| Gastric Cancer(GC)is one of the most common gastrointestinal tumors and the third leading cause of cancer-related death.The incidence and mortality rate of gastric cancer in China ranks second among all malignant tumors,and the 5-year overall survival rate is lower than 30%.Patients with advanced gastric cancer are at an increased risk of recurrence,metastasis,and resistance to treatment.In recent years,with the development of surgery combined with chemoradiotherapy,immune agents,and biologic-targeted therapies,the survival and prognosis of gastric cancer patients have been extended and improved to a certain extent,but they still face the problems of recurrence and metastasis and resistance to chemotherapy.Cancer Stem Cells(CSCs)refer to a small subset of cells with self-renewal,multilineage differentiation,and tumorigenicity,which determines various malignant biological properties such as tumorigenesis and heterogeneity.The characteristics of CSCs can stimulate the formation,recurrence,and metastasis of cancer,and generate drug resistance,which significantly affects the effectiveness of tumor treatment.It can be seen that the existence of CSCs is crucial to the formation and development of tumors,and research on CSCs is expected to fundamentally uncover the mechanism of tumorigenesis and achieve a breakthrough in treatment effect.Gastric cancer is recognized as "regurgitation" and "stomach pain" in the theory of traditional Chinese medicine.The main causes of gastric cancer mainly include positive deficiency,toxic pathogens,and spittoon coagulated blood stasis.In addition,Qi and blood stagnation,spleen and stomach disorders,improper diet,and mental disorders are also common causes.The pathogenesis of gastric cancer is generally attributed to the evil excess and positive deficiency,and cancer toxicity accumulates in the stomach.The general principles of treatment are anti-cancer,detoxifying,strengthening,and tonifying deficiency.The treatment is based on strengthening the spleen and stomach,Guben Peiyuan,Invigorating the spleen regulating Qi,detoxifying and dispersing the nodules,together with the regimen and psychological guidance.The diagnosis and treatment of gastric cancer should be based on the dynamic balance of zang-fu organs,Yin and Yang,Qi and blood,cold and heat,and focus on fuzheng and dispelling evil.Compared with the single use of Western medicine or Chinese medicine,the advantages of combining Chinese and Western medicine in the treatment of gastric cancer are very obvious,mainly focusing on reducing patients’clinical symptoms,accelerating post-operative recovery,increasing the sensitivity to radiotherapy,reducing the toxic side effects of radiotherapy and chemotherapy,improving the immunity,and preventing the recurrence and metastasis of gastric cancer.Therefore,Chinese medicine combined with surgery,chemoradiotherapy,biological therapy,immunotherapy,and other comprehensive treatment means can achieve the purpose of reducing toxicity and increasing effectiveness,improving patients’ survival rate and quality of life.Celastrus Orbiculatus Thunb.is a deciduous rattan shrub of the genus Celastraceae.It is effective in dispelling wind and relieving pain,promoting blood circulation and reducing swelling,detoxifying and dispelling blood stasis,treating bruises and injuries,carbuncles and abscesses,and rheumatic arthritis,etc.Its stem,leaves,roots,and rattan can be used as medicine.Our research group conducted pharmacological studies on anti-digestive tract tumors by focusing on Celastrus Orbiculatus Extract(COE)and found that it has highly effective anti-tumor activity.Previous studies of our research group have confirmed that COE can inhibit the Epithelial-Mesenchymal Transition(EMT)of gastric cancer cells,weaken their invasion and migration ability and increase their sensitivity to chemotherapy.EMT refers to the process in which epithelial cells obtain mesenchymal cell phenotype by blocking intercellular contact and reshaping the cytoskeleton.It is a key step in mediating tumor invasion and metastasis and one of the important ways for cancer cells to obtain CSCs malignant phenotype.Relevant studies have also shown that CSCs also possess mesenchymal cell characteristics,and inhibition of EMT can attenuate the stemness phenotype of CSCs.However,it is not clear whether COE can inhibit the recurrence