ABSTRACT
The novel protooncogene Golgi phosphoprotein (GOLPH)3 is overexpressed in a variety of tumor tissues and is associated with poor prognosis. The authors previously demonstrated that GOLPH3 gene is overexpressed in colorectal cancer tissues and promotes the proliferation of colonic cancer cells by activating the phosphatidylinositol3kinase/protein kinase B/the mammalian target of rapamycin and Wnt/ßcatenin signaling pathways. However, to the best of the authors' knowledge, if and how the GOLPH3 gene is involved in inducing resistance to colonic cancer chemotherapy has not been reported. In the present study, the association between the overexpression of the GOLPH3 gene and resistance of HT29 colonic cancer cells to 5fluorouracil (5FU) was investigated. Following confirmation of the effective silencing of the GOLPH3 gene, proliferation and apoptosis of colonic cancer cells were detected by MTT assay, colony formation assay and flow cytometry, and then the mechanism of GOLPH3induced resistance to 5FU chemotherapy in colonic cancer cells was investigated by western blotting. The results demonstrated that the expression of phosphorylated (p)glycoprotein and GOLPH3 was increased in HT29 cells following treatment with 5FU, which resulted in the development of drug resistance. Silencing GOLPH3 increased the sensitivity of HT29 cells to 5FU, reduced their tumorigenicity and partly reversed their resistance to 5FU. The expression of pextracellular signalregulated kinase (pERK)1/2 and ßcatenin was decreased, which indicated that its mechanism was associated with the activation of the mitogenactivated protein kinase/ERK and Wnt/ßcatenin signaling pathways. Therefore, GOLPH3 may be a potential, novel target for reversing chemotherapy resistance in colon cancer.
Subject(s)
Drug Resistance, Neoplasm/genetics , Fluorouracil/pharmacology , Gene Expression , Membrane Proteins/genetics , Signal Transduction/drug effects , Apoptosis/drug effects , Cell Proliferation/drug effects , HT29 Cells , Humans , TransfectionABSTRACT
OBJECTIVE: Overexpression of GOLPH3 in colorectal cancer tissue may promote cell proliferation and activate the Wnt signaling pathway. We investigated the correlation between GOLPH3 gene expression and the Wnt signaling pathway to explore the mechanism of the overexpression of GOLPH3 gene which promotes proliferation in human colon cancer cells. METHODS: We measured expression of GOLPH3 mRNA in the human colon cancer cell lines HCT116, HT29, SW480 and SW620 by RT-PCR, and the cells with the highest expression were selected and divided into four groups: negative control, GOLPH3 siRNA transfection (siRNA-GOLPH3), Akt inhibitor (Tricinbine), and glycogen synthase kinase (GSK)-3ß inhibitor (TWS119). After human colon cancer cells were transfected with siRNA-GOLPH3, we used RT-PCR to investigate the silencing effect of GOLPH3 gene. We assessed the activity of the Wnt signaling pathway in all groups using the Topflash method. Proliferation and apoptosis of colon cancer SW620 cells were detected by MTT assay, colony formation assay and flow cytometry. Expression of Golgi phosphoprotein (GOLPH)3, ß-catenin, GSK-3ß and pS9-GSK-3ß in cancer cells was determined by Western blotting. RESULTS: SW620 cells expressed the highest level of GOLPH3 mRNA, and the silence effect was good after they were transfected with siRNA-GOLPH3. The relative luminescence units (RLU) values in the experimental groups were significantly lower than in the negative control group (P<0.001). There was no significant difference in the RLU values among the experimental groups (P> 0.05). The growth inhibition ratio and apoptosis rate of cancer cells in each experimental group were significantly higher than those in the control group, and the cell colony count in the experimental group was significantly lower than in the control group (P<0.05). In addition, the RLU value, proliferation and apoptosis rate of cancer cells did not differ significantly between each two experimental groups. Western blotting showed that, compared with the control group, expression of ß-catenin and pS9-GSK3 proteins were significantly decreased in the experimental group. Expression of GSK-3ß in the experimental group did not different from that of the control group. CONCLUSIONS: Overexpression of GOLPH3 gene activated the Wnt signaling pathway, as well as increasing expression of ß-catenin, promoting proliferation and inhibiting apoptosis in human colon cancer cells. The mechanism of action was that overexpression of GOLPH3 gene activated Akt, which may also further activate the Wnt signaling pathway via GSK-3ß, and promote proliferation in human colon cancer cells.
ABSTRACT
AIM: To investigate the effect of Golgi phosphorylation protein 3 (GOLPH3) expression on cell apoptosis, angiogenesis and prognosis in colorectal cancer (CRC). METHODS: The expression of GOLPH3 in CRC tissues and normal colorectal mucosae was determined by immunohistochemistry in 62 patients. In addition, immunohistochemistry was also carried out to detect the expression of vascular endothelial growth factor (VEGF), CD34 and microvessel density (MVD). Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay was used to determine the apoptotic index (AI). The Kaplan-Meier method was used to analyze the relationship between GOLPH3 expression and survival in another 123 CRC cases. RESULTS: Compared with normal colorectal mucosae, a notably higher level of GOLPH3 protein expression was identified in CRC tissues (53.2% vs 24.2%, P < 0.05). Positive GOLPH3 expression was significantly associated with tumor invasion depth, TNM stage, and lymph node metastasis (P = 0.001; P = 0.020; P = 0.020; P < 0.05, respectively), but not with tumor length, tumor site, and age (P = 0.363; P = 0.819; P = 0.599; P > 0.05, respectively). VEGF expression and MVD in GOLPH3-positive CRC was significantly higher than in GOLPH3-negative CRC (VEGF: 69.7% vs 31.0%; MVD: 21.45 ± 9.39 vs 14.24 ± 8.97; P < 0.05). GOLPH3 expression was negatively correlated with AI in CRC as shown by Spearman correlation analysis (r = -0.320, P < 0.05). The 5-year survival rate in GOLPH3-negative CRC (69.4%) was significantly higher than in GOLPH3-positive CRC (48.6%) (log-rank test, P < 0.05). CONCLUSION: High expression of GOLPH3 is found in CRC tissues. GOLPH3 expression may be a novel prognostic marker for CRC patients.