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Wip1 gene silencing enhances the chemosensitivity of human colon cancer cells.

ABSTRACT: Colon cancer is one of the most common cancers in the world. Multidrug resistance is one of the main reasons for failure of therapy in patients with advanced colon cancer. In previous studies, multiple methods were investigated to reverse the multidrug resistance of colon cancer cells. However, to date, no clinical method has been identified to be satisfactory. Therefore, successful reversal of drug resistance in colon cancer cells still requires new therapeutic strategies or pharmaceuticals. Wild-type p53-induced phosphatase (Wip1), a member of the 2C type serine/threonine protein phosphatase family, is closely associated with the p53 gene, which is the most important tumor-suppressor gene. Wip1 was reported to be associated with the chemosensitivity of breast cancer cells. However, the correlation between the expression of Wip1 gene and the chemosensitivity of colon cancer cells has not been reported yet. In the present study, Wip1-811 small interfering RNA (siRNA) targeting Wip1 was investigated to reverse the multidrug resistance of colon cancer cells. The siRNA duplexes were transfected into RKO colon cancer cells. The messenger RNA (mRNA) expression of Wip1 was measured by reverse transcription-quantitative polymerase chain reaction. The protein level of Wip1 was detected by western blotting. The cell viability was measured by MTS assay. The cell apoptosis and cell cycle were analyzed by flow cytometry. Intracellular adriamycin cumulative concentration was determined using flow cytometry. Wip1-811 siRNA efficiently inhibited the expression of Wip1 at the mRNA and protein levels, and enhanced the sensitivity of RKO colon cancer cells towards chemotherapy, which was accompanied by increased cell apoptosis, following the inhibition of Wip1 gene expression. These results indicate that Wip1 gene silencing could enhance the chemosensitivity of colon cancer cells, which may provide a new potential approach for the reversal of multidrug resistance in colon cancer cells.

SUBMITTER: Xia ZS

PROVIDER: S-EPMC5530189 | biostudies-other | 2017 Aug

REPOSITORIES: biostudies-other

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Project description:BackgroundMicrotubules are considered major therapeutic targets in patients with breast cancer. In spite of their essential role in biological functions including cell motility, cell division and intracellular transport, microtubules have not yet been considered as critical actors influencing tumor cell aggressivity. To evaluate the impact of microtubule mass and dynamics on the phenotype and sensitivity of breast cancer cells, we have targeted tubulin binding cofactor C (TBCC), a crucial protein for the proper folding of alpha and beta tubulins into polymerization-competent tubulin heterodimers.MethodsWe developed variants of human breast cancer cells with increased content of TBCC. Analysis of proliferation, cell cycle distribution and mitotic durations were assayed to investigate the influence of TBCC on the cell phenotype. In vivo growth of tumors was monitored in mice xenografted with breast cancer cells. The microtubule dynamics and the different fractions of tubulins were studied by time-lapse microscopy and lysate fractionation, respectively. In vitro sensitivity to antimicrotubule agents was studied by flow cytometry. In vivo chemosensitivity was assayed by treatment of mice implanted with tumor cells.ResultsTBCC overexpression influenced tubulin fraction distribution, with higher content of nonpolymerizable tubulins and lower content of polymerizable dimers and microtubules. Microtubule dynamicity was reduced in cells overexpressing TBCC. Cell cycle distribution was altered in cells containing larger amounts of TBCC with higher percentage of cells in G2-M phase and lower percentage in S-phase, along with slower passage into mitosis. While increased content of TBCC had little effect on cell proliferation in vitro, we observed a significant delay in tumor growth with respect to controls when TBCC overexpressing cells were implanted as xenografts in vivo. TBCC overexpressing variants displayed enhanced sensitivity to antimicrotubule agents both in vitro and in xenografts.ConclusionThese results underline the essential role of fine tuned regulation of tubulin content in tumor cells and the major impact of dysregulation of tubulin dimer content on tumor cell phenotype and response to chemotherapy. A better understanding of how the microtubule cytoskeleton is dysregulated in cancer cells would greatly contribute to a better understanding of tumor cell biology and characterisation of resistant phenotypes.

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Wip1 gene silencing enhances the chemosensitivity of human colon cancer cells.

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