Study On The Mechanism Of Salinomycin Combined With Sulforaphane Or Oxaliplatin In Inhibiting Colorectal Cancer

BackgroundColorectal cancer(CRC)is a global health problem,and it is one of the most aggressive tumors with the highest diagnosis rate.According to the latest global cancer incidence and mortality statistics,the incidence of colorectal cancer ranks third,and the mortality rate ranks second.In 2020,about 1.93 million new cases and 935,000 people died of colorectal cancer worldwide,accounting for about one-tenth of the incidence and mortality of cancer.The incidence and mortality of colorectal cancer in China from 2000 to 2015 are on the rise.The incidence and mortality of colon cancer have risen significantly,seriously endangering people's health.Although early diagnosis and treatment have improved,about 20%of patients have metastases at diagnosis,and up to 50%of non-metastatic colorectal cancers have metastases at a later stage,and approximately one-third of patients after treatment relapsed.The latest "NCCN Guidelines" recommends that patients with advanced colorectal cancer should take a comprehensive treatment plan with surgery as the mainstay and chmotherapy as the supplement.FOLFOX(oxaliplatin,5-fluorouracil and leucovorin)and CapeOX Or XELOX regimen(oxaliplatin and capecitabine)is listed as the same preferred chemotherapy regimen.Oxaliplatin is a first-line anti-colorectal cancer chemotherapy drug.It has a high application advantage in the treatment of metastatic colorectal cancer.However,nearly half of the patients have a reduced survival rate due to chemotherapy resistance.Due to the development of chemotherapy resistance and acute or persistent nerve damage,the overall success of oxaliplatin-based treatment options is limited.Therefore,looking for new drug treatments or improving the chemosensitivity of oxaliplatin and other original chemotherapeutic drugs,so as to sensitize cancer cells and obtain higher curative effects,while minimizing adverse side effects,and improving the treatment of colorectal cancer.It is very necessary to be efficient,to extend the disease-free survival of patients and to improve the prognosis.Sulforaphane(SFN)is an isothiocyanate contained in cruciferous plants.It can block the growth and development of cancer cells in the three stages of cancer initiation,development and proliferation,and reduce the risk of cancer.SFN Has a certain value in tumor prevention and treatment.SFN is one of the natural active substances with the best anti-cancer and anti-cancer effects in plants.It not only exerts anti-cancer effects in multiple stages of tumorigenesis,but also has anti-cancer,anti-proliferation,pro-apoptosis,anti-metastasis and anti-angiogenesis properties.The role of it is a potential tumor treatment drug.It has been demonstrated that SFN mediates the protective effect of NF-E2-related factor 2(Nrf2)against oxidative damage caused by toxic drugs.The same SFN concentration has a protective effect in normal cells and is harmful to tumor cells,which indicates its potential for application in chemotherapy.We suggest SFN as a new type of anticancer drug for the treatment of human colorectal cancerSalinomycin(SAL)is a high-efficiency,broad-spectrum,low-resistance,rapid excretion and low-residue monocarboxylic acid polyether antibiotic isolated from the strain of Streptomyces albicans.Salinomycin can overcome the resistance of tumor cells to chemotherapy drugs,induce apoptosis of cisplatin-resistant colon cancer cells,and significantly promote the apoptosis of gemcitabine-resistant pancreatic cancer cells.It has little effect on normal cells,can kill drug-resistant cancer cells and tumor stem cells,and can reduce the invasion and metastasis of cancer cells.It has been identified as a powerful chemosensitizer and can induce apoptosis of multidrug resistant tumor cells.In this study,a model of salinomycin combined with sulforaphane or oxaliplatin chemotherapy was designed.The effect of salinomycin,sulforaphane and oxaliplatin on the survival rate of colonrectal cancer cells was further explored through the use of salinomycin,sulforaphane and oxaliplatin alone.In vitro experiments study the effects of salinomycin and oxaliplatin on cell growth,cell morphology,cell cycle,cell apoptosis,cell migration ability,etc.and explore its related mechanism,in vivo experiments on subcutaneous xenograft tumors in nude mice were further verified.Part ? Study