| Objective:Ficus carica Linn is an ancient plant with rich edible and medicinal properties.We aimed to summarize the understanding of colorectal cancer in Chinese medicine,as well as the ancient and modern application of Ficus carica Linn through theoretical research.Through experimental research,the anti-colon cancer effect and mechanism of psoralen,one of the main antitumor components of Ficus carica Linn,were observed.Finally,the effects of psoralen on tumors were discussed based on tumor organoid culture techniques.Methods:1.Theoretical research: summarized the understanding of colorectal cancer in traditional and modern Chinese medicine by consulting ancient books and modern literature,such as etiology,pathogenesis and differential treatment.The ancient and modern applications of Ficus carica Linn(such as fruits,leaves and latex,etc.)were also summarized,especially the active components of Ficus carica Linn and their pharmacological effects.2.Experimental research:(1)HCT116 and HT29 colon cancer cells were selected.Psoralen stock solution was prepared by DMSO.(2)Different concentrations of psoralen(5μM,10μM,20μM,40μM,80μM)were used to act on colon cancer HCT116 and HT29 cells for 24 h,48h,72 h,and the effects of psoralen on cell survival were detected by CCK-8 test.Based on the results of CCK-8 test,the appropriate concentrations were selected for subsequent experiments.(3)Different concentrations of psoralen(10μM,20μM,40μM)were used to act on colon cancer HCT116 and HT29 cells for 24 h,and the effects of psoralen on the proliferation of colon cancer cells were observed by plate cell clone formation and Ed U.(4)Different concentrations of psoralen(10μM,20μM,40μM)were used to act on colon cancer HCT116 and HT29 cells for 24 h.The effects of psoralen on the migration and invasion ability of colon cancer cells were detected by scratch test and Transwell.(5)Different concentrations of psoralen(10μM,20μM,40μM)were used to treat colon cancer HCT116 and HT29 cells for 24 h,and the effects of psoralen on apoptosis and mitochondrial membrane potential of colon cancer cells were observed by flow cytometry.(6)Colon cancer HCT116 and HT29 cells were treated with different concentrations of psoralen(10μM,20μM,40μM)for 24 h.The effects of psoralen on the expression of proteins such as invasion,migration(E-cadherin,N-cadherin,Vimentin,MMP2,MMP9)and apoptosis(Bcl-2,Bax,PARP,Caspase-3,cleaved Caspase-3,Caspase-9,cleaved Caspase-9)of colon cancer cells were detected by western blotting.(7)The target proteins directly acting on psoralen were obtained using the Swiss Target Prediction website.The TCGA-COAD data were used to enrich the GSEA signaling pathway.The signaling pathways that psoralen could target in colorectal cancer were analyzed.Finally,The TNF-α/NF-κB signaling pathway was selected for the further mechanism study.(8)Different concentrations of psoralen(10μM,20μM,40μM)were applied to colon cancer HCT116 and HT29 cells for 24 h.The expression levels of key proteins of TNF-α/NF-κB signaling pathway,TNF-α,P65 and p-P65,were detected by western blotting.(9)Based on the preliminary work of our research group,tumor organoids with stable construction were selected as the experimental model.Different concentrations of psoralen(80μM,160μM,320μM)were applied to a variety of patient-derived tumor organoids,and drug sensitivity experiments were performed to calculate the tumor organoid growth inhibition rate.Results:1.Theoretical research:(1)There was no specific name for colorectal cancer in traditional Chinese medicine.According to the symptoms,it probably belonged to the category of “intestinal accumulation”,“accumulation”,“intestinal qi”,“intestinal fetish”,“intestinal knot”,“intestinal swimming”,“intestinal wind”,“diarrhea”,“lower blood”,“lock tuberculosis”and “dirty poison”.In terms of etiology,pathogenesis and dialectical treatment,traditional Chinese medicine and modern Chinese medicine held similar views.Most of them believed that due to the deficiency of healthy qi,external cold and evil feeling,damage to the spleen and stomach mobilization function,containing humidifying heat,humid heat evil bet on the large intestine.Finally,poor flow of qi and blood,humid heat stasis for a long time and then formed stubborn tumors.The symptoms could be roughly divided into five types: moist heat and intestines,stasis inner node,qi and blood deficiency,spleen and kidney deficiency and liver and kidney deficiency.In terms of treatment,in early stage,more evil facts were mainly used to dispel evil spirits.In later stage,more false evidence or false and real mixed with real evidence,mainly to support the righteous.