| Objective: Colorectal cancer(CRC)is one of the most common malignant gastrointestinal tumors,which can display high morbidity and mortality worldwide.In recent years,with rapid changes in the dietary structure,social environment and lifestyle,the global incidence rate of CRC is increasing significantly.For most CRC patients,surgery remains the preferred treatment option,but postoperative recurrence and distant metastasis become the main factors restricting the prognosis of CRC patients.With the rapid development of systemic comprehensive therapy such as chemotherapy,targeted therapy and immunotherapy,the prognosis of patients has been greatly improved.At present,the various drugs used in clinic for therapy of CRC are toxic towards the normal cells and even life-threatening at higher doses.It has also been found that with longer application of drugs,tumor cells can gradually become drug resistant or even multidrug resistant,leading to the tumor recurrence and metastasis.Therefore,there remains an unmet need to find novel drugs that are more efficient,less toxic and antagonizing drug-resistance.Edible and medical foods are substances that are rich in medicinal value and can be taken as food safety.The natural products extracted from Edible and medical foods have been an important source for the development of various new drugs because of their anti-tumor,anti-oxidant,anti-inflammatory and other effects.Wedelolactone is a small molecule belonging to furanocoumarin family which has been extracted from the edible and medical plant Eclipta prostrata L.,which has various pharmacological activities such as anti-inflammatory,hepatoprotective,bone differentiation promotion and anti-immunosuppressive.In recent years,accumulating evidences have indicated that wedelolactone can exhibit anti-tumor activities against various types of tumors through diverse mechanisms such as inhibition of the cell proliferation,inducing cell apoptosis and increasing the sensitivity to chemotherapy.Nevertheless,the anti-tumor effects of wedelolactone on CRC cells and its potential molecular mechanisms remain unclear.Methods:1.Effects of wedelolactone on proliferation and apoptosis of CRC cells:Treatment with various concentrations of wedelolactone(0、20、40、60、80、100μM)for 24 and 48 h,CCK-8 was used to analyze the effect of wedelolactone on the cell viability of CRC cell HCT116 and normal colonic epithelial cell CCD841 Co N.According to the above results,in all following experiments,HCT116 cells were treated with wedelolactone(0,40,50 and 60 μM)for 24 h.Cell morphological changes were observed by optical microscopy,cell proliferation was detected by Ed U staining,cell clone formation was detected by plate colony formation assay,cell nuclear morphology was observed by DAPI staining,apoptotic rate were analyzed by Annexin V-FITC/PI double staining,the expression levels of apoptosis-related proteins(Cleaved PARP,Cleaved caspase-3,Bcl-2 and Bax)were detected by western blot.2.Molecular targets and mechanism of wedelolactone in inhibiting proliferation and inducing apoptosis in CRC cells.After treatment with wedelolactone(50 μM)for 24 h,HCT116 cells were collected by adding appropriate amount of Trizol and transcriptome sequencing was performed to screen the potential molecular pathways of wedelolactone by enrichment analysis of differential genes.HCT116 cells were treated with 0,40,50 and 60 μM wedelolactone for 24 h.The total β-catenin protein and its phosphorylation level were detected by western blot.The m RNA and protein levels of downstream target genes of β-catenin(c-Myc and survivin)were analyzed by real-time PCR and western blot.The binding mode of wedelolactone and β-catenin was further explored by using computerized molecular docking and molecular dynamics simulation,and the binding relationship between wedelolactone and β-catenin in HCT116 cells was examined using cellular thermal transition analysis assay.In addition,cells were transfected with β-catenin overexpression plasmid and western blot was used to verify the overexpressed effect ofβ-catenin.After transfection,wedelolactone(50 μM)was added and continued to be treated for 24 h,the cell viability was analyzed by CCK-8,apoptosis rate was detected by flow cytometry,and the expression level of apoptosis-related proteins were detected by western blot.3.The study on the anti-tumor effect of wedelolactone in CRC tumor-bearing nude mice.The transplantation tumor nude mice model was constructed by subcutaneous injection of HCT116 cells,and wedelolactone was given by gavage every three days.The status of nude mice was observed during the experiment,and