and invasion of gastric cancer by reversing the EMT of gastric cancer cells and destroying the stem-like cell phenotype of gastric cancer,and few domestic and international studies have been reported on the inhibition of gastric cancer stem-like cells by COE.Based on the previous experiments,we propose to further investigate the effect of COE on the stemness phenotypic characteristics of gastric cancer stem-like cells.Firstly,we propose to use magnetic-activated cell sorting combined with flow cytometry to isolate gastric cancer stem-like cells and construct a gastric cancer stem-like cell model by serum-free medium culture;Then,high throughput transcriptomic sequencing technology was used to screen the target molecules of COE to interfere with the stemness phenotype of gastric cancer stem-like cells;Finally,the role and influence of the target molecule in mediating the stem-like cell characteristics of gastric cancer were investigated in vitro and in vivo by molecular biology experiments combined with clinical tissue microarrays,so as to elucidate the role and potential mechanism of COE on the stem-like cell characteristics of gastric cancer mediated by this target molecule,and provide an experimental basis for the development of novel drugs for the treatment of gastric cancer,and provide a new strategy for the treatment of gastric cancer stem-like cells to solve the clinical dilemma of recurrence,metastasis and resistance to chemotherapy.Part Ⅰ:CD133+/CD166+human gastric cancer cell subpopulation presents cancer stem-like cell phenotypic characteristics.Objective:The purpose of this part of the study is to identify whether there is a subpopulation of gastric cancer cells that can be labeled by specific transmembrane molecules(CD 133,CD 166)and have cancer stem-like cell characteristics,and to provide a target and basis for the treatment of gastric cancer.Methods:CD133+/CD166+cell subpopulations were isolated from human gastric adenocarcinoma cell lines by magnetic-activated cell sorting,and the sorting efficiency was identified by flow cytometry.The stemness phenotypes of the cell subpopulations,including sphere-forming ability,proliferative ability,resistance to chemotherapy,clonogenic ability,and tumorigenic ability in vivo,were evaluated.The expression levels of cancer stem cell markers(Nanog,Sox2,Oct-4,and c-Myc)in each cell subpopulation were measured by qRT-PCR and Western Blot assays.Results:Compared with CD133-/CD166-,CD133-/CD166+,and CD133+/CD166-cell populations and parental cells,the CD133+/CD166+cell subpopulation had the highest sphere-forming ability,proliferative ability,chemotherapy tolerance,clonogenic ability,and tumorigenic ability in vivo.In CD133+/CD166+cells,the mRNA and protein expression levels of Oct-4 and c-Myc were higher than in parental cells or other cell populations.Conclusion:The CD133+/CD166+gastric cancer cell subpopulation has cancer stem-like cell characteristics and can be used as a gastric cancer stem-like cell model.Part Ⅱ:PTBP1 promotes the proliferation and maintains the stemness phenotype of gastric cancer cells.Objective:To elucidate the role of PTBP1 in maintaining the stemness phenotype of gastric cancer cells and to investigate its potential mechanisms.Methods:Clinical tissue microarrays were used to investigate the association between the protein expression of PTBP1 and the prognosis and survival of gastric cancer patients.Western blot assay was used to investigate the differences in PTBP1 expression in gastric cancer cell lines and gastric epithelial cells.To compare the differential expression of cancer stem cell markers between monolayer gastric cancer cells and 3D cultured gastric cancer stem-like cells using qRT-PCR and western blot assays.PTBP1 knockdown and overexpression were constructed using small interfering RNA,plasmid,and lentiviral vector,respectively.The effects of PTBP1 knockdown and overexpression on cell proliferation were detected by cell counting kit-8 assays,colony formation assays,and EdU assays.The effect of PTBP1 knockdown and overexpression on cell cycle proteins was detected by western blot assay;the effect of PTBP1 knockdown and overexpression on stemness phenotype of gastric cancer cells was detected by sphere-forming assays,ALDH1 activity assays,side population assays,and high-content imaging system.PTBP1 knockdown and overexpression