| BackgroundColorectal cancer(CRC)is one of the most common malignant tumors in the human digestive system.Due to the insidious clinical symptoms in early patients,tumor metastasis usually occurs at the first diagnosis.Surgery and chemoradiotherapy are the main methods for the treatment of colorectal cancer.However,because of the trauma,risk and limitations of surgical resection,systemic toxic side effects and drug resistance of chemotherapy,and local normal tissue damage caused by radiotherapy,the treatment effect and prognosis of colorectal cancer are still not ideal.In recent years,the anticancer and immunomodulatory effects of traditional Chinese herbal medicine and its active components have gradually attracted people’s attention.Lupeol is a pentacyclic triterpenoid derived from lupine,dandelion and other herbs.Lupeol has anti-inflammatory,antioxidant,anti-infection,heart protection,liver protection,nerve protection and immunity regulation effects,suggesting that lupeol has different pharmacological activities and multiple targets.Lupeol has a good therapeutic effect on a variety of tumors,safe doses and protective effects on body organs.However,lupeol is a lipid-soluble drug with low water solubility and insoluble in organic solvents,which limits the study of its anti-tumor activity and hinders its development to clinical use.ObjectiveTo improve the solubility of lupeol and increase its anticancer activity,the chemical structure of lupeol was modified in the early stage of this study,and a quaternary ammonium salt derivative of lupeol pyridine(lupeol derivative LD-1)which was easily soluble in dimethyl sulfoxide(DMSO),ethanol and other organic solvents were obtained.In this study,the antitumor activity of lupeol derivative LD-1 was clarified by in vivo and in vitro experiments,and its mechanism of action was further explored,so as to provide a theoretical and experimental basis for the development of clinical drugs for the treatment of colorectal cancer.MethodsDifferent concentrations of LD-1 were used to treat tumor cells in vitro.CCK-8 assay,clonogenesis assay,wound healing assay,transwell assay and flow cytometry were used to evaluate the effects of LD-1 on tumor cell proliferation,clonogenesis ability,migration and invasion ability,and the effect of LD-1 on cell apoptosis.Subcutaneous tumorigenesis in nude mice was established to verify the antitumor effect of LD-1 in vivo.The potential molecular mechanism of LD-1 inhibition of HCT116 cells was further analyzed by Tandem Mass Tags(TMT)quantitative proteomic analysis technology and bioinformatics technology,and the expression of related proteins was verified by western blotting.Results1.LD-1 could inhibit the proliferation of colorectal cancer,renal cancer,nasopharyngeal cancer,liver cancer and other cell lines,among which,it has the strongest inhibitory effect on colorectal cancer HCT116 cell lines,and LD-1 has little toxicity on a variety of normal cell lines.2.LD-1 significantly inhibited the clonogenic ability,migration ability,invasion ability and induced apoptosis of HCT116 cells.3.LD-1 at low,medium and high concentrations could significantly inhibit the growth of subcutaneous xenografts in nude mice.4.TMT analysis showed that compared with the control group,a total of 195 differentially expressed proteins were found in the lupeol derivative LD-1 group,including 128 up-regulated proteins and 67 down-regulated proteins.The results of biological information analysis suggested that LD-1 may inhibit the proliferation and metastasis of HCT116 cells through HIF-1 signaling pathway.5.Western blotting results confirmed that LD-1 could inhibit the proliferation and metastasis of HCT116 cells by inhibiting the expression of HIF-1α,HK2,ENO1 and LDHA proteins in the HIF-1 signaling pathway.ConclusionLupeol derivative LD-1 could inhibit the growth of colorectal cancer in vitro and in vivo by interfering with the HIF-1 signaling pathway and had low toxicity to a variety of normal cell lines,which is expected to become a new anticancer drug. |
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