Enhance The Sensitivity Of Sorafenib In Renal Carcinoma By Regulating Autophagy And Akt Pathway

Part 1 Therapeutic effect and adverse reaction of sorafenib in the treatment of advanced renal cancerBackgroundRenal cancer is one of the most common clinical urologicaltumors,accounting for 4%of adult malignancies and 80-90%of adult renal malignant diseases.In China,renal cancer affects453,000 people and causes 396,000 deaths every year.Incidence of renal cancer is higher in developed countries than indeveloping countries,and is higher in males than in females.Main factors leading to renal cancer include tobacco consumption,alcohol abuse and obesity.Renal cancer at early stageshas no obvious symptoms,and patients showing hematuria,Pain,and mass are usually in advanced stages with distanttumor metastasis.At present,surgical treatment is still the main treatment for renal cancer.However,recurrence andmetastasis still occurs in 32%patients with renal cancer after operation.Survival period of patients in advanced stages is6-12 months,and the 5-year survival rate is<10%.Traditional treatment methods such as radiotherapy,chemotherapy,and interferon are effective for only 10-30%patients.In recent years,a large number of clinically open phase Ⅲ trials have demonstrated that targeted drugs are superior to radiochemotherapy and interferon in the treatment of advanced renal cancer,and have good tolerance and minimal adverse reactions.Cancer treatment has entered the age of targeted therapy.As a kinase inhibitor,sorafenib inhibits many kinds of extracellular and intracellular kinases and has dual antitumor and anti-angiogenic effects.It can inhibit MRK and RAF signaling pathways as well as VECFR,PDGFR and tumor neovascularization,and has become the first-line targeted drug for the treatment of advanced renal cancer.In this study,clinical data of patients with advanced renal cancer treated with sorafenib in Shandong Provincial Hospital Affiliated to Shandong University(Jinan,China)were analyzed,and treatment effcacy,adverse events and prognosis were analyzed as well.Our study provided references for the treatment of advanced renal cancer.Objective:Effcacy and safety of sorafenib in patients with advanced renal cancer were evaluated.Materials and methods:Seventy-four patients with advanced renal cancer treated with sorafenib+interferon from January 2010 to August 2013 were included as the observation group.Another 53 renal cancer patients treated with interferon alone were included in the control group.Clinical data of those patients were retrospectively analyzed.Treatment plan:initial dose of solafenib was 400 mg,twice a day.All patients were treated with another 300 MU every other day.Effcacy was evaluated according to RECIST criteria,and progression-free survival(PFS),overall survival(OS),and incidence of adverse reactions were recorded.Results:In the observation group,a median OS was 15.3 months(range,9-60 months),and a median PFS was 8.2 months(range,2-36 months).There were 4 cases of complete remission(CR)(5.41%),16 cases of partial remission(PR)(21.62%),42 cases of stable disease(SD)(56.76%),12 cases of disease progression(16.22%),and disease control rate(DCR)was 83.78%(62 cases).In the control group,median OS time was 12.5 months(range,8-60 months),and the median PFS time was 9.3 months(range,2-40 months).There were 2 cases of CR(3.77%),11 cases of PR(20.75%),20 cases of SD(37.74%),20 cases of disease progression(37.74%),and DCR was 62.26%(33 cases).Disease control rate in the observation group was signifcantly higher than that in the control group(P<0.05).Main adverse events in the groups were skin reaction,fever,diarrhea,fatigue,rash,loss of appetite,hypertension,hair loss and liver function abnormality.Conclusion:Sorafenib-based targeted therapy for the treatment of advanced renal cancer has a higher rate of disease control.and the adverse reactions are controllable and tolerable.Part 2 Ubenimex improve the sorafenib treatment sensitivity of RCC by increasing expression via regulating autophagyBackground:In this paper,we mainly studied the effect of autophagy on the sensitivity of sorafenib in renal cell carcinoma,and explored the effect of umbrel on renal cancer cells by experimental demonstration.The drug can enhance the sensitivity of sorafenib.It is generally believed that autophagy plays a double-edged sword role in the occurrence and development of tumors.When the tumor