The Role And Underlying Mechanism Of Cucurbitacin E On Chemosensitization Of Colon Cancer Cells

Colorectal cancer(CRC)is one of the most common malignant tumors worldwide.It is estimated that 1.8 million new cases occur each year,and 880 thousand people die of CRC.5-fluorouracil(5-FU)and oxaliplatin(L-OHP)are first-line chemotherapeutic drugs for the treatment of CRC.Chemotherapy resistance has been the biggest obstacle to tumor treatment,which limits the clinical application of chemotherapeutic drugs seriously.Natural products are valuable sources for screening and developing compounds with anticancer activity because of their structural diversity and minimal side effects.Therefore,looking for active small molecules from natural products to improve chemosensitivity is a significant way to overcome chemotherapy resistance.Cucurbitacin E(CE)is a kind of natural highly oxidized tetracyclic triterpenoid with a variety of biological activities,mainly found in cucumbers,melons,watermelons,pumpkins and other cucurbitaceous plants.It is also found in higher plants such as Cruciferae and some macro fungi.At present,CE has been proved to possess immunomodulatory,anti-inflammation,liver protection,anti-diabetes and anti-tumor effects.The latest studies have showed that cucurbitacin B can not only induce cancer cell apoptosis and inhibit tumor growth,but also reverse tumor drug resistance.However,CE is an important member of the cucurbitacin family,and the role CE in regulating chemotherapy resistance of CRC is still unclear.In this study,CE was used in combination with chemotherapeutic drugs to explore the effect of CE in regulating the sensitivity of CRC cells to chemotherapy.At the same time,magnetic nanoparticles were used to identify the intracellular direct target protein of CE,and we further clarify the molecular mechanism of CE-induced senescence to enhance the sensitivity of CRC cells to chemotherapy.The research contents of this study are as follows in three parts:1.MTT assay showed that CE combined with chemotherapeutic drugs inhibited the viability of CRC cells.The inhibitory effect of CE combined with chemotherapeutic drugs on colony growth of CRC cells was detected by clonogenic formation assay.The flow cytometry about Annexin V/PI staining showed that CE combined with chemotherapeutic drugs could enhance L-OHP-induced apoptosis of CRC cells.The effect of CE on intracellular accumulation of Rh123 was detected by high performance liquid chromatography(HPLC),and it was found that CE could significantly inhibit the drug efflux function of CRC cells.Moreover,the results of q PCR and Western blot further showed that CE could reduce the m RNA and protein levels of multidrug resistance proteins ABCC1 and MDR1.These results suggest that CE increases the sensitivity of CRC cells to chemotherapeutic drugs via down regulation of ABCC1 and MDR1.2.Solid-phase solid phase fishing experiments were carried out by using the synthesized CE-functionalized magnetite nanoparticles.The intracellular binding proteins of CE were analyzed by proteomics.The results of Gene Ontology showed that the intracellular binding protein of CE was enriched in Wnt/?-catenin signal pathway.The results of Western blot,q PCR and immunofluorescence staining showed that CE combined with 5-FU could significantly reduce the expression level of ?-catenin.After knockdown or overexpression of ?-catenin in CRC cells,it was found that overexpression of ?-catenin could inhibit the chemosensitivity mediated by CE.In contrast,knockdown of ?-catenin could enhance the chemosensitivity mediated by CE.In addition,the results of MTT,immunofluorescence staining,Western blot and flow cytometry further showed that CE could promote the killing effect of chemotherapeutic drugs on drug-resistant cells.Transcription factor activating enhancer-binding protein 4(TFAP4)is one of the intracellular binding proteins of CE.The combination of CE and 5-FU can significantly reduce the expression of TFAP4.Overexpression of TFAP4 abrogated the down-regulation of ?-catenin,ABCC1 and MDR1 proteins mediated by the combined treatment with CE and 5-FU,and significantly decreased the chemosensitivity to CRC cells.Therefore,CE sensitized CRC cells to the chemotherapeutic drugs that rely on the suppression of TFAP4/Wnt/ ?-catenin signaling.3.The results of MTT,clonogenic formation and Ed U assay showed that high dose of CE could significantly inhibit the proliferation of HCT-116 and DLD1 cells.Trypan blue staining showed that low dose of CE had no effect on the proliferation of CRC cells.Low dose of CE can increase the activity of ?-galactosidase and cause the senescence of CRC cells.CE significantly inhibited the expression of TFAP4,p Rb and Cyclin D1,and promoted the expression of senescence-associated genes such as p16,p21 and p53.Improtantly,the senescence of CRC cells caused by CE can be reversed by overexpression of TFAP4.Further studies found that CE-mediated senescence of CRC cells could be selectively eliminated by resveratrol rather than control cells.In addition,CE treatment causes little senescence of normal hepatocytes HL-7702,and resveratrol had no effect on the proliferation of HL-7702 cells treated with CE.In summary,CE enhances the sensitivity of CRC cells to chemotherapeutic drugs by inhibiting TFAP4/Wnt/?-catenin signaling pathway,while CE selectively induces senescence of CRC cells by targeting inhibition of TFAP4 expression and up-regulation of senescence-associated genes.Meanwhile,resveratrol can selectively kill senescent CRC cells and promote the response of CRC to chemotherapy.The results of this study reveal that CE has a certain application potential in chemosensitization of CRC,and also provides new strategies and opinions for the treatment of CRC.