| Objective: With the increasing of the incidence of cancer,human health is seriously threatened.The incidence rate of colorectal cancer is the third in the world.The clinical data shows that the five-year survival rate of patients with colorectal cancer is less than 10%.Therefore,prevention and treatment of colorectal cancer gradually attract people’s attention.Patients with early stage of colorectal cancer can be cured by surgical treatment.However,due to the early stage of colorectal cancer is not obviously to be found,and the lack of attention to early screening,most patients are now found to be late.The late stage of metastatic colorectal cancer is more malignant and progresses rapidly,so the primary goal of treatment in advanced patients is to improve quality of life and prolong survival.With the development of society and science,we have entered an era of targeted precision therapy,and the emergence of targeted drugs has played an important role in patients with advanced colorectal cancer.BRAF is the core molecule downstream of the RAS in the MAPK pathway,which plays an important role in the regulation of intracellular signal transduction and in tumor growth,development and prognosis.BRAF mutation rates vary with tumor types.BRAF mutations occur in about 8% of malignant tumors,with a mutation rate of approximately 50% for BRAF in malignant melanoma,30%-70% in thyroid carcinomas,8%-10% in colorectal cancers and NSCLC is 0.5%-4.9%.However,the majority of BRAF mutations are BRAFV600 E mutations.Vemurafenib(RG7204,PLX4032)is a small molecule inhibitor targeting BRAFV600 E that inhibits the proliferation of tumor cells by inhibiting BRAFV600 E and effectively blocking the MAPK pathway.The response rate was 50% in the BRAF600 E mutant malignant melanoma and only 5% in the colorectal cancer.The malignant melanoma patients with Vemurafenib treatment have significantly improved in the efficiency and overall survival rates.In addition,the clinical treatment has surpassed the effect of chemotherapy.Because the complex and diverse genotypes of colorectal cancer,the poorer response to Vemurafenib.Thus,the treatment of colorectal cancer will be combined with other targeted drugs or chemotherapeutic that agent to sensitize Vemurafenib,which achieving a synergistic therapeutic effect.However,as the treatment progressed,it will produce primary or acquired resistance to Vemurafenib whether is malignant melanoma or colorectal cancer,which greatly limit the clinical application of Vemurafenib.Even though the same tumor tissue may have different gene expression levels,it is difficult to detect the entire tumor tissue in the clinic.Therefore,there are primary drug-resistant cells in the tumor tissue.At the same time,the sensitive tumor cells are gradually eliminated during the single-agent or combination treatment with Vemurafenib,the primary drug-resistant cells will be retained selectively.On the other hand,under the continuous stimulation of drugs,the sensitive cells can also be mutated,which resulting in drug resistance consequently.It has been reported that the over expression of cyclin D1 in tumor cells is one of the mechanisms of Vemurafenib resistance.In the study of acquired drug resistance mechanism of colorectal cancer,the current reactivity of EGFR is mainly reason that lead to the continuous activation of MAPK pathway,and even activation of PI3K-AKT-m TOR signaling pathway,thereby promoting the growth and proliferation of tumor cells.In line with the EGFR pathway,other tyrosine kinases of receptor(RTKs),such as the hepatocyte growth factor receptor MET,which are involved in drug resistance to BRAF inhibitors.For a variety of drug resistance mechanisms,the current clinical trials have begun to increase the effective of Vemurafenib,such as BRAF inhibitor which combined with MEK inhibitor that double-targeted inhibition of MAPK pathway activation,which can inhibit tumor growth effectively.The BRAF inhibitor combined with anti-EGFR monoclonal antibody—cetuximab,which can prevent the feedback activation of EGFR effectively,which achieving the purpose of synergistic treatment.The latest study found that it is different for BRAF inhibitor single drug resistance mechanism and combination drug resistance mechanism.No matter what kind of treatment,it is difficult to achieve a complete response rate.Thus,we can protect that