is under stress from chemotherapy or targeted therapy.Autophagy can often achieve the goal of maintaining cell homeostasis by mobilizing the implementation of intracellular protein degradation and recycling of cytoplasmic components,and enable cells to pass the energy crisis so as to avoid treatment.And excessive autophagy will start cell apoptosis,this kind of programmed death is different from the necrotic death and apoptosis,its typical characteristics of performance for autophagosome formation and autophagy the expression of related genes,the apoptosis 1s called type II programmed death,that 1s to say,excessive autophagy,can lead to cell death.Ubumex is a commonly used anti-tumor drug.It is an APN inhibitor.It is widely used in clinical practice and has played an important role.It has also received widespread attention from scholars.Its pharmacological mechanism is currently under investigation.Some scholars have studied this drug and found that it can exert an effective anti-tumor effect in renal cell carcinoma,which is closely related to its blocking of the 5-LO-LTA4 hydrolase pathway.This study consisted of a study on the mechanism of action of umbryi,which was used in renal cancer cells.It was found that the drug can induce autophagic death in the latter,the proliferation of cancer cells is greatly reduced,and the invasive ability is obviously insufficient.Both are associated with the Akt pathway,a key signaling pathway for drug inhibition of autophagy.It can be seen that the drug can regulate autophagy of tumor cells and has regulatory potential for radiotherapy and targeted therapy.Recent studies abroad have pointed out that autophagy can not only prevent the accumulation of damaged or unnecessary components,but also recycle degraded components to maintain metabolic homomorphism.Beclin-1 plays an important role in the formation of the beclin-1-vps34-vps15 core complex,which is necessary for autophagy induction.MIR regulates the autophagy pathway.For example,mir-30a and mir-376b have been shown to inhibit beclin-1 activity,thereby preventing autophagy vesicle nucleation and autophagy initiation.In addition,mir-101 is an effective autophagy inhibitor,although it inhibits rab5a,a small gtpase that regulates early autophagosome formation.In the present study,we found that in renal cell carcinoma tissues and cell lines of some people,autophagy related mir-30a was down-regulated,which may lead to beclin-1 up-regulation in renal cell carcinoma.High levels of autophagy activation were found in cancer cells stimulated by chemotherapeutic drugs or not,and were involved in chemotherapeutic resistance.Therefore,blocking autophagy is becoming a new strategy to improve the sensitivity of chemotherapy to various cancers.For example,Yu et al.showed that mir-30a-mediated inhibition of autophagy significantly increased the activity of human chronic myeloid leukemia cells induced by imatinib.Previously,we demonstrated that the treatment of RCC cells by sorafenib induces autophagy activation,which is also involved in chemotherapy resistance.Pharmacological or genetic approaches to block autophagy can significantly enhance the cytotoxicity of sorafenib on RCC cells.Chloroquine(CQ)is a lysosomal drug that increases the ph of lysosomes in vivo to destroy autophagy protein degradation.3-methyl adenine(3-ma)is known to block the transformation from lc3b-i to lc3b-ii and inhibit the formation of antophagosomes.B-afiatoxin A1(baf-al)prevents autophagic vesicle maturation by inhibiting fusion between autophagosomes and lysosomes.These different inhibitors targeted at different stages of autophagy all enhanced the activity of sorafenib on RCC cells,suggesting that the autophagy activation of sorafenib is an important chemotherapy resistance factor promoting survival.This conclusion is further supported by the fact that RNAi silencing of sensitized sorafenib by beclin-1 or atg-5 induces cytotoxicity in RCC cells.In summary,these findings suggest that mir-30a dysregulation in RCC may interfere with the efficacy of sorafenib mediated apoptosis through autophagy dependent pathways,thus representing a new potential therapeutic target in RCC.Combined with our preliminary test results and previous studies by researchers,it has been proved that both chemotherapy and radiotherapy,as well as targeted therapy,may lead to the up-regulation of protective autophagy.More studies have proved that