Decreased chemosensitivity of platinum in human colon cancer cells by overexpression of Golgi phosphorylation protein 3 and possible mechanisms

Abstract

Objective To investigate the influence of overexpression of Golgi phosphorylation protein 3 (GOLPH3) gene on the chemosensitivity of Cisplatin in human colon cancer HT29 cell line. Methods HT29 cells were divided into four groups: control group, small interfering RNA (siRNA) transfection group, experimental group 1 (10 μmol/L Cisplatin), and experimental group 2 [small interfering RNA (siRNA)-GOLPH3+ 10 μmol/L Cisplatin]. The silencing effect of GOLPH3 gene was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. The tumor cell proliferation and formation ability of HT29 cells were measured respectively by methyl thiazol tetrazolium (MTT) assay and tumorsphere formation assay. Annexin V-fluoresceine isothiocyanate (FITC)/propidium iodide (PI) flow cytometry was used to detect the apoptosis of each group. The protein expression levels of GOLPH3, permeability glycoprotein (P-gp) and β-catenin in HT29 cells were detected by Western blotting. Sixteen nude mice were randomly divided into 4 groups (4 mice in each group): control group, transfection group, experimental group 1 and experimental group 2. The corresponding colon cancer cells were injected subcutaneously into the axillary tissue. The tumor formation in nude mice was observed within 30 days. Results The relative expression level of GOLPH3 mRNA and protein in the transfection group was significantly lower than in the control group (0.162±0.062 vs. 1.002±0.223; 0.099±0.112 vs. 1.003±0.094) (P<0.01). The cell apoptosis rate in the transfection group was significantly higher than in the control group [(15.520±2.921)% vs. (1.843±1.298)%, P<0.01]. Under cisplatin treatment, the A value (0.746±0.085) and the tumorsphere number (212.800±30.380) in the experimental group 1 were significantly higher than those in the experimental group 2 (0.236±0.071 and 30.750±14.500) (P<0.01). Annexin V-FITC/PI flow cytometry showed that the apoptosis rate of the experimental group 1 was significantly lower than that of the experimental group 2 [(23.890±6.363)% vs. (59.400±2.392)%, P<0.01]. The volume of the axillary subcutaneous tumor in the siRNA transfection group, the experimental group 1 and the experimental group 2 was significantly lower than that in the control group [(1.738±0.121), (1.573±0.224), (0.625±0.189) mm3 vs. (2.083±0.110) mm3,P<0.05 for all]. In the experimental group 2, the tumor volume of the nude mice was significantly less than that in the experimental group 1 in the same period after 15 days (P<0.01). Compared to the control group, the expression levels of GOLPH3, β-catenin and P-gp in the experimental group 1 were significantly upregulated (1.000±0.173 vs. 2.734±0.440, 1.000±0.078 vs. 2.472±0.444, and 1.014±0.346 vs. 4.269±0.454) (P<0.01), and the expression levels of these proteins in the experimental group 2 were significantly down-regulated in the experimental group 1 (0.249±0.084 vs. 2.734±0.440, 0.993±0.052 vs. 2.472±0.444, and 1.842±0.383 vs. 4.269±0.454) (P<0.01). Conclusion The overexpression of GOLPH3 can decrease the chemosensitivity of Cisplatin in colon cancer HT29 cells by activating Wnt/β-catenin signal pathway. Key words: Golgi phosphorylation protein 3; Colon cancer cell; Wnt/β-catenin signal pathway; Chemosensitivity; Cisplatin