Prolonged survival of patients with colorectal cancer is associated with a higher regucalcin gene expression: Overexpression of regucalcin suppresses the growth of human colorectal carcinoma cells in�vitro

Abstract

Regucalcin plays a crucial role as a regulator of transcriptional signaling activity, and its decreased expression or activity may contribute to the promotion of human carcinogenesis. A higher regucalcin expression in the tumor tissues has been demonstrated to prolong the survival of patients with various types of cancer, including pancreatic cancer, breast cancer, liver cancer and lung adenocarcinoma. The involvement of regucalcin in human colorectal cancer was investigated in the current study. Regucalcin gene expression and the survival data of 62 patients with colorectal cancer were obtained though the Gene Expression Omnibus (GEO) database (GSE12945) for outcome analysis. The data of gene expression revealed that the prolonged survival of patients with colorectal cancer was associated with a higher regucalcin gene expression in tumor tissues. The overexpression of regucalcin suppressed colony formation and proliferation, and induced the death of human colorectal carcinoma RKO cells cultured in a medium containing fetal bovine serum in vitro. Mechanistically, the overexpression of regucalcin induced the G1 and G2/M phase cell cycle arrest of the RKO cells through the suppression of multiple signaling pathways, including Ras, Akt, mitogen-activated protein (MAP) kinase and SAPK/JNK. Of note, the overexpression of regucalcin induced an increase in the levels of the tumor suppressors, p53 and Rb, and the cell cycle inhibitor, p21. Moreover, the levels of the transcription factors, c‑fos, c‑jun, nuclear factor (NF)‑κB p65, β-catenin and signal transducer and activator of transcription 3 (Stat3), were suppressed by the overexpression of regucalcin. On the whole, the findings of this study suggest that regucalcin plays a crucial role as a suppressor in human colorectal cancer, and that the suppressed expression of the regucalcin gene may predispose patients to the promotion of colorectal cancer. The overexpression of regucalcin by gene delivery may thus prove to be a novel therapeutic strategy for colorectal cancer.

2,3,7,8‑tetrachlorodibenzo‑p‑dioxin suppresses the growth of human colorectal cancer cells in vitro: Implication of the aryl hydrocarbon receptor signaling

“…Overexpression of regucalcin has been demonstrated to repress the enhanced proliferation and death of RKO cells in vitro (43). Therefore, the present study investigated whether the effects of TCDD were attenuated in regucalcin-overexpressing RKO cells in vitro .…”

“…TCDD treatment on transfectants cells did not appear to have a significant effect on CYP1A1 expression, since the effect of TCDD treatment on AHR-dependent CYP1A1 levels were depressed by regucalcin overexpression. Regucalcin overexpression has been demonstrated to increase the levels of p53, Rb and p21 in RKO cells (43) and other human cancer cells (50,51). Notably, the effects of TCDD in increasing p53, Rb and p21 levels were potentiated by regucalcin overexpression.…”

“…To generate the regucalcin-overexpressing RKO cells, the RKO wild-type cells were transfected with empty pCXN2 vector (Addgene, Inc., Cambridge, MA, USA; 600 µ g/ml) or pCXN2 vector (Addgene, Inc.; 600 µ g/ml) expressing a cDNA encoding the human full-length (900 bp) regucalcin (regucalcin cDNA/pCXN2) (42,43). For transfection, the RKO cells (1×10 5 /well per ml of DMEM) were grown on 24-well plates to reach subconfluency.…”

“…For transfection, the RKO cells (1×10 5 /well per ml of DMEM) were grown on 24-well plates to reach subconfluency. Regucalcin cDNA/pCXN2 (1 µ g/well) or empty pCXN2 vector (1 µ g/well) alone was transfected into the RKO cells using the synthetic cationic lipid Lipofectamine ® reagent, according to the manufacturer’s protocols (Promega Corporation, Madison, WI, USA) (43). Following overnight incubation after transfection, Geneticin (600 µ g/ml G418; Sigma-Aldrich; Merck KGaA) was added to the culture wells to select transfectants, and the cells were cultured in an atmosphere containing 5% CO 2 at 37°C for 3 weeks to produce transfected cells.…”

“…no. ab213459; dilution 1:1,000), and it was used as described previously (42,43,46). For immunoblotting with the aforementioned specific antibodies, the membranes were incubated with each primary antibody overnight at 4°C, followed by horseradish peroxidase-conjugated secondary antibody (cat.…”