there are intricate network of tumor cell resistance mechanisms,which not only alone play their respective roles,but also may exist in various channels crosstalk,There is not just a single mechanism.Therefore,as much as possible to grasp the mechanism of Vemurafenib resistance in colorectal cancer cells,which plays an important role in further increasing the response rate of Vemurafenib and improving the curative effect.STAT3 is a member of the Signal transducers and activators of transcription.STAT3 plays an important role in tumorigenesis and development.Some studies have shown that STAT3 activation up-regulates the expression of anti-apoptotic proteins(BCL-Xl,BCL-2,MCL-1,Survivin),while the knockdown of STAT3,which reduces the expression of anti-apoptotic proteins and can activate the apoptotic pathways that consist with apoptotic molecule Bax,resulting in inducing apoptosis.In addition,STAT3 can also regulate the cell cycle molecules(Cyclin D1,c-myc)and promote its transcription as a transcription factor,therefore affecting the cell cycle progression.Some Studies have shown that STAT3 regulate the expression of PD-L1,which help to suppress immunity and promote tumorigenesis,invasion and metastasis.Some studies have found that BRAF inhibitors can lead to the activation of STAT3 in some malignant melanoma.Therefore,the generation of cell resistance may be related to the activation of STAT3.Searching for the target of secondary drug-resistant cells is an important means to solve the resistance of Vemurafenib,and it is also critical to screen the superiority of vemurafenib in the treatment of people and to improve the efficacy of the drug.Studies have confirmed that different colorectal cancer cells have different drug sensitivity to Vemurafenib,which may be related to tumor cell heterogeneity,gene expression,and even about half of the sources.Therefore,it is important for the treatment of Vemurafenib in the screening of sensitive cells,and the dominant population.Therefore,to further explore the mechanism of resistance to Vemurafenib can not only provide effective help for targeted combination therapy,but also can help clinical screening of the dominant patients.This study confirmed by cell experiments BRAFV600 E mutant colorectal cancer cells on the sensitivity of Vemurafenib is different.We have in-depth exploration in Vemurafenib activates signaling molecules leading to drug resistance.Clarifying the cause of activation,as well as downstream regulatory target molecules.In addition,confirmed by adding inhibitors can significantly enhance the inhibitory effect of Vemurafenib on colorectal cancer cells.The results provide a theoretical basis for the determination of the resistance of Vemurafenib,and help us to screen the best patients in subsequent clinical treatment.Methods: The cell proliferation activity was detected by MTS assay.The content of IL-6 in cell supernatant was measured by ELISA.The use of colony formation assay to determine cell proliferation.Flow cytometry(Annexin V/PI double staining)to detect apoptosis.Liposome-mediated si RNA transfection.The protein expressions of STAT3,p-STAT3,ERK,p-ERK,BCL-2,Actin,Ca-3,c-Ca3,PUMA,BIM and PARP were detected by Western blotting.Using TRANSWELL method to detect cell migration ability.Statistical Processing: Each experiment was repeated 3 times and data analyzed using SPSS version 21.0(IBM,Armonk,NY,USA).All values are expressed as mean ± standard deviation(x± s).The difference between the two groups was confirmed by t-test,P <0.05 was statistically significant.Results: 1.The colorectal cancer of BRAFV600 E mutant cells have different sensitivity to Vemurafenib.HT-29 and RKO cells were treated with different concentrations of Vemurafenib(0,0.2,1,5 μM)for 24,48 and 72 hours.The viability of HT-29 cells was significantly higher than that of RKO cells by MTS assay,And the inhibition rate is in a time-dependent and concentration-dependent manner.The apoptosis rate of Vemurafenib-treated cells was detected by flow cytometry(Annexin V / PI double staining).The results showed that apoptosis of HT-29 cells was higher than that of RKO cells after treatment with Vemurafenib(5μM).The apoptotic proteins(caspase3,cleaved-Caspase3,BIM,PUMA,PARP,cleaved-PARP)after treatment with Vemurafenib(5μM)for 12 h were detected by Western blotting,RKO apoptosis protein upregulation