protective autophagy plays an important role in the development of drug resistance in tumors.This fully demonstrates the high complexity and background dependence of autophagy.Therefore,it is of great significance to fully elucidate the role of autophagy in the occurrence and development of renal cancer,as well as the role of drug resistance.In addition,we need to further confirm whether this effect of ubenemide is related to the regulation of autophagy level.Only by fully understanding the role of autophagy in tumor resistance can we make better use of this drug to assist the targeted treatment of renal cancer.Objective:To investigate the role of autophagy in enhancing sorafenib sensitivity in the treatment of renal cell carcinoma.Materials and methods:(1)We try to construct a sorafenib-resistant cell line under gradient pressure.IC50 demonstrates drug resistance of drug-resistant cell lines.To observe the changes of drug-resistant and Non-drug-resistant cell lines under different concentrations of sorafenib.(2)Western blot was introduced to detect the autophagy-related markers P62 and LC3B of cell lines under different concentrations of sorafenib,and to clarify their expression level.(3)To observe the changes of susceptibility to sorafenib of drug-resistant cell lines before and after treatment with ubenimex.(4)To detect the apoptotic changes of drug-resistant cell lines before and after treatment with ubenimex.(5)The number of autophagosomes was different before and after treatment with ubenimex.The changes could be detected by electron microscopy,and autophagy could be verified by the above methods.(6)The cancer cell lines needed were identified by the above methods,and the resistant plants to sorafenib were selected to construct the tumor-bearing mice model.The mice were given intragastric administration and divided into three groups:sorafenib combined with ubenimex group,sorafenib group and control group(DMSO).Subsequently,the tumor volume was measured,which confirmed that the combination of ubenimex and sorafenib could enhance the tumor inhibition effect.Results:(1)It is successful to construct sorafenib-resistant cell lines under gradient pressure.The resistance of drug-resistant cell lines was verified by IC50.Under the same concentration of sorafenib,drug-resistant cell lines showed higher activity than Non-drug-resistant cell lines.(2)Under different concentrations of sorafenib,the autophagy-related indexes P62 and LC3B of cell lines changed in a dose-dependent manner,and the higher the concentration,the higher the autophagy level,which indicated that sorafenib could cause the increase of autophagy level of tumor cells.(3)The susceptibility of drug-resistant cell lines to sorafenib decreased,but increased with the addition of ubenimex.(4)The apoptotic degree of drug-resistant cell lines increased significantly after treatment with ubenimex,suggesting that ubenimex promotes apoptosis of drug-resistant cell lines,thereby reversing the resistance to sorafenib.(5)The number of autophagosomes decreased significantly after treatment with ubenimex,suggesting that ubenimex can reduce the autophagy of tumor cells.(6)After intragastric administration,the tumor volume of the sorafenib group was smaller than that of the control group,while that of the sorafenib combined with ubenimex group was more obvious,which confirmed that the combination of ubenimex and sorafenib had better antitumor effect than sorafenib alone.Conclusions:(1)The formation of acquired drug resistance of sorafenib in renal cancer cells may be associated with up-regulation of autophagy.(2)Up-regulated autophagy of renal cancer cells can lead to down-regulated sensitivity of renal cancer cell lines to sorafenib.(3)The treatment of renal cancer cells with ubenemex can reduce the level of autophagy in renal cancer cells and restore the sensitivity of drug-resistant renal cancer cell line sorafenib.