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  • br caption on next page br Fig AD

    2020-03-24


    (caption on next page)
    Fig. 6. AD reverses the resistance of VR colon cancer AZD7687 to vemurafenib. (A, B) HCT116-CDK1 and HT29-CDK1 cells were treated with vemurafenib (10 μM), AD (1 μM), or the combination of both, and cell viability and colony formation ability were measured by WST-1 (A) and colony-formation assays (B). (C) Nude mice bearing HT29-CDK1-derived tumor xenografts were treated with vemurafenib (20 mg/kg), AD (10 mg/kg) or a combination of vemurafenib and AD daily (n = 6 per group). (D) Immunohistochemical analysis of Ki-67 proliferative index. (E) Expression of p-CDK1 and CDK1 was compared among tumors from mice treated with vemurafenib, AD, or a combination of vemurafenib and AD by Western blot. (F) Body weight of nude mice during the experiment period suggested the safety of the single and combined treatments. Bars, SD; n.s., nonsignificance; *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared to control group unless otherwise indicated.
    interactions using the ELISA screening system [50]. Our previous study demonstrated that the KCTD12-CDK1 interaction was a prerequisite to CDK1 activation and promotes cell division. During the cell cycle, proper G2/M transition is important for cell division, and CDK1 acti-vation is a precondition for G2/M transition [20]. Although some CDK1 inhibitors have been developed, such as Ro-3306, this compound has not been used for clinical therapy, and the side effects are unclear [20]. By performing ELISA screening and a series of functional assays, we found that AD inhibits the KCTD12-CDK1 interaction and suppresses growth of colon cancer cells in vitro and in vivo (Figs. 3–5), further suggesting that targeting protein-protein interactions is a promising strategy for developing novel therapeutic agents.
    The indication of many FDA-approved drugs includes various dis-eases other than cancer. Given that the side effects of these “old drugs” and the corresponding countermeasures for clinical treatment are known [51], “old drugs for novel use” is a promising strategy for de-veloping anticancer drugs. AD was the focus of this study. A clinic trial launched in Japan demonstrated that antiviral therapy with AD con-veyed longer survival for postoperative HBV-related hepatocellular carcinoma (HBV-HCC) patients [52]. Another study claimed that AD treatment was effective in normalizing liver function, decreasing AZD7687 HBV-HCC recurrence, and improving postoperative survival [53]. In the present study, a series of experiments demonstrated that AD suppresses colon cancer cell proliferation by inactivating CDK1 and inducing G2 phase arrest. Moreover, inactivation of CDK1 by AD reversed CDK1 expression-induced vemurafenib resistance in colon cancer cells, thereby sensitizing VR colon cancer cells to vemurafenib (Figs. 6–7). Furthermore, combined use of AD and vemurafenib exhibited a more potent effect on colon cancer growth inhibition.
    Colon cancer is one of the most common malignancies worldwide
    [1,54]. As most “front-line proteins” are evolutionarily conserved and rarely mutated, these proteins represent more suitable targets for de-velopment of novel treatment agents. Our study demonstrated that the activation of CDK1, one of the “front-line proteins”, is significantly inhibited by AD, a nucleoside analog approved by the FDA to treat HBV infection. For the first time, our data provide solid evidence that AD used as a single agent or in combination with vemurafenib significantly induces G2 cell cycle arrest to inhibit tumorigenesis of colon cancer cells by disrupting the interaction between CDK1 and KCTD12, thus supporting the hypothesis that AD administration may be a potential strategy for colon cancer therapy.
    Conflicts of interest
    The authors declare no conflicts of interest.
    Acknowledgements
    Appendix A. Supplementary data
    Fig. 7. Schematic diagram summarizing the mechanism for AD sensitizing colon cancer cells to vemurafenib treatment. (A) Colon cancer cells show no response to vemurafenib after a period of vemurafenib treatment. AD sensitizes colon cancer cells to vemurafenib, and the combined use of vemurafenib and AD induces growth ar-rest in colon cancer cells. (B) CDK1 activation induces unresponsiveness of colon cancer to vemurafenib, while AD inactivates CDK1 by disrupting the KCTD12-CDK1 interaction, thus sensitizing colon cancer cells to vemurafenib.
    References
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