is relatively small.The above results confirmed that HT-29 cells are Vemurafenib relatively sensitive cell lines,RKO cells are Vemurafenib relative drug-resistant cell lines.2.Vemurafenib causes RKO cells to secrete IL-6,which up-regulate BCL-2 to activate STAT-3 cells to produce drug resistance.The expression of p-ERK,p-STAT3,STAT3,p-Akt,Akt,p-ERK,BCL-2,and Actin protein expression were detected by immunoblotting after fixing the concentration of Vemurafenib(5μM),RKO cells in the first 6 hours after treatment began to activate STAT3,and anti-apoptotic protein BCL-2 up regulate at the same time.HT-29 cells did not activate STAT3 signal,while BCL-2 Not expressed.At the same time,we found that Vemurafenib inhibits the activation of ERK in HT-29 cells relatively high extent and inhibits RKO relatively weakly.The secretion of IL-6 in the supernatant of the cells treated with vemurafenib was detected by ELISA to determine whether the up-regulation of STAT3 was related to the activation of the classical IL-6 / STAT3 pathway.The cells were seeded in six-well plates,plus 10% fetal bovine serum culture to promote cell growth and adherent,the serum to antibiotics to wash cells 3 times after 24 hours,the culture system replaced by double no culture the liquid was added to Vemurafenib(5μM)for 12 hours,the supernatant of the cells was subjected to ELISA to detect the secretion of IL-6.As a result,it was confirmed that RKO cells secrete increased IL-6,while HT-29 cells do not have IL-6 secretion.The above results confirm that up-regulation of BCL-2 expression by secreted IL-6-activated STAT3 signal leads to drug resistance in RKO cells.3.Neutralization of RKO secretion of IL-6,STAT3 activation was inhibited while not BCL-2 upregulation,which cell viability decreased.The expression of p-STAT3,STAT3 and BCL-2 in RKO cells after IL-6 neutralizing antibody(50 ng / ml)and Vemurafenib(5 μM)for 12 hours were detected by Western blotting.The results showed that in serum-free medium,RKO cells treated with Vemurafenib(5μM)for 12 hours could activate STAT3 and up-regulate the expression of BCL-2 simultaneously.When combined with IL-6 neutralizing antibody,STAT3 was inactive and BCL-2 not up.At the same time,it was detected that the secretion of IL-6 by Vemurafenib was not detected by ELISA,and there was no increase of IL-6 in the cell supernatant after IL-6 neutralizing antibody was combined.MTS assay showed that combination of Vemurafenib and IL-6 neutralizing antibody could further decrease cell viability and sensitize RKO cells to Vemurafenib to a certain extent.The above results further suggest that in RKO cells,activation of STAT3 is due to the secretion of IL-6,resulting in the relative resistance of cells.4.STAT3 inhibitor effectively sensitized the drug-resistant cell RKO to Vemurafenib.MTS assay STAT3 inhibitor stattic combined with Vemurafenib and si RNA transiently transfected knockout STAT3 combined with Vemurafenib on cell viability and found that after 72 hours combined with cell viability was significantly inhibited.Colony formation assay showed that the proliferation of RKO cells was more obvious than that of monotherapy in the RKO cells treated with Vemurafenib(1μM)and stattic(1μM).Western blotting demonstrated that the signals of apoptosis proteins(Caspase3,Cleaved-Caspase3,BIM,PUMA,PARP and cleaved-PARP)were significantly upregulated after treatment with Vemurafenib(5μM)and stattic(2μM)for 12 h.Flow cytometry(Annexin V / PI double staining)test also confirmed a significant increase in apoptosis rate after 12 hours of combined treatment.In order to further explore the signal changes after combined treatment,we detected the expression of p-ERK,ERK,p-STAT3,STAT3,BCL-2 and Actin after treatment with Vemurafenib(5μM)(2μM)pretreatment for 2 hours,Vemurafenib failed to activate STAT3 effectively after 12 hours and no significant up-regulation of BCL-2.The same trend was observed with combination of Vemurafenib after si RNA knockdown of STAT3 signal.The above results confirm that Vemurafenib can affect the resistance of RKO cells which by activating STAT3 and affecting the expression of BCL-2.5.Activation of STAT3 signal in HT-29 cells can up-regulate the expression of BCL-2 and promote the resistance of sensitive cells.Immunoblotting confirmed that STAT3 was continuously activated in HT-29 cells treated with IL-6(1 ng / ml)for 1 hour to 24 hours.BCL-2 was not up-regulated at the beginning of STAT3 activation,whereas BCL-2 expression Increase after 12 hours.IL-6 increased HT-29 cell viability.MTS confirmed that IL-6 decreased the inhibitory effect of Vemurafenib on HT-29 cells.However,no colony formation assay was found after 7 days of IL-6 treatment Significantly promote cell proliferation.However,in the subsequent cell migration assay,it was confirmed by the TRANSWELL assay that migration of HT-29 and RKO cells was promoted after IL-6(50 ng / ml,100 ng / ml)treatment for 72 hours.To further verify the changes of signal transduction pathways,the signal changes of HT-29 cells after IL-6(1ng / ml),Vemurafenib(5μM),stattic(2μM)IL-6(1ng / ml)failed to activate STAT3 after 2 hours pretreatment with stattic(2μM),while BCL-2 was absent.However,activation of STAT3 by IL-6 promoted BCL-2 expression.The above results show that the sensitive cells HT-29 through the activation of STAT3 IL-6,BCL-2 up-regulation,can make sensitive cells into resistant cells.6.Vemurafenib feedback activates EGFR signaling to promote drug resistance.Western blotting have confirmed that p-EGFR was up-regulated in RKO cells after treatment with Vemurafenib(2μM)for 48 hours,and p-EGFR was still expressed in HT-29 cells treated with Vemurafenib(2μM),indicating EGFR activation may cause colorectal cancer cells resistant to Vemurafenib,which need further anti-EGFR treatment to prove.7.Cetuximab can effectively sensitize the responsiveness of Vemurafenib to VEGF by inhibiting the activation of EGFR.Colony formation assays showed that the combination of Vemurafenib(1 μM)and Cetuximab(10 μg / ml)for 7 days can act synergistically to inhibit cell proliferation.Signaling pathways were verified by immunoblotting and showed that EGFR signaling activated after Cetuximab(50 μg / ml)treatment was inhibited.At the same time,MTS demonstrated that the inhibitory effect of monotherapy Cetuximab(50μg / ml)on cells was not obvious,while Cetuximab(50μg / ml)combined with Vemurafenib(72μM)obvious inhibition.Flow cytometry(Annexin V / PI double staining)showed that 48 hours after combination,apoptosis rate increased significantly.This also demonstrates that Cetuximab can sensitize the effect of Vemurafenib on tumors by inhibiting the activation of EGFR signaling.8.Cetuximab activates STAT3 signaling.Immunoblotting showed that RKO cells could induce activation of STAT3 signal after 48 hours of treatment whether Vemurafenib(2μM)or Cetuximab(50μg / ml),further ELISA validation,both through the promotion IL-6 secretion to activate STAT3 signal.9.STAT3 inhibitors inhibit the activation of Vemurafenib and Cetuximab STAT3 signal to further improve the cell sensitivity and enhance the role of the three drugs combined so that the increasing possible of combination of three drugs.Western blotting confirmed that Cetuximab could downregulate the expression of p-EGFR to sensitize the inhibitory effect of Vemurafenib on tumor cells.STAT3 inhibitor could inhibit the effect of Vemurafenib and Cetuximab double activation STAT3 signal,so that when combined with the three drugs play a synergistic effect of each other ring.At the same time,MTS,colony formation assay and flow cytometry(Annexin V / PI double staining)test all showed that the combination of three drugs can significantly inhibit cell viability and cell proliferation,and significantly increase the rate of apoptosis.CONCLUSION: 1.colorectal cancer cells of BRAFV600 E mutant have different sensitivity to Vemurafenib.2.Vemurafenib causes RKO cells to secrete IL-6 and up-regulation BCL-2 to activate STAT-3 cells to produce drug resistance.3.STAT3 inhibitor sensitized the resistance of RKO to Vemurafenib effectively.4.Activation of STAT3 signal in HT-29 cells can up-regulate the expression of BCL-2 and promote the resistance of sensitive cells.5.Vemurafenib feedback activates EGFR signaling to promote drug resistance.6.Cetuximab can enhance the responsiveness of Vemurafenib effectively by inhibiting the activation of EGFR.7.Cetuximab activates STAT3 signaling.8.STAT3 inhibitor inhibits the activation of Vemurafenib and cetuximab STAT3 signal to further improve the cell sensitivity and enhance the role of the three drugs combined so that the incresing possible of combination of three drugs. |
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