(4)In vivo experiments further confirmed that the combination of ubenimex and sorafenib can enhance the effect of tumor inhibition and control tumor growth(delaying tumor growth and inhibiting tumor metastasis).Part 3 Role of Akt pathway in Ubenimex enhanced sorafenib sensitivity and inhibiting invasion of RCC cellsBack ground:Autophagy is closely related to the occurrence and development of tumors,and is regulated by many signaling pathways,including Akt pathway,which is closely related to it,but also affects the susceptibility of sorafenib.Although some studies have investigated the molecular mechanism of acquired cell resistance to molecular targeted drugs(including sunitinib),it is still unclear for renal cell carcinoma(RCC).It has been pointed out that the maintenance of protein kinase activation during sunitinib therapy may involve the acquisition of a phenotype resistant to sunitinib in RCC.Targeted activated protein kinase therapy may be a promising way to overcome the resistance of RCC to sunitinib.Previous studies have shown that signal transduction pathways are involved in the generation of drug resistance to a variety of molecular targeted drugs.By studying the status of major signal transduction pathways in ACHN sublines before and after targeted therapy,it was confirmed that although the expression levels of phosphorylated Akt and MAPK in ACHN sublines before and after treatment were not significantly different,the phosphorylation of ACHN/R Akt and MAPK was significantly inhibited;however,the activation of Akt and MAPK pathways could be detected in ACHN/R after treatment.In addition,the acquired drug resistance of ACHN/R cells to targeted therapy can be overcome by adjuvant therapy with PI3K specific inhibitor LY294002.Similarly,previous studies have demonstrated the potential role of inhibiting Akt pathway in overcoming acquired resistance to various cytotoxic drugs;for example,Peng et al.suggested that cisplatin-resistant cells express higher levels of activated Akt than cisplatin-sensitive controls,and the inhibition of Akt or mTOR pathway makes these resistant cells sensitive to cisplatin-induced apoptosis.Overall.these results suggest that the activation of Akt signaling pathway during treatment,especially PI3K/Akt pathway,may be related to the drug resistance of RCC cells to this reagent and may be overcome by specific inhibitors of these pathways.Objective:To investigate the role of Akt pathway in ubenimex enhanced sorafenib sensitivity and inhibiting invasion of RCC.Materials and methods:(1)The Akt signaling pathway of renal cancer cells is affected by ubenimex.After treatment with ubenimex,the phosphorylation level of Akt cells changes.Weston Blot method can be used to detect the extent of the effect.(2)Ubenmex can help to increase the susceptibility of renal cell carcinoma to sorafenib,and Akt signaling pathway plays a role in this process,which can be demonstrated by experiments.Akt agonists can act on renal cell carcinoma cells and change Akt signal,thus playing a regulatory role.After the combination of ubenimex and sorafenib,Akt agonists were added to observe the changes of sensitization of the former to sorafenib,in order to confirm that inhibition of Akt signal can enhance the positive effect of sorafenib.(3)Ubenmex can affect the invasion of renal cell carcinoma,which can be verified by Transwell experiment.Ubenmex can act on renal cell carcinoma cells to change the invasive ability of cancer cells,and add Akt agonist to observe the changes.Results:(1)The changes of Akt signaling pathway were observed when Ubenmex was used to treat renal cancer cells.The results showed that the phosphorylation level of Akt decreased significantly.(2)After using Akt agonist IGF-1(10nM),the drug resistance to sorafenib of tumor cells was restored.Therefore,it is suggested that ubenimex can increase the sensitivity of sorafenib,and the inhibition of Akt signaling pathway by ubenimex is an important mechanism to exert the above effects.(3)For the invasive ability of renal cell carcinoma,the Transwell experiment did not find that sorafenib had obvious promoting effect,but when combined with ubenimex,the invasive ability of renal cell carcinoma decreased and was inhibited to a certain extent.(4)Upregulation of Akt signal in renal cell carcinoma cells will enhance the invasiveness of tumor cells,which requires the use of Akt agonists.Therefore,ubenimex has a definite inhibitory effect on the invasion of tumor cells,thus inhibiting its metastasis,and inhibiting Akt signal plays an important role in this process.Conclusions:(1)Ubenmex can enhance the susceptibility of renal cell carcinoma cells to sorafenib,and inhibiting Akt signaling pathway is an important mechanism to achieve the above purpose.(2)Tumor cells are invasive.Combination of ubenimex and sorafenib can inhibit the invasion of other tissues,which is closely related to the inhibition of Akt signaling pathway.