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Induction of G1 Arrest by SB265610 Involves Cyclin D3 Down- Regulation and Suppression of CDK2 (Thr160) Phosphorylation

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Induction of G1 Arrest by SB265610 Involves Cyclin D3 Down- Regulation and Suppression of CDK2 (Thr160) Phosphorylation ANTICANCER RESEARCH 35: 3235-3244 (2015) Induction of G1 Arrest by SB265610 Involves Cyclin D3 Down- regulation and Suppression of CDK2 (Thr160) Phosphorylation AHMED E. GODA1,3, RAYMOND L. ERIKSON1,2, JONG-SEOG AHN4 and BO-YEON KIM1 1World Class Institute, Incurable Diseases Therapeutics Research Center, and 4Chemical Biology Research Center, Korea Research Institute of Bioscience and Biotechnology, Ochang, Cheongwon, Republic of Korea; 2Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, U.S.A.; 3Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tanta University, Tanta, Egypt Abstract. Background/Aim: The current study investigated inflammatory chemokine interleukin-8 (IL8, CXCL8) the mechanisms underlying the antitumor activity of compared to the surrounding normal tissues. Cancer cells can SB265610, a cysteine-amino acid-cysteine (CXC) chemokines further increase IL8 synthesis and secretion following receptor 2 (CXCR2) antagonist. Materials and Methods: chemotherapy or as a result of hypoxia (1). Two G-protein- Cell-cycle progression and regulatory molecules were coupled receptors namely, cysteine-amino acid-cysteine assessed by flow cytometry, immunoblotting, real-time PCR (CXC) chemokine receptor 1 (CXCR1) and CXCR2 are and immunoprecipitation. Target validation was achieved via expressed on cancer cells, as well as tumor-associated RNA interference. Results: G1 arrest induced by SB265610 vascular endothelial cells and macrophages, and occurred at concentrations lacking CXCR2 selectivity, differentially mediate the complex autocrine/paracrine persisted upon interleukin 8 (IL8) challenge, and did not signaling of IL8 and other Glu-Leu-Arg tripeptide motif + affect IL8 downstream target expression. Profiling of G1 (ELR ) CXC chemokines within the tumor micro - regulators revealed cyclin-dependent kinase 2 (CDK2) environment where both CXCR1 and CXCR2 receptors are (Thr160) hypophosphorylation, cyclin D3 gene down- activated by IL8 and granulocyte chemotactic peptide-2 regulation, and p21 post-translational induction. However, (CXCL6). However, only CXCR2 receptors are activated by + only cyclin D3 and CDK2 contributed towards G1 arrest. several other ELR CXC chemokines, such as those with Furthermore, SB265610 induced a sustained phosphorylation melanoma growth-stimulatory activities (CXCL1, CXCL2, of the p38MAPK. Pharmacological interference with CXCL3), epithelial neutrophil-activating protein (CXCL5) p38MAPK significantly abrogated SB265610-induced G1 and neutrophil-activating protein (CXCL7) (1, 2). Elevated arrest and normalized the expression of cyclin D3, with levels of IL8 and other ELR+ CXC chemokines have been restoration of its exclusive binding to CDK6, but with weak shown to drive the proliferation, angiogenesis and migration recovery of CDK2 (Thr160) hypo-phosphorylation. of cancer cells and tumor-associated vascular endothelial Conclusion: The present study described the mechanisms for cells, with a relatively higher contribution of CXCR2 the anti-proliferative activity of SB265610 which may be of receptors to these processes (1, 3). value in IL8-rich tumor microenvironments. Over the past two decades several small-molecule receptor antagonists belonging to different chemical classes have been Human cancers of different origins, including breast, gastric, developed to target IL8 receptors in a competitive or colon, melanoma, ovarian, pancreatic and prostatic, have allosteric manner and with varying degrees of selectivity been shown to express elevated levels of the pro- towards CXCR1 or CXCR2 receptors or acting as dual CXCR1/2 inhibitors (2). In our previous work we identified novel off-target antitumor mechanisms for the compound SB225002, a highly selective CXCR2 antagonist of the Correspondence to: Bo-Yeon Kim, Ph.D., World Class Institute, diarylurea nucleus, which enable this compound to dually- Incurable Diseases Therapeutics Research Center, Korea Research target cancer through microtubule destabilization and Institute of Bioscience and Biotechnology, 685-2 Ochang, inhibition of IL8-driven cancer progression (4). In the current Cheongwon 363-883, Republic of Korea. Tel: +82 432406100, Fax: study, we investigated whether SB265610, a highly selective +82 432406259, e-mail: [email protected] CXCR2 antagonist and a close structural analog of Key Words: SB265610, G1 arrest, cyclin D3, phospho(Thr160)- SB225002, may exert antitumor activity through IL8- CDK2, p21, p38MAPK. independent mechanism(s). 