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∗ Corresponding author. Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou, 510632, China. ∗∗ Corresponding author. Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou, 510632, China.
E-mail addresses: [email protected] (B. Li), [email protected] (Q.-Y. He).
activation of CDK1 signaling in VR cells led us to hypothesize that SP 600125 checkpoints are associated with the sensitivity of cancer cells to vemurafenib.
Cell cycle dysregulation is a common feature in human cancers leading to at least two hallmarks of cancer development: uncontrolled cell proliferation and genomic/chromosomal instability [17,18]. Proper progression through the cell cycle is mediated by members of the cy-clin-dependent kinase (CDK) family, and activity of members of this family is regulated by specific activators (cyclins) and inhibitors (Ink 4 and Cip/Kip family members) . Our previous study identified that the interaction between potassium channel tetramerization domain containing 12 (KCTD12) and CDK1 is necessary for CDK1 activation and G2/M transition . Constitutive CDK1 activation causes sig-nificant changes in protein phosphorylation and drives tumor cell cycle progression, resulting in dysregulated cell proliferation and tumor-igenicity [21,22]. These findings suggest that CDK1 may be a ther-apeutic target for the treatment of human cancer.
In the field of drug discovery, the concept of “old drugs for new applications” is gaining increasing recognition . When a new function for a clinical drug is identified, it increases the opportunity for that drug to be clinically utilized. In this study, a drug library consisting of 616 Food and Drug Administration (FDA)-approved compounds was used to screen for drugs with the potential to block the CDK1 and KCTD12 interaction. We found that Adefovir dipivoxil (AD), a nucleo-side analog approved by the FDA for clinical therapy of hepatitis B virus (HBV) infections [24–26], disrupted the CDK1-KCTD12 interaction. AD is an oral prodrug of adefovir, an analog of AMP . Previous studies demonstrated that AD suppressed viral DNA synthesis by targeting re-verse transcriptase in host cells [28,29]. However, the role of AD in cancer therapy remains unclear. In this study, the eﬀects of AD on cell cycle progression, proliferation and tumorigenicity of colon cancer cells were investigated both in vitro and in vivo. Furthermore, we assessed whether targeting CDK1-KCTD12 with AD enhances the sensitivity of colon cancer cells to vemurafenib.
2. Materials and methods
Human colon cancer cell lines HCT116 and HT29 (ATCC, Rockville, MD, USA) were maintained in DMEM supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C in 5% CO2. Vemurafenib-resistant sublines, designated HCT116-vemurafenib resistant (VR) and HT29-VR, were established by treating HCT116 and HT29 cells, respectively, with high concentrations (200 μM) of vemurafenib. All cell lines were authenticated by short tandem repeat profiling and were tested for mycoplasma contamina-tion. Vemurafenib and adefovir dipivoxil were purchased from Selleck Chemicals (Huston, TX, USA) and were dissolved in dimethyl sulfoxide (DMSO).
2.2. Plasmids, transfection and infection
The CDK1 lentiviral expression plasmid was generated by PCR amplification from a colon cancer cDNA library and cloned into the pLVX vector. Primers for generating the CDK1 plasmid were as follows: forward (5′-GCGAATTCATGGAAGATTATACCAAAA-3′) and reverse (
5′-GCGGATCCTTACTTATCGTCGTCATCCTTGTAATCCA TCTTCTTAAT CTGAT-3′). Transfection, infection and establishment of stable cell lines were performed as described previously [30,31]. The Lipofectamine™ 3000 reagent (Thermo Fisher Scientific) was used for transfection ac-cording to the manufacturer's recommendations.