(2)Ficus carica Linn was a traditional mulberry plant with a long history,which was considered sweet,flat and non-toxic in traditional medicine.It could be used to treat thirst quenching,cough,asthma,hemorrhoids and other diseases.Modern studies had shown that their fruits,peels,leaves and roots contained a variety of active components,such as flavonoids,organic acids,phenols,enzymes,amino acids,fatty acids,soluble fiber and trace elements,etc.,with anti-inflammatory,antioxidant,immune regulation,antitumor,digestion,antimicrobial and parasitic effects.From this point of view,Ficus carica Linn was a safe,edible and medicinal food.At present,there were few studies on the antitumor mechanism of Ficus carica Linn bioactive components.In future,researchers should focus on the antitumor mechanism of Ficus carica Linn bioactive components.2.Experimental research:(1)Different concentrations of psoralen(5μM,10μM,20μM,40μM,80μM)were used to act on colon cancer HCT116 and HT29 cells,and the results showed that psoralen inhibited the survival of colon cancer cells(P<0.05)in a concentration-dependent manner.The IC50 of 24 h,48h and 72 h were 60.3μM,31.8μM,21.3μM,52.4μM,32.2μM and23.4μM,respectively.Therefore,10μM,20μM,and 40μM concentration gradients were used for subsequent experiments.(2)The results of plate cell clone formation and Ed U showed that psoralen significantly inhibited the clone formation and DNA replication ability of colon cancer HCT116 and HT29 cells(P<0.05).(3)The results of scratch test and Transwell showed that with the increase of concentration,psoralen significantly inhibited the migration(P<0.01)and invasive ability(P<0.0001)of colon cancer HCT116 and HT29 cells.(4)The results of flow cytometry showed that psoralen induced apoptosis of colon cancer HCT116 and HT29 cells(P<0.05).When the concentrations of psoralen were20μM and 40μM,the mitochondrial membrane potential could be significantly reduced(P<0.05).(5)Western blotting results showed that psoralen significantly inhibited the expression of N-cadherin,Vimentin,MMP2 and MMP9 in colon cancer HCT116 and HT29 cells,and enhanced the expression of E-cadherin,P<0.05.In addition,psoralen also significantly inhibited the expression of Bcl-2 anti-apoptotic protein and enhanced the expression of Bax,PARP,cleaved Caspase-3 and cleaved Caspase-9 apoptotic protein,P<0.05.(6)Network pharmacology results showed that the effect of psoralen against colorectal cancer was related to multiple signaling pathways,such as inflammatory response,interferon γ response,hypoxia,epithelial-mesenchymal transition,P53,TNF-α/NF-κB and other signaling pathways.(7)According to the results of network pharmacology,the TNF-α/NF-κB signaling pathway was selected for the next mechanism study.The results of western blotting showed that psoralen significantly inhibited the expression of TNF-α and p-P65 in colon cancer HCT116 and HT29 cells(P<0.05),indicating that psoralen could achieve antitumor effects by inhibiting the TNF-α/NF-κB signaling pathway.(8)Psoralen had different degrees of inhibitory effect on the growth of tumor organoids in 237# rectal cancer,244# sigmoid colon cancer,325# gastric cancer,346#gastric cancer,386# lung cancer,407# ascending colon cancer,413# cholangiocarcinoma,and 421# breast cancer.At the concentration of 320μM,the growth inhibition rates of tumor organoids were 98.2%,-81.5%,55.0%,-19.7%,-19.7%,-3.4%,19.7%,-197.5%,and-3.0%,respectively.237# rectal cancer,325# gastric cancer and 407# ascending colon cancer showed positive inhibitory effect under this concentration gradient.244# sigmoid colon cancer,346# gastric cancer,386# lung cancer,413# cholangiocarcinoma,421#breast cancer organoids showed negative inhibition.However,according to dynamic observations,with the increase of concentration,the growth rate of organoids slowed down.This indicated that different tumor species organoids or different individual-derived organoids of the same tumor species had different drug sensitivity to psoralen,which also reflected the tumor heterogeneity of patients.Conclusion:According to the symptoms,colorectal cancer probably belonged to the category of“intestinal accumulation”,“accumulation”,“bowel disease”,“bowel addiction” and other diseases.Traditional Chinese medicine and modern Chinese medicine mostly believed that colorectal cancer was due to the deficiency of healthy qi,external cold and evil,damage to the spleen and stomach mobilization function,containing humidification heat,humid heat evil bet on the large intestine,poor flow of qi and blood,and stubborn tumor body formed by humid heat stasis for a long time.It can be divided into five types: moist heat and intestines,stasis inner node,qi and blood deficiency,spleen and kidney deficiency and liver and kidney deficiency.In the early stage,more evil facts were mainly used to dispel evil spirits,and in the later stage,more false evidence or mixed with false and real were mainly supported by righteousness.Both traditional Chinese medicine and modern pharmacological research had shown that Ficus carica Linn was a plant with great edible and medicinal properties.This study showed that psoralen,the main antitumor component of Ficus carica Linn,could inhibit the TNF-α/NF-κB signaling pathway,inhibit the proliferation,invasion and migration of colon cancer HCT116 and HT29 cells,and induce apoptosis of tumor cells.In addition,homogeneous tumor cell lines and tumor organoids derived from different tumors showed different drug susceptibility to psoralen,which may be due to the heterogeneity of the patient’s tumor.In future,the factors affecting the sensitivity of tumor organoids to psoralens need to be further analyzed... |
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