were constructed in a nude mice xenograft model,and the effect of PTBP1 on tumorigenic ability in vivo was analyzed using IVISTM live imaging system.Results:PTBP1 was highly expressed in gastric cancer tissues compared with adjacent tissues(P<0.001),and high PTBP1 expression was negatively correlated with the overall survival of gastric cancer patients(P=0.002),which was consistent with the analysis of PTBP1 in the TCGA database.Compared with gastric epithelial cells,PTBP1 was highly expressed in gastric cancer cell lines MKN28,MKN45,AGS,and HGC-27,suggesting that PTBP1 expression is closely related to gastric carcinogenesis.The expression of PTBP1 and tumor markers were higher in spheroid-cultured gastric cancer stem-like cells than in the monolayer gastric cancer cells,suggesting that the expression of PTBP1 correlates with the stemness phenotype of the cancer cells.The knockdown of PTBP1 induced cell cycle arrest in the G1 phase and reduced the proportion of cells in the S phase,thus inhibiting cell proliferation.In contrast,overexpression of PTBP1 caused cells to accumulate in the G2 phase and promoted cell proliferation.The results of western blot assay showed that knockdown of PTBP1 could inhibit the expression of the cell cycle-related protein Cyclin D1 and promote the expression of p21 and p27,while overexpression of PTBP1 showed the opposite effect,suggesting that PTBP1 could affect the proliferation of gastric cancer cells by regulating the cell cycle.The results of sphere-forming assay,ALDH1 activity assay,and side-population assay indicated that knockdown of PTBP1 could inhibit cell sphere-forming ability,reduce ALDH1 activity and down-regulate the proportion of drug-resistant cells,while the opposite results were observed for overexpression of PTBP1.The results of the high-content imaging system analysis showed that the migration ability of PTBP1 knockdown was diminished,while overexpression of PTBP1 promoted migration ability.Knockdown of PTBP1 down-regulated the expression of cancer stem cell markers(c-Myc,Oct-4,Sox2,Nanog),while overexpression of PTBP1 up-regulated the expression of cancer stem cell markers.The results from the nude mice xenograft model showed that the proliferation of xenograft in the PTBP1 knockdown group was inhibited,while the proliferation of xenograft in the PTBP1 overexpression group was significantly accelerated,provided that the body weights of tumor-bearing nude mice were essentially comparable.Conclusion:PTBP1 can promote the proliferation of gastric cancer by maintaining the stemness phenotype of gastric cancer.Part Ⅲ:PTBP1 mediates the stability of c-Myc through the ubiquitin protease pathway to maintain the stemness phenotype of gastric cancer.Objective:To explore the potential mechanism of PTBP1 on the function of maintaining the stemness phenotype of gastric cancer.Methods:The co-localization of PTBP1 and c-Myc in cells was detected by immunofluorescence assay.The regulatory effect of PTBP1 on c-Myc expression was examined by qRT-PCR and western blot assay.After the addition of the protein synthesis inhibitor CHX,cellular proteins were extracted at time intervals to determine the effect of PTBP1 on the rate of c-Myc degradation.In the presence or absence of PTBP1,the protease inhibitor MG132,the autophagic vesicular ATPase inhibitor Bafilomycin A1 and the lysosomal inhibitor Leupeptin were respectively added to uncover the key pathway of the degradation effect of PTBP1 on c-Myc.The regulatory role of PTBP1 on the ubiquitination level of c-Myc was analyzed by immunoprecipitation assay.The regulatory effects of PTBP1 on the expression of two key phosphorylation sites(Ser62,Thr58)of c-Myc ubiquitination,E3 ubiquitin ligase FBW7,and deubiquitinated protein USP28 were analyzed by western blot.In PTBP1 wild expression and overexpression cell lines,a specific inhibitor 10058-F4 targeting c-Myc synthesis was added to analyze the effect of c-Myc deletion on PTBP1 expression.Overexpression of c-Myc in PTBP1 knockdown cell lines was conducted in vitro and in vivo experiments to observe whether c-Myc overexpression could rescue the effect of PTBP1 deletion on cancer stem-like cell phenotype.Results:The results of immunofluorescence assay suggested that PTBP1(red)was co-localized with c-Myc(green)in the nucleus,and the immunoprecipitation results also revealed that