significantly reduced.5.VER promotes the apoptosis of gastric carcinoma cells obviously in the presence of ADM which overexpression of GCS genes.Silencing of GCS gene(siR-GCS)expression,the cell apoptosis rates were reduced by VER.6.After overexpression of GCS gene(pEGFP-GCS),the expression levels of Bcl-2 and ERK were significantly reduced by VER.Conclusion:GCS mediated the difference in VER's ability to reverse ADM chemotherapy resistance in gastric cancer cells,and VER may significantly alter its ability to reverse ADM chemotherapy resistance and promote cell apoptosis by changing the expression level of GCS gene.Chapter 4.Clinical study on the expression of P-gp and GCS in tissue samples of gastric cancer casesObjective:The expression of P-gp and GCS genes in the tissue samples of extreme cases of gastric carcinoma(treatment group/disease progression group).Methods:1.A total of 11 cases of gastric carcinoma in middle and terminal stages were selected in our hospitals.Lesion samples were extracted using endoscopy,and all cases were diagnosed as gastric carcinoma.Each patient received one intervention every month or a total of two to four interventions.The therapeutic effects of these interventions to the patients were evaluated into two groups,namely,7 cases of VER effective group(high cure rate,CR)and 5 cases of VER ineffective group(progression of disease,PD).2.P-gp,GCS protein expression test in cancer tissue samples of GC patents by immunohistochemical method.Result:1.The P-gp protein was mainly expressed in the cytoplasm of cancer cells.In the cancer tissue and adjacent tissues,the average P-gp protein density of the effective group and the ineffective group(p>0.05).2.The GCS protein is mainly expressed in the nucleus and cytoplasm of cancer cells.In cancer tissues,the IOD/area of P15 of the VER effective group is significantly higher than that of the VER ineffective group(p<0.01)Conclusion:1.There was no significant difference in the P-gp protein expression between the effective group and the ineffective group in verapamil resistance treatment.2.The expression level of GCS protein in verapamil resistance treatment effective group was significantly lower than that in verapamil resistance treatment ineffective group.