NPRL2 Enhance Sensitivity To Chemotherapy Drugs And Mechanism Of Action Studies In Colon Cancer Cells

Background and PurposeColorectal cancer(CRC)ranks third in worldwide cancer incidence,while the mortality in various malignant tumor disease ranks second place,the trend of morbidity and mortality increased in recent years.Among the chemotherapeutic compounds used for the treatment of CRC,chemotherapy drugs like oxaliplatin(L-OHP)and 5-fluorouracil(5-FU)improves colorectal cancer survival.However,some patients are resistant to treatment with these chemotherapy drugs.This is the major obstacle of the curative effect of chemotherapy and prognosis,so to explore a new therapeutic strategy has important significance for improving the clinical curative effect of treatment.The nitrogen permease regulator like-2(NPRL2)gene is a candidate tumor suppressor gene that resides in a 120-kb homozygous deletion region on chromosome 3p21.3.NPRL2 has a high degree of amino acid sequence homology,and its inactivation may promote tumorigenesis.The aims of this study were to explore the mechanism by which NPRL2 affects L-OHP and 5-FU sensitivity and to determine its clinical significance in CRC.MethodsThe colon cancer cell line HCT116 was used for lentiviral-mediated transduction of the NPRL2 gene.Cytotoxicity and apoptosis were assessed using the Cell Counting Kit-8(CCK-8)assay and flow cytometry(FCM),respectively.FCM was used to examine changes in cell cycle distribution,the rate of apoptosis and the expression of membrane cluster differentiation antigen 24(CD24).Phosphatidylinositol 3-kinase(PI3K)/Akt/ mammalian target of rapamycin(m TOR)network proteins were extracted,and western blot analysis was performed to examine the expression of related proteins.ResultsLentiviral-mediated transduction of the NPRL2 gene successfully transfected in HCT116 cells,the expression of NPRL2 was significantly increased in cells after transfection.NPRL2 over-expression increased the chemotherapy drugs(L-OHP and 5-FU)sensitivity of HCT116 cells.The IC50 of chemotherapy drugs was decreased in cells transduced with NPRL2 compared with negative control(NC)cells(P<0.05),and the NPRL2-mediated chemotherapy drugs sensitivity was time dependent.The cell cycle was arrested in G1 phase,and there was a remarkable decrease in the S phase population after NPRL2-transduced cells treated with chemotherapy drugs(P<0.01).FCM revealed an increase in apoptotic cells in NPRL2-transduced cells treated with chemotherapy drugs compared with NC cells(P<0.01).NPRL2 over-expression led to the down-regulation of CD24,which could significantly reduce tumor invasiveness by reducing the metastatic capacity of HCT116 cells(P<0.01).NPRL2 over-expression enhances chemotherapy drugs sensitivity by down-regulating the functions of the PI3K/Akt/m TOR network.Furthermore,chemotherapy drugs up-regulated caspase-3 and caspase-9 expression to promote remarkable apoptosis in NPRL2-transduced cells compared with negative control cells,NPRL2-transduced cells,or cells treated with chemotherapy drugs(P<0.01).ConclusionsNPRL2 have obvious inhibitory effect on HCT116 cells.NPRL2 down-regulate PI3K/Akt/m TOR signaling pathways,which could increase the rate of apoptosis,make the stagnation of the cell cycle at G1 phase,lower expression of CD24 and reduces the ability of invasion and metastasis of the tumor,so as to improve the sensitivity of the colorectal cancer cells to chemotherapy drugs.This may become a new colorectal cancer targeted therapy strategies and a new way to improve the effect of tumor treatment.