on the mechanism of salinomycin combined with sulforaphane in inhibiting colorectal cancerObjetive1.To study the effect of salinomycin combined with sulforaphane on the inhibitory effect and biological function of colorectal cancer cells.2.To study the molecular mechanism of salinomycin combined with sulforaphane to inhibit the proliferation of colorectal cancer cells and promote apoptosis.3.To study the inhibitory effect of salinomycin combined with sulforaphane on colorectal cancer in vivo.Methods1.Detection of colorectal cancer cell activity:gradient concentrations of salinomycin and sulforaphane act on colorectal cancer cells respectively,and the MTT experiment is used to detect the cell activity of each drug concentration.According to the results,a total of 12 drug combinations were selected,and the MTT experiment was used to detect changes in cell activity.2.Detect the effects of combination drugs on the biological behaviors of colorectal cancer cells:EdU experiment was used to detect the effects of the control group,single drug group and combination drug group on the proliferation ability of colorectal cancer cells;flow cytometry was used to detect the effects of each group on the colorectal cancer cell apoptosis;Transwell experiment was used to detect the influence of each treatment group on the migration and invasion ability of colorectal cancer cells.3.Detection of the mechanism of colorectal cancer cell apoptosis caused by the combination therapy:Western Blotting was used to detect the effects of each treatment group on the expression of PI3K/Akt pathway proteins and apoptosis-related proteins;immunofluorescence was used to detect the expression of p-Akt;RT-qPCR method was used to detect the effect of each treatment group on the Bcl-2 and Bax mRNA;Western Blotting was used to detect the effect of PI3K/Akt pathway inhibitor treatment on the apoptotic proteins and cell activity in the combined drug group.4.Detect the effect of combination medication on the growth of subcutaneous xenograft tumor in nude mice:construct an animal model of subcutaneous xenograft tumor in nude mice,measure tumor diameter,draw tumor growth curve,and further verify the effects of combination medication on the growth of subcutaneous xenograft tumor in nude mice.Results1.Changes in cell activity:salinomycin and sulforaphane treated colorectal cancer cells alone,and the effect of salinomycin and sulforaphane on cell activity showed a concentration and time dependence;the 12 groups of salinomycin and sulforaphane drug combinations showed a synergistic effect.2.The biological behavior of colorectal cancer cells.SAL 5 ?M and SFN 10 ?M are selected for the next experiment,and the results are as follows:?Colorectal cancer cells were divided into control group,single-drug groups and combination group.EdU experiment detected that the cell proliferation ability of combination group was significantly lower than that of single-drug group and control group,and the difference was statistically significant.?Flow cytometry results show that SAL combined with SFN significantly promoted the apoptosis of colorectal cancer cells;The results of Western Blotting showed that the expression of anti-apoptotic protein Bcl-2 in the combination group was significantly reduced,while p53,Bax and cleaved PARP proteins was significantly increased;The results of RT-qPCR showed that the expression of Bcl-2 mRNA in the combined drug group was significantly reduced,and the expression of Bax mRNA was significantly increased,and the differences were statistically significant.?The results of Transwell showed that the combined drug group significantly inhibited the migration and invasion of colorectal cancer cells compared with the single drug groups and the control group,and the difference was statistically significant.3.Detect the effect of salinomycin and sulforaphane on PI3K/Akt pathway?Compared with the control group and the single-drug treatment groups,the combination of SAL and SFN significantly inhibited PI3K and p-Akt.?p-Akt was significantly reduced in the combined drug group detected by immunofluorescence method.?The results showed that pre-treatment with LY294002 can significantly reduce p-Akt,increase the apoptotic protein cleaved PARP,and significantly reduce cell activity in the combination group.The differences were statistically significant.4.The anti-tumor effect was detected in vivo:salinomycin or sulforaphane treatment alone can inhibit tumor growth,but the combination of the two drugs can significantly block tumor growth and reduce tumor volume and weight.There are statistical differences.In the control group,single-drug treatment groups,and combination drug group,the weight of nude mice did not change significantly,and the difference was not statistically significant.Conclusions1.The combination of salinomycin and sulforaphane synergistically inhibited the activity of colorectal cancer cells.2.The combination of salinomycin and sulforaphane significantly inhibited the proliferation of colorectal cancer cells,induced apoptosis,inhibited migration and invasion.3.The combination of salinomycin and sulforaphane promoted cell apoptosis and inhibited cell proliferation by inhibiting the PI3K/Akt signaling pathway.4.The combination of salinomycin and sulforaphane significantly inhibited the ability of colorectal cancer to form tumors in vivo.Part ? Study on the mechanism of salinomycin combined with oxaliplatin in inhibiting colorectal cancerObjetive1.To study the effects of salinomycin combined with oxaliplatin on the cytotoxicity and biological functions of colorectal cancer cells.2.To study the molecular mechanism of salinomycin combined with oxaliplatin in inhibiting the proliferation and inducing apoptosis of colorectal cancer.3.To study the effect of salinomycin combined with oxaliplatin on the growth of colorectal cancer in vivo.Methods1.Detect the cytotoxic effects of salinomycin or oxaliplatin on colorectal cancer:MTT was used to detect the survival rate of salinomycin or oxaliplatin at different concentrations on colorectal cancer cells.2.The effects of salinomycin and oxaliplatin on the biological behavior of colorectal cancer:MTT experiment was used to detect the cell survival rate of colorectal cancer cells with different concentrations of salinomycin and oxaliplatin,and the combination index was calculated,the single drug concentrations were selected;the plate clone formation experiment was used to detect the plate clone formation ability of the control group,salinomycin group,oxaliplatin group and the combination group;flow cytometry was used to detect cell apoptosis;flow cytometry was used to detect the changes in cell cycle distribution;the cell scratch test and the Transwell test were used to detect the migration ability of cells.3.Observe the morphological changes of colorectal cancer cells after treatment with drugs in each group by microscope.4.The combination of salinomycin and oxaliplatin caused changes in the reactive oxygen species and mitochondrial membrane potential of colorectal cancer cells:after the colorectal cancer cells are grouped and processed,the DCFH-DA fluorescent probe was used to detect the intracellular reactive oxygen species;JC-1 staining kit was used to detect mitochondrial membrane potential.5.The effects of salinomycin and oxaliplatin on the expression of apoptosis-related proteins in the mitochondrial pathway:the expression of apoptosis-related proteins in the mitochondrial pathway were detected by Western blotting.6.The effect of combination of salinomycin and oxaliplatin on MAPK pathway:the colorectal cancer cells were divide into control group,salinomycin group,oxaliplatin group and NAC,salinomycin and oxaliplatin group.Flow cytometry was used to detect the changes of cell apoptosis in each group;colorectal cancer cells were divided into control group,salinomycin group,oxaliplatin group,salinomycin and oxaliplatin group,and NAC,salinomycin and oxaliplatin group.the expression of MAPK pathway proteins were detected by western blotting.7.Study on the effects of salinomycin and oxaliplatin on transplanted tumors of colorectal cancer in nude mice:After nude mice have subcutaneously formed tumors,they were randomly divided into groups,and corresponding drugs were injected intraperitoneally,tumor volume was calculated,and tumor growth curve was drawn;immunohistochemistry was used to detect the expression of Ki-67 and p-ERK in tumor tissues of each group.Results1.Detection of cell viability:different concentrations of salinomycin or oxaliplatin acted on colorectal cancer cells,the viability of colorectal cancer cells decreased,and it is dose-and time-dependent manner.MTT detected 15 groups of drug combinations,and all showed that the two drugs were synergistic.Compared with the control group and the single-drug groups,the cell viability of the salinomycin and oxaliplatin was significantly reduced,and the difference was statistically significant(P<0.001).SW480 was more effective than Caco-2 in the combination treatment.2.Detection of cell proliferation:Compared with the control and monotherapy groups,the number of clones in the cotreatment group was significantly reduced,and the cell proliferation was significantly slowed down(P<0.001).3.Detection of apoptosis:Flow cytometry detected that the apoptosis of the two cell lines combined drug group was significantly higher than that of the other groups(P<0.001).4.Detection of cell cycle:compared with the control group,the number of cells in the G0/G1 phase of the cotreatment group increased significantly,while the number of cells in the G2/M phase and S phase decreased.It shows that salinomycin combined with oxaliplatin can block colorectal cancer in G0/G1 phase.5.Detection of cell migration ability:The cell scratch test showed that the cell scratch healing rate of the monotherapy groups were not significantly different from that of the control group at 24 hours.Compared with the control group and the monotherapy groups,the cotreatment group was significantly reduced at 24 and 48 hours,and the difference was statistically significant(P<0.05);the results of Transwell experiment showed that:the number of migrated cells was markedly decreased in the combination groups compared to the control and monotherapy groups(P<0.001).6.Detection of cell morphology:the number of cells in the cotreatment group was significantly reduced,the size was reduced and deformed,the connection between the cells disappeared,the surrounding cells were detached,and the adherent cells shrank,rounded and shedding.7.Detection of cellular reactive oxygen:The intracellular reactive oxygen content of the cotreatment group was significantly higher than that of the control group and the monotherapy groups.8.Detection of mitochondrial membrane potential:The green fluorescence in the combination of salinomycin and oxaliplatin increased significantly,the mitochondrial membrane potential decreased,and the depolarization ratio increased.9.Detection of apoptosis-related proteins in the mitochondrial pathway:Compared with the control group,the expression of Bax,cytochrome c,cleaved caspase-9,cleaved caspase-3 and cleaved PARP increased in the salinomycin group or the oxaliplatin group,but the expression of cotreatment was increased significantly(P<0.05),while the decrease in Bcl-2 expression was more significant.10.Detection of MAPK pathway protein:Pre-added NAC can reduce cell apoptosis caused by cotreatment(P<0.01);The expression of p-ERK,p-p38 and p-JNK were decreased in the combination of salinomycin and oxaliplatin with pre-added NAC,and the difference was statistically significant(P<0.05).It showed that the massive production of reactive oxygen species could cause the activation of the MAPK pathway.11.Detection of anti-tumor effect in vivo:Compared with the control group and the monotherapy groups,salinomycin combined with oxaliplatin could more significantly inhibit the volume and weight of transplanted tumors in nude mice,and inhibited the growth of colorectal cancer significantly;immunohistochemical experiments showed that the expression of Ki-67 in the cotreatment group was significantly reduced,while the expression of p-ERK was significantly increased.Conclusions1.Salinomycin or oxaliplatin reduced the viability of colorectal cancer cells in a dose-and time-dependent manner.The combination of salinomycin and oxaliplatin can synergistically inhibited colorectal cancer cells.2.The combination of salinomycin and oxaliplatin could significantly inhibit the proliferation of colorectal cancer,promote apoptosis,block cell cycle and inhibit migration.3.The combination of salinomycin and oxaliplatin inhibited the growth of colorectal cancer by inhibiting the proliferation of colorectal cancer cells and inducing apoptosis.4.The combination of salinomycin and oxaliplatin inducing cell apoptosis may be achieved by generating a large amount of reactive oxygen species to induce apoptosis in the mitochondrial pathway,and at the same time to cause the activation of the MAPK pathway.5.The combination of salinomycin and oxaliplatin can significantly enhance the effect of oxaliplatin against colorectal cancer,and increase the sensitivity of colorectal cancer cells and transplanted tumors in nude mice to oxaliplatin.