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Co?expression of Axin and APC gene fragments inhibits colorectal cancer cell growth via regulation of the Wnt signaling pathway.

ABSTRACT: Adenomatous polyposis coli (APC) and Axin interactions serve an important role in colorectal cancer (CRC) pathogenesis. The aim of the present study was to assess the combined effects of Axin and APC co?expression in CRC cells, and to determine the underlying mechanisms involved. SW480 cells were divided into the following groups: Untransfected (SW480 group), transfected with pEGFP?N3plus pCS2?MT (SW480/vector?vector), transfected with pEGFP?N3?APC5 (SW480/APC5), and transfected with pEGFP?N3?APC5 pluspCS2?MT?Axin (SW480/APC5?Axin). APC5 and Axin mRNA levels were determined by reverse transcription?polymerase chain reaction. MTT assays and flow cytometry analysis were performed to assess cell growth and cell cycle distribution, respectively. Quantitative PCR and western blot analyses were conducted to evaluate the mRNA and protein levels, respectively, of Wnt signaling effectors, including ??catenin, c?myc and survivin. Successful transfection of SW480 cells was determined with APC and APC?Axin plasmids as indicated by the green fluorescence signals. Notably, SW480/APC5 cell growth was inhibited by 40.33%, and cells co?expressing APC5 and Axin demonstrated 61.27% inhibition of cell growth compared with SW480 control cells. The results demonstrate that APC5 may induce G1/S arrest in SW480 cells, and Axin may enhance cell growth arrest induced by APC5. The mRNA and protein levels of ??catenin, c?myc and survivin were significantly reduced in SW480/APC?Axin cells when compared with the SW480/APC group. In conclusion, co?expression of APC5 and Axin genes significantly downregulated Wnt signaling in human SW480 CRC cells and inhibited cell growth, when compared with cells transfected with APC5 alone. These results may provide experimental evidence to support combined gene therapy in CRC.

SUBMITTER: Xu M

PROVIDER: S-EPMC5646956 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Co‑expression of Axin and APC gene fragments inhibits colorectal cancer cell growth via regulation of the Wnt signaling pathway.

Xu Meili M Liu Xianling X Xu Yan Y Zhu Shicong S Gao Yawen Y

Molecular medicine reports 20170721 4

Adenomatous polyposis coli (APC) and Axin interactions serve an important role in colorectal cancer (CRC) pathogenesis. The aim of the present study was to assess the combined effects of Axin and APC co‑expression in CRC cells, and to determine the underlying mechanisms involved. SW480 cells were divided into the following groups: Untransfected (SW480 group), transfected with pEGFP‑N3plus pCS2‑MT (SW480/vector‑vector), transfected with pEGFP‑N3‑APC5 (SW480/APC5), and transfected with pEGFP‑N3‑AP ...[more]

PMID: 28731177

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Project description:BACKGROUND:The role and mechanism of the nicotinamide adenine dinucleotide (NAD+) salvage pathway in cancer cell proliferation is poorly understood. Nicotinamide phosphoribosyltransferase (NAMPT), which converts nicotinamide into NAD+, is the rate-limiting enzyme in the NAD+ salvage pathway. Here, we assessed the role of NAMPT in the proliferation of colorectal cancer. METHODS:Real-time PCR, immunohistochemistry, western blotting, and analyses of datasets from Oncomine and Gene Expression Omnibus were conducted to assess the expression of NAMPT at the mRNA and protein levels in colorectal cancer. The Kaplan Meier plotter online tool was used to evaluate the prognostic role of NAMPT. Knockdown of NAMPT was performed to assess the role of NAMPT in colorectal cancer cell proliferation and tumorigenesis both in vitro and in vivo. Overexpression of NAMPT was used to evaluate impact of NAMPT on colorectal cancer cell proliferation in vitro. NAD+ quantitation, immunofluorescence, dual luciferase assay and western blot were used to explore the mechanism of colorectal cancer proliferation. Transwell migration and invasion assays were conducted to assess the role of NAMPT in cell migration and invasion abilities of colorectal cancer cells. RESULTS:Our study indicated that the inhibition of NAMPT decreased proliferation capacity of colorectal cancer cells both in vitro and in vivo. Conversely, overexpression of NAMPT could promote cell proliferation in vitro. NAMPT inhibition induced ?-catenin degradation by increasing Axin expression levels; this resulted in the inhibition of Wnt/?-catenin signaling and cell proliferation in colorectal cancer. The addition of nicotinamide mononucleotide, the enzymatic product of NAMPT, effectively reversed ?-catenin protein degradation and inhibited growth. Similarly, the knockdown of Axin also decreased the cell death induced by the inhibition of NAMPT. In addition, we showed that colorectal cancer tissues harbored significantly higher levels of NAMPT than the levels harbored by paired normal tissues, especially in colorectal cancer stages I and II. And the overexpression of NAMPT was associated with unfavorable survival results. CONCLUSIONS:Our findings reveal that NAMPT plays an important role in colorectal cancer proliferation via Wnt/?-catenin pathway, which could have vital implications for the diagnosis, prognosis and treatment of colorectal cancer.

Dataset Information

Co?expression of Axin and APC gene fragments inhibits colorectal cancer cell growth via regulation of the Wnt signaling pathway.

Publications

Co‑expression of Axin and APC gene fragments inhibits colorectal cancer cell growth via regulation of the Wnt signaling pathway.

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