| With an estimate of 104,270 new cases and 52,980 deaths in 2021 according to the American Cancer Society,colorectal cancer(CRC)is one of the most prevalent malignancies with a high mortality,ranking third in all cancers of either sex.Although the enormous effort has been made on early screening or detection,surgical resection and chemotherapy those years to make a great extent improvement of CRC treatment,the 5-year survival rate of patients with advanced CRC remains less than 10%.Currently,there still lacks optimal therapeutic approaches for CRC,and chemotherapy in combination with anti-EGFR drugs(zivaflibercept,ramucirumab,regorafenib and bevacizumab)is generally adopted as the first-line treatment of metastatic CRC.However,despite those drugs designed to repress tumor progress successfully,they can also bring about severe toxic effects on normal tissues and even disrupt therapeutic efficiency and life quality of patients.Hence,the new and effective strategies are urgently required to ameliorate CRC treatment and prognosis.Epithelial-mesenchymal transition(EMT)is described as a process whereby non-motile epithelial cells obtain mesenchymal characteristics such as loss of cell polarity and adhesion as well as acquirement of motility and invasiveness,thereby leading to cytoskeletal remodeling and morphological changes.A numerous works have determined that EMT,regulated by various molecules and pathways,plays a critical part in facilitating metastasis comprising invasion and migration of diverse tumors including CRC.Therefore,investigation on EMT and relevant mechanisms may provide some helpful ideas for CRC management.As the essential source of bioactive natural products,traditional Chinese medicine,used for a long history,contributes to modern medicine with unique advantages.Sophoridine is an active quinolizidine alkaloid component extracted from Radix Sophorae Flavescentis fulfilling multiple pharmacological functions,which shows a variety of biological effects like anti-inflammatory,anti-fibrotic,antimicrobial,antiviral and anticancer activities or so.The protective role of sophoridine against human CRC has been revealed in a previous study.Nevertheless,whether and how it acts on EMT in CRC has not been expounded yet.Belonging to cyclin family,CCND1 intimately connects with the modulation of cell cycle and it has been proven to have a relationship with EMT in breast and nasopharyngeal carcinoma.ZO-1 is a main cytoplasmatic protein related to tight junction which serves as a hallmark of epithelial cell-cell junction.Someone once proposed that sophorodine is another major component of Sophora flavescens,which can upregulate ZO-1 levels in patients with encephalomyelitis.Moreover,it has been found that the down-regulation of ZO-1 is accompanied by the upregulation of CCND1 and able to promote EMT.Through bioinformatics tools,we discovered that both of CCND1 and ZO-1 has been predicted as the targets of sophoridine.Thus,this paper tried to explore whether sophoridine targeted ZO-1/CCND1 pathway to mediate EMT in CRC.At the same time,we successfully cultivated organoid models of colorectal cancer from different patient sources using 3D methods,and tested the drug efficacy of different concentrations of sophoridine,oxaliplatin,5-FU,gemcitabine,and paclitaxel.We compiled drug sensitivity curves and measured the IC50 values of the drugs.Objective:The exact mechanism of action of sophoridine on epithelial mesenchymal transition(EMT)in colorectal cancer(CRC)is not yet clear.Observing the effect of sophoridine on the biological behavior of colorectal cancer(CRC)cells and revealing its role in colorectal cancer(CRC).Simultaneously,construct an vitro colorectal cancer research model that is closer to the human body in clinical practice,and use this model for drug sensitivity testing.Methods:Potential targets of sophoridine and relevant biological functions of targets were analyzed through bioinformatics.CRC cells were treated with sophoridine or transfected with short hairpin RNA Cyclin D1(shCCND1)/Zonula occludens-1(ZO-1)overexpression plasmid.The expressions of epithelial-mesenchymal transition(EMT)-related factors(ZO-1,vimentin,snail,α-SMA,E-cadherin)and molecules associated with Wnt signaling pathway(CCND1,β-catenin)were determined by quantitative reverse transcription-polymerase chain reaction(qRT-PCR)and Western blot.Cell migration and invasion were tested through wound healing and Trans well assays.CRC tumorigenesis was assessed by xenograft construction in vivo.Simultaneously,fresh tumor specimen tissues from patients undergoing colorectal cancer surgery were selected to construct colorectal cancer organoids using 3D culture method.Determine the inhibitory effect of drugs on colorectal cancer organoids using chemiluminescence method,draw a semi inhibitory concentration curve,and preliminarily establish a drug sensitivity database.Results:CCND1 and ZO-1 were predicted as targets of sophoridine.Sophoridine restrained migration,invasion and levels of vimentin,snail,α-SMA,CCND1 as well as β-catenin whereas promoted E-cadherin and ZO-1 expressions in CRC cells(p<0.05).ZO-1 overexpression and CCND1 knockdown inhibited CRC cell migration,invasion and expressions of vimentin,snail,α-SMA,CCND1 as well as β-catenin while advanced E-cadherin and ZO-1 levels in CRC cells(p<0.05).ZO-1 overexpression and CCND1 knockdown inhibited CRC tumor growth in mice(p<0.01).At the same time,through the detection of the colorectal cancer organoid model,we selected 5 drugs that all showed certain inhibitory effects on colorectal cancer organoids,and based on the drug sensitivity results,we plotted the drug sensitivity curve and detected the IC50 value.Conclusion:Sophoridine inhibited EMT,migration and invasion in CRC via ZO-1/CCND1 pathway.Colorectal cancer organoids have a good preclinical model,which helps to detect the effectiveness of anti-tumor drugs in preclinical settings. |
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