Caffeic Acid Phenethyl Ester Inhibits The Growth Of U87 Glioma Cells In Vitro And In Vivo

Background:Glioblastoma is the most malignant tumor in the central nervous system,with an extremely unfavorable prognosis.Improving the treatment efficacy and quality of life for glioblastoma patients has been a hot topic among scholars worldwide.In recent years,the anti-tumor effects of traditional Chinese medicine have attracted attention from researchers.Caffeic acid phenethyl ester(CAPE),an active polyphenolic compound derived from traditional Chinese medicine propolis,has been proven to exhibit anti-cancer effects in various central nervous system tumors,including glioblastoma,due to its ability to cross the blood-brain barrier and cause significant toxicity to tumor cells while having no significant impact on normal cells.However,the anti-tumor mechanism of CAPE is not yet clear.The Wnt/β-catenin signaling pathway is one of the important dysregulated signaling pathways in glioblastoma,and its activation is closely related to the pathogenesis of glioblastoma,including proliferation,apoptosis,invasion,and other biological processes.Due to its complexity,the regulatory mechanism of the Wnt/β-catenin signaling pathway has not been clearly elucidated,but it has been found that the combination of β-catenin and the transcription factor TCF to form a complex and translocate into the cell nucleus is the initiating factor for pathway activation,while the expression of downstream target genes is a key factor in the occurrence and development of tumors.Additionally,research has found that CAPE can inhibit the activation and regulation of downstream target genes c-myc and VEGF in U251 cells via the Wnt/β-catenin signaling pathway,thus playing a role in inhibiting glioblastoma.Connective tissue growth factor(CTGF),as a downstream target gene of the Wnt/p-catenin signaling pathway,is involved in fibrotic diseases,childhood bronchial dysplasia,and other diseases,but research on its relevance to glioblastoma is lacking.Therefore,this study selected human glioblastoma cell line U87 as the research object and constructed a subcutaneous tumor model in nude mice.Through relevant experiments such as CCK8,flow cytometry apoptosis detection,HE,and WB detection,the anti-glioblastoma effects and mechanisms of CAPE in vivo and in vitro were investigated.Purpose:1.To clarify the effects of CAPE on the morphology and biology of U87 glioma cells.2.To observe the in vivo anti-glioma effect and relative safety of CAPE after oral administration.3.To verify whether CTGF can participate in glioma progression through mediating Wnt/β-catenin signaling pathway.4.To explore whether CAPE can inhibit the in vivo and in vitro growth of U87 glioma cells by downregulating CTGF expression through regulating Wnt/β-catenin pathway.Materials and Methods:1.In vitro experiments:CCK8 assay was used to detect the effect of CAPE on the proliferation of U87 glioma cells.Flow cytometry was used to detect the positive effect of CAPE on inducing apoptosis of U87 glioma cells.WB was used to detect the expression of Wnt/β-catenin signaling pathway-related proteins after CAPE acted on U87 glioma cells.2.In vivo experimental part:12 male nude mice aged 4 weeks and weighing about 20 g were used U87 cells were injected subcutaneously into the right axilla to construct a subcutaneous tumor model.When the tumor grew to about 100mm3,the mice were randomly divided into control group and experimental group,with 6 mice in each group.The experimental group was given 20mg/kg CAPE dissolved in 0.1ml organic solvent by gavage,while the control group was given the same organic solvent by gavage,once a day.A caliper was used to measure the length and width of the tumor.The tumor volume was calculated by the tumor formula,twice a week,to evaluate the effect of CAPE on tumor growth.Body weight was monitored once a week to evaluate the safety of the drug.After 3 weeks of experimental intervention,the mice in each group were euthanized,and the tumor tissues were taken.HE staining was used to observe the pathological changes of CAPE on the tumor tissues.WB was used to detect the expression of Wnt/β-catenin signaling pathway-related proteins after CAPE acted on the tumor-bearing mice orally.Results:1.CCK8 proliferation assay showed: CAPE could inhibit the proliferation of U87 glioma cells in a concentration and time-dependent manner within a certain concentration range.2.Flow cytometry apoptosis assay showed: Within a certain concentration range,the apoptosis rate of U87 glioma cells increased with increasing CAPE concentration.3.WB detection of Wnt/β-catenin pathway-related proteins in cells showed:Compared with the control group,the expression of β-catenin,TCF4,and CTGF proteins was significantly decreased in the experimental group,and the difference was statistically significant(p<0.05).4.Subcutaneous tumor volume measurement results showed: The volume of the experimental group was significantly smaller than that of the control group from the16 th day of drug intervention,and the difference was statistically significant(p<0.05).5.HE results showed: The area of tumor necrosis in the tumor tissue of the experimental group given 20mg/kg gavage was significantly increased compared with the tumor tissue of the control group,and apoptosis and fragmentation of tumor cells in the tissue were obvious.6.WB detection of Wnt/β-catenin pathway-related proteins in tumor tissues showed: Compared with the control group,the expression of β-catenin,TCF4,and CTGF proteins was significantly decreased in the experimental group,and the difference was statistically significant(p<0.05).