the body weight and volume of transplantation tumor were measured.The histopathological changes in the vital organs of nude mice were observed by HE staining,cell apoptosis in the transplanted tumor tissues was analyzed by TUNEL staining,and the expression of proliferation-related protein Ki67,apoptosis-related protein and Wnt/β-catenin pathway-related protein(p-β-catenin(ser675),c-Myc and survivin)were detected by immunohistochemistry.Results:1.Wedelolactone inhibits proliferation and induces apoptosis in CRC cellsIn cell experiments,treatment with increasing concentrations(0-100 μM)and time(0-48 h)of wedelolactone,the cell viability of HCT116 cells was gradually decreased in a dose-and time-dependent manner.In contrast,within the effective concentration range of wedelolactone,it had no significant cytotoxicity to CCD841 Co N cells.Based on the above results,40,50 and 60 μM wedelolactone were subsequently selected to treat HCT116 cells for 24 h for the following experiment.Compared with the control group,after the treatment with wedelolactone,HCT116 cells gradually became rounded and the cell proliferation ability decreased with the increasing of the concentration.Meanwhile,wedelolactone induced apoptosis in HCT116 cells in a dose-dependent manner,and this induction could be reversed by the apoptosis inhibitor Z-VAD-FMK.Detection of apoptosis-related proteins revealed that wedelolactone could up-regulate the expression of Cleaved PARP,Cleaved caspase-3 and Bax,and down-regulate the expression of Bcl-2.2.Wedelolactone inhibits proliferation and induces apoptosis by targeting β-catenin in HCT116 cells.The transcriptome sequencing results of HCT116 cells treated with wedelolactone showed that there were 2767 differential genes,of which 1457 genes were up-regulated and 1310 genes were down-regulated.KEGG pathway enrichment analysis suggested that the anti-tumor effect of wedelolactone on CRC cells was related to Wnt signaling pathway.After treatment with wedelolactone,the expression of total β-catenin,which is the key protein of Wnt signaling pathway,was not significantly changed,but the expression of p-β-catenin(ser675)was significantly down-regulated.Meanwhile,wedelolactone could significantly down-regulate the m RNA and protein levels of c-Myc and survivin.The results of molecular docking were suggested that wedelolactone could directly bind to β-catenin,and the conformation of the complex formed by them was relatively dynamic and stable.In addition,wedelolactone could increase the thermal stability of β-catenin protein,indicating that wedelolactone interacts with β-catenin in intact cells.The overexpression plasmid of β-catenin was successfully constructed,and transient transfection could effectively up-regulate the expression of β-catenin.β-catenin overexpression counld significantly reverse the proliferation inhibition and apoptosis induction of HCT116 cells after wedelolactone treatment.Compared with the group treated with wedelolactone alone,the expression of Bax,cleaved caspase-3 and cleaved PARP was significantly decreased,and the expression of Bcl-2 was significantly increased.3.Evaluation of the anti-tumor effect of wedelolactone in HCT116 human CRC tumor-bearing nude mice.Wedelolactone was able to inhibit the growth of transplanted tumors of CRC cells,and had no significant effects on body weight and vital organs such as heart,liver and kidney in nude mice.The expression of Ki67 in transplanted tumor tissues was down-regulated and the apoptosis was up-regulated in the wedelolactone-treated group.Compared with the control group,the expression of Bax,Cleaved caspase-3 and Cleaved PARP were significantly increased and the expression of Bcl-2 was significantly decreased in the transplanted tumor tissues after treatment with wedelolactone.In addition,wedelolactone could also significantly down-regulate the expression of p-β-catenin(ser675),c-myc and survivin.Conclusion:1.In vitro experiments showed that wedelolactone significantly inhibited proliferation and induced apoptosis in CRC cells,while exhibited relatively no obvious toxicity to normal colonic epithelial cells.2.Wedelolactone may exert anti-CRC effects by directly binding to β-catenin and inhibiting β-catenin activity,significantly down-regulating the expression of its downstream target genes.3.In vivo experiments confirmed that wedelolactone significantly inhibited the growth of CRC transplantation tumors and induced apoptosis of tumor cells,but had no significant toxic effects on nude mice,and its mechanism of action may involve the inhibition of Wnt/β-catenin signaling pathway. |
PDF Full Text Request