PTBP1 was bound to c-Myc protein,and the expression of PTBP1 was positively correlated with c-Myc at the protein level.In contrast,at the mRNA level,knockdown or overexpression of PTBP1 was unable to cause changes in c-Myc,suggesting that the regulation of c-Myc by PTBP1 is at the post-transcriptional level.With the administration of the protease inhibitor CHX,PTBP1 deficiency accelerated the degradation of the c-Myc protein,while PTBP1 overexpression maintained the stability of the c-Myc protein.By comparing the effect of three inhibitors of protein degradation pathways,it was confirmed that only MG132 could block the effect of c-Myc protein degradation caused by PTBP1 deficiency.Immunoprecipitation assays confirmed that PTBP1 regulates c-Myc protein degradation through the ubiquitin-proteasome pathway and that knockdown of PTBP1 promotes ubiquitinated degradation of c-Myc,while overexpression of PTBP1 inhibits ubiquitinated degradation of c-Myc,and all of these results are rescued by MG132.With the administration of 10058-F4,a specific antagonist of c-Myc protein,the protein expression of PTBP1 was not affected in PTBP1 overexpression group compared with the control group,suggesting that c-Myc is a downstream of PTBP1.Overexpression of c-Myc was constructed in PTBP1 knockdown cell lines,and results of both in vitro and in vivo confirmed that overexpression of c-Myc could rescue the effects of impaired stem cell-like phenotype in gastric cancer caused by PTBP1 knockdown.Conclusion:PTBP1 maintains the sternness phenotype of gastric cancer by mediating the stability of c-Myc.Part Ⅳ:Celastrus orbiculatus extract inhibits the stemness phenotype of gastric cancer stem-like cells via TGF-β/Smad signaling pathway.Objective:To investigate the effect of COE on the stemness phenotype of gastric cancer stem-like cells and explore its possible role and mechanism.Methods:A gastric cancer stem-like cell model was constructed using spheroid culture;the expression of CD 133 and CD 166 in gastric cancer stem-like cells was detected by immunofluorescence assay,western blot assay,and flow cytometry.Different concentrations of COE were applied to treat gastric cancer stem-like cells,and the effects of COE on stem-like cell phenotypes such as cancer stem cell markers,proliferation,and apoptosis were detected by CCK-8 assay,sphere-forming assay,western blot assay,and flow cytometry.The expression of PTBP1 was knocked down by using small interfering RNA,and the effect on the TGF-β signaling pathway after the knockdown of PTBP1 was examined by western blot and a high-content imaging system.By setting up the control group,TGF-β group,COE group,and TGF-β+COE group,the effect of COE on the TGF-β signaling pathway and the role of PTBP1 in it were examined by western blot assay,to clarify whether PTBP1 was involved in the inhibition of gastric cancer stem-like cell characteristics by COE through TGF-β/Smad signaling pathway.Results:The expression of CD133,CD166,and tumor markers such as Oct-4,Sox2,ALDH1,and Nanog were significantly higher in spheroid-cultured gastric cancer stem-like cells compared with monolayer gastric cancer cells.(P<0.05);COE downregulated the expression of cancer stemness biomarkers in gastric cancer stem-like cells in a dose-dependent manner.(P<0.05)and inhibited the proliferation and induced apoptosis of gastric cancer stem-like cells,suggesting that COE could inhibit the stemness characteristics of gastric cancer stem-like cells.Knockdown of PTBP1 expression in gastric cancer stem-like cells could down-regulate the expression of stemness markers and TGF-β/Smad signaling pathway-related proteins(P<0.05);Meanwhile,comparing the results of TGF-βand COE alone and in combination,it was concluded that TGF-β alone could stimulate the activation of Smad signaling pathway and upregulate the expression of cancer stem cell markers,while COE alone had the opposite effect.The combination showed that COE could partially suppress the effect of TGF-β,suggesting that COE could inhibit the stemness phenotype of gastric cancer stem-like cells through the TGF-β/Smad signaling pathway.Conclusion:COE can reduce the stemness phenotypic properties of gastric cancer stem-like cells,and the mechanism may be related to the inhibition of TGF-β/Smad signaling pathway activation by COE. |
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