0250-7005/2015 $2.00+.40 3235 ANTICANCER RESEARCH 35: 3235-3244 (2015) Materials and Methods After washing in 5% non-fat dry milk (BioRad) in TBS-T, membranes were probed for 1 hour at room temperature with the Chemicals. SB265610 (Tocris, Bristol, UK) was dissolved in appropriate secondary antibodies: horseradish peroxidase (HRP)- dimethyl sulfoxide (DMSO), aliquoted and stored at −80˚C for no conjugated anti-rabbit IgG, HRP-conjugated anti-mouse IgG (Cell more than one week. Recombinant human IL8 (Biolegend, San Signaling Technologies). The resulting chemiluminescence (Pierce) Diego, CA, USA) was prepared in sterile phosphate buffered saline was recorded on X-ray films (Agfa, Mortsel, Belgium). (PBS) containing 0.5% bovine serum albumin, aliquoted and stored at −80˚C. Two specific chemical inhibitors of p38 mitogen-activated Immunoprecipitation (IP). All IP steps were carried out on ice. PC- protein kinase (p38MAPK), namely SB202190 and SB203580 3 cells were harvested in IP lysis buffer (Pierce) supplemented with (Tocris), were dissolved in DMSO, aliquoted and stored at −80˚C. protease and phosphatase inhibitor cocktails (Pierce) and were Freeze-thaw cycles were avoided for all chemicals. disrupted by repeated aspiration through a 21-gauge needle. Cell lysates were recovered by centrifugation at 21000xg at 4˚C. Five Cell culture. PC-3 androgen-independent human prostate hundred micrograms of protein of the cell lysates were adenocarcinoma cell line (American Type Culture Collection, immunoprecipitated by incubation with 2 μg of antibodies for Manassas, VA, USA) was cultured in RPMI 1640 medium CDK4, CDK6 or IgG control antibody in a total volume of 750 μl (Hyclone, Logan, Utah, USA) supplemented with 10% fetal bovine with rotation at 4˚C overnight followed by incubation with 40 μl of serum (Hyclone, Logan, Utah, USA), L-glutamine (2 mmol/l), protein A/G-agarose beads (Santa Cruz Biotechnology) for 4 h at penicillin (100 units/ml), and streptomycin (100 μg/ml) (Gibco, Life 4˚C. Immunoprecipitates were collected by centrifugation at 600 xg Technologies, Grand Island, NY, USA) and maintained at 37˚C in for 5 min at 4˚C. After washing five times with IP lysis buffer, immunoprecipitates were resuspended and boiled in 40 μl SDS a humidified atmosphere of 5% CO2. Cell cultures at 50% confluence level were treated with SB265610, IL8 or p38MAPK sample buffer and resolved by SDS-PAGE. Co-immunoprecipitated chemical inhibitors as detailed in legends to figures. cyclin D3 was detected by immunoblotting. Analysis of the cell cycle by flow cytometry. At the end of drug siRNA-mediated knockdown of gene expression. To knockdown treatments, cells were harvested for cell-cycle analysis as described CDKN1A (p21) expression, two commercially available and previously (5), where PC-3 cells were washed once with PBS, validated siRNA sequences #1029367 and #1029372 were trypsinized and harvested. After washing twice with PBS at 500xg and purchased from Bioneer Corporation (Daejeon, Republic of Korea). 4˚C, cells were resuspended in PBS containing RNAse (100 μg/ml) In addition, a third previously described CDKN1A (p21) siRNA Triton-X-100 (1 μl/ml) and propidium iodide (2.5 μg/ml) (all Sigma- sequence (6) was synthesized by Bioneer Corporation. These three Aldrich, St. Louis, MO, USA) and were analyzed (after 30 min and different CDKN1A (p21)-targeting sequences are referred to herein protected from light) on a BD FACSCalibur flow cytometer using as sip21#1, sip21#2 and sip21#3, respectively. For the knockdown CellQuest software package (BD Biosciences, San Jose, CA, USA). of CCND3 (cyclin D3) and CDK2, commercially available and Data representative of three independent experiments (triplicates in validated CCND3 (cyclin D3) siRNA sequence (#1027310) and each) are presented as the mean±SEM. Differences were considered CDK2 siRNA sequence (#1029167), as well as the non-targeting statistically significant at p