Abstract
Purpose: To determine the association between phosphatidylethanolamine binding protein 1, which is an Raf kinase inhibitor protein (RKIP), and 5-fluorouracil (5-FU) via analysis of the association between RKIP and clinical responses in individuals treated using fluorouracil-based chemotherapy.Methods: Human gastric cancer cell lines MGC-803 and SGC-7901 were used in this study. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis and migration were determined with flow cytometry and Transwell chamber assays, respectively. The mRNA and protein expressions of apoptosis-related factors were assayed using realtime polymerase chain reaction (RT-PCR) and Western blotting, respectively, while the expression of RKIP was determined by immunohistochemical staining.Results: Chemotherapeutic drug (5-FU) treatment induced low RKIP expression levels in tumorigenic GC cells, thereby sensitizing the cells to apoptosis (8.57 vs 1.25 %, p < 0.01). The highest RKIP level correlated well with initiation of apoptosis (4.20 vs 1.25 %, p < 0.01). Following in vitro downregulation of RKIP, there was increase in the viability and proliferation of RKIP-inhibited cells over time, and these changes were linked to alterations in cell cycle phases and increased optical density in MTT proliferation assay (1.55 vs 1.18, p < 0.01). In vitro Transwell assay measurement revealed an association between RKIP downregulation and enhancement of cell migration potential (652 vs 436, p < 0.01). Ectopic RKIP expression restored the apoptotic sensitivity of resistant cells (14.30 vs 1.36 %, p <0.01). This sensitization was annulled by upregulation of survival routes. Reduction of RKIP by expression of antisense and siRNA conferred resistance on cancer cells sensitive to 5-FU-mediated apoptosis (6.88 vs 2.13 %, p < 0.01).Conclusion: Thus, RKIP is a promising therapeutic strategy for improving the efficacy of clinically relevant chemotherapeutic drugs for GC.
 Keywords: Gastric cancer, Raf kinase inhibitor protein, Cell proliferation, Invasion, Apoptosis, Chemotherapy, Phosphatidylethanolamine binding protein 1
Highlights
Overexpression plasmid and shRNAGastric cancer (GC) is the second most prevalent cancer all over the globe
This study has demonstrated that Raf kinase inhibitor protein (RKIP) inhibited cell colony formation and invasion of GC cells
The results of the present study suggest that cancer cell metastasis may be suppressed using druginduced expression of RKIP, leading to enhancement of apoptosis
Summary
Gastric cancer (GC) is the second most prevalent cancer all over the globe. China is among several Asian nations with a high incidence of GC and high level of mortality from the disease. The Raf kinase inhibitor protein (RKIP) is a globular protein with molecular weight of 20-25 kDa, and it belongs to the PEBP family made up of over 400 members [3]. The identification of the signaling pathways and elucidation of the effector genes regulated by RKIP will enhance knowledge of the mechanism of suppression of metastasis, but will be beneficial for inhibition of metastasis in the clinics. The expressions of RKIP protein in GC cells and normal cells were assayed, and the effects of RKIP suppression on the malignancy of GC and sensitivity to chemotherapy were determined. Human GC cell lines MGC-803 and SGC-7901 were obtained from Cell Bank of Chinese Academy of Sciences They were maintained in RPMI-1640 having 10 % FBS and 1 % penicillinstreptomycin at 37 oC in a 5 % CO2 humidified incubator. Values of p 0.05 were assumed as indicative of significant differences
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