The Mechanism Study Of Periplogenin Suppresses The Growth Of Esophageal Squamous Cell Carcinoma By Targeting STAT3

Background and research purposeThe global incidence and mortality rate of esophageal cancer ranks sixth and seventh most common cancer in the world,the 5-year survival rate remains less than20%.The occurrence of esophageal cancer is regional,especially in the provinces near the Taihang mountains in China.There mainly exist two histological types:esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EAC),and 90% of esophageal cancer patients in China account for esophageal squamous cell carcinoma.Esophageal cancer is a kind of cancer with a high degree of malignancy.Due to the lack of early screening and diagnostic markers,the esophageal cancer is often diagnosed at an advanced stage or the tumors have already metastasized.Currently,palliative chemotherapy,radiotherapy,targeted therapy combined with surgery are the mainly treatment methods of esophageal cancer.However,the toxic and side effects caused by chemotherapy and radiotherapy such as vomiting,liver and kidney damage,nerve damage,hair loss and other problems are serious.Targeted therapy lacks effective therapeutic targets and targeted drugs.In addition,recurrence and drug resistance are prominent.Therefore,it is urgent to develop new therapeutic targets and anti-tumor drugs with high efficiency,low toxicity and resisting to drug resistance.STAT3 is an important transcription factor involved in almost all cancer hallmark related activities such as proliferation,metastasis,angiogenesis,immunosuppression,inflammation,tumor microenvironment,metabolic reprogramming,drug resistance,cancer stemness,and exosome-mediated tumorrelated activity.It has been reported that STAT3 is abnormally activated in a variety of cancer types including solid tumors such as breast cancer,colon cancer,and nonsmall cell lung cancer and hematological tumors such as lymphoma and leukemia.And constitutive activation of STAT3 is often positive correlated with clinical stage,pathological grade,lymph node metastasis,and poor survival.Therefore,STAT3 is a potential therapeutic target for developing antitumor drugs.STAT3 is composed of N-terminal domain(NTD),coiled-coiled domain,DNA binding domain,linker domain,SH2 domain and transactivation domain(TAD).Since STAT3 lacks the targetable enzymatic active site,structure-based development has become the main strategy for the STAT3 drug discovery.Especially the DNAbinding domain and SH2 domain have the functions of protein-DNA interaction and protein-protein interaction,separately.Making it possible to directly target STAT3.The SH2 domain has dual functions,one is being recruited to membrane receptors for phosphorylation,and the other is binding to the phosphorylated tyrosine 705 residue of another STAT3 monomer,forming STAT3 dimers and facilitating the subsequent nuclear importing,the DNA binding ability with the promoter region of target genes involved in cancer-related activities.However,there exists two problems in the development of STAT3 inhibitors based on the SH2 domain,one is specificity,and the other is that non-phosphorylated STAT3 still has transcriptional activity.Although there are hundreds of STAT3 inhibitors have been identified,no FDA-approved drugs have entered the market.There still has a long way to go for the STAT3 drug discovery.There are diverse natural medical resources in China.The natural small molecular compounds extracted from traditional Chinese medicine resources have made great contributions to the treatment of intractable diseases.Here,we study periplogenin,a glycoside compound which is derived from the root of Streptocaulon juventas.Cardiac glycoside compounds have been identified to have great potential in the treatment of cancer in recent years.Periplogenin is traditionally treated for arrhythmia and heart failure.Compelling evidences reveal that periplogenin reduces the tumor volumes in tumor-bearing mice.Periplogenin has been shown to trigger cancer cell apoptosis via ROS-ER stress and death receptor pathways.Whereas some researchers also show that periplogenin ameliorates skin lesions in psoriasis-like mouse models.However,whether periplogenin inhibits the growth of ESCC are unknown,and the mechanism of periplogenin inhibits the tumor cell proliferation also remains further studies.In summary,this project elucidates the effects and mechanism of periplogenin inhibits the proliferation of esophageal squamous cell carcinoma via the following four aspects:(1)Periplogenin inhibits the ESCC cell growth and promotes the ESCC cell apoptosis in vitro;(2)Periplogenin directly binds to STAT3 and inhibits the phosphorylation of STAT3,leading to significant suppression of subsequent dimerization,nuclear importing,transcription activities,binding to the promoter region of target genes and their expression.(3)p-STAT3 is highly expressed in ESCC tissues,and periplogenin inhibits the proliferation of ESCC is dependent on STAT3.(4)Periplogenin inhibits the tumor growth in ESCC PDX models,and the therapeutic effect is better than that of other STAT3 inhibitors.Methods1.Detecting the inhibitory effect of periplogenin on the proliferation of ESCC in vitroThe IC50 values of periplogenin in ESCC cells and Shantou human embryonic esophageal cell SHEE are detected to compare the cytotoxicity of periplogenin using MTT assay.The effect of periplogenin on the proliferation and clony formation of ESCC cells and Shantou human embryonic esophageal cell SHEE is detected by cell proliferation assay and soft agar assay;The early phase apoptosis of periplogenin on ESCC cells and SHEE cells are detected by FITC-Annexin V assay combines with Western blot.2.Screening of the target of periplogeninPredicting the potential targets of periplogenin using Swiss PredictionTarget software;Identifying the targets of periplogenin using periplogenin-conjugated sepharose 6B beads pull-down assay and computer docking model;The highthroughput kinase profiling assay,in vitro kinase assay,Western blot and immunofluorescence assays are performed to detect that periplogenin directly binds to STAT3 and inhibits the phosphorylation of STAT3 Tyr705 rather than acting on the upstream kinases of STAT3.3.Exploring the effect of periplogenin on STAT3 functionNative-PAGE and Western blot experiments are performed to identify the effects of periplogenin on the dimerization of STAT3;Multiple immunofluorescence experiments and Western blot are performed to detect the effects of periplogenin on nuclear importing of STAT3 and p-STAT3;The dual-luciferase reporter assay is performed to detect the effects of periplogenin on transcriptional activity of STAT3;Ch IP-PCR is performed to detect the effects of periplogenin on the DNA binding ability of STAT3 with the promotor region of target genes;Western blot experiment is performed to detect the effects of periplogenin on the expression of STAT3 downstream target genes.4.Exploring the expression levels and clinical significance of STAT3 and pSTAT3 Tyr705 in ESCCThe expression levels of STAT3,other members of the STATs family,and STAT3 downstream genes in ESCC are analyzed though TCGA data mining;Immunohistochemical staining of ESCC tissue array are performed to analyze the expression levels of p-STAT3 Tyr705 in ESCC and its correlation with pathological grade,age,gender,lymph node metastasis,tumor size and survival rate.5.Exploring periplogenin inhibits the ESCC growth is dependent on STAT3Western blot is performed to screen the cell lines with high expression levels of STAT3 and p-STAT3 Tyr705 in ESCC for STAT3 knockdown experiments.The sensitivity of periplogenin on mock group and STAT3 knockdown group are detected using MTT and soft agar assay.Western blot is performed to screen the cell lines with low expression levels of STAT3 and p-STAT3 Tyr705 in ESCC for STAT3 overexpression experiments.The sensitivity of periplogenin on mock group and STAT3 overexpressing group are detected using MTT and soft agar assay.6.Detecting the tumor inhibitory effects of periplogenin in ESCC PDX modelsThe effects of periplogenin on the proliferation of esophageal squamous cell carcinoma are detected in ESCC PDX models;The expression levels of ki67,pSTAT3 Tyr705,MCL-1,MYC and other genes in tumor tissue post-treatment with periplogenin are detected using immunohistochemistry.7.Comparing tumor inhibitory effects of periplogenin and other STAT3 inhibitors C188-9 and BBI-608 on ESCC cells in vitro and in vivo.The IC50 values of periplogenin,C188-9 and BBI-608 in ESCC and SHEE are compared through MTT assay;The expression level of p-STAT3 Tyr705 in ESCC post-treatment with periplogenin,C188-9 and BBI-608 with the same condition are detected using Western blot;The tumor inhibitory effects of periplogenin,C188-9 and BBI-608 on tumor growth are compared through the ESCC PDX models.Results1.The IC50 values in KYSE30,KYSE70,KYSE450 are 1.707±0.275 μM,2.898±0.959 μM,1.201±0.167 μM,separately.The IC50 value of Shantou human embryonic esophageal cell SHEE is 10.95±1.259 μM;Periplogenin inhibits ESCC cells growth in a time-and concentration-dependent manner;Periplogenin inhibits the colony formation in ESCC cells;1 μM,2 μM,4 μM of periplogenin have no significant inhibitory effects on cell proliferation and colony formation of Shantou human embryonic esophageal cell SHEE.2.The results of Swiss Prediction Target prediction,computer docking model and pull-down assay indicate that periplogenin specifically binds to STAT3,and the binding sites are located in the linker and SH2 domains;The results of highthroughput kinase profiling experiment,in vitro kinase assay,immunofluorescence,and Western blot show that periplogenin directly binds to STAT3 and inhibits the phosphorylation of STAT3 Tyr705 instead of acting on the upstream kinase activity of STAT3.3.Native PAGE-Western blotting result reveals that periplogenin inhibits the dimerization of STAT3,The results of immunofluorescence and nucleocytoplasmic separation-Western blot show that periplogenin inhibits nuclear importing of STAT3 and p-STAT3 Tyr705;The result of dual luciferase reporter experiment show that periplogenin inhibits the transcriptional activity of STAT3;The result of ChIP-PCR experiment show that periplogenin inhibits the DNA binding ability of STAT3 with the promoter region of downstream genes such as Cyclin D1,Myc,and VEGFA;Western blot result shows that periplogenin inhibits the expression levels of STAT3 downstream genes such as anti-apoptosis gene MCL-1,proliferation-relates genes MYC,Cyclin D1,and survivin.4.STAT3 and STAT3 downstream genes including MYC,MCL-1,Cyclin D1,Survivin,PIMI1,JUNB,COX-2,TGF-β are significantly highly expressed in ESCC tissues in the TCGA database;p-STAT3 Tyr705 is highly expressed in ESCC tissues,and the higher expression level of p-STAT3 Tyr705 is correlated with poor prognosis,however,there are no correlation between the expression of p-STAT3 Tyr705 with pathological grade,age,gender,lymph node metastasis or tumor size.5.The mock group and STAT3 knockdown group are treated with periplogenin,the MTT and soft agar results indicate that the STAT3 knockdown group becomes less sensitive to the periplogenin than the mock group;Conversely,the mock group and the STAT3 overexpressing group are treated with periplogenin,the STAT3 overexpressing group becomes more sensitive to periplogenin than the mock group.6.Periplogenin significantly decreases the tumor volume in the ESCC PDX models;Periplogenin has no significant inhibitory effect on the body weight of mice;Periplogenin significantly inhibits the expression levels of ki67,p-STAT3 Tyr705,MCL-1,MYC in tumor tissues.7.Periplogenin shows the most potent tumor inhibitory effects in KYSE30 and KYSE450 when compared with C188-9 and BBI-608 in the side-by-side experiments;Periplogenin shows a moderate cell inhibitory activities in the SHEE when compared with C188-9 and BBI-608.Western blot results show that periplogenin attenuates the phosphorylation levels of STAT3 Tyr705 most significantly in all ESCC cell lines compared with C188-9 and BBI-608.And Periplogenin shows the most robust tumor inhibitory effect among these three drugs in the PDX models.ConclusionIn this study,we find that STAT3 and p-STAT3 Tyr705 are highly expressed in ESCC and associate with poor prognosis.Periplogenin specifically binds to STAT3 and inhibits the phosphorylation,dimerization,nuclear importing,transcriptional activities,DNA binding ability of STAT3 with the promotors of downstream target genes,the transcription and expression of downstream target genes,thus inhibiting the proliferation of ESCC in vitro and in vivo.Moreover,the anti-tumor effects of periplogenin are better than those of other STAT3 clinical trial inhibitors C188-9 and BBI-608.This study provides a basis for the effects and mechanism of periplogenin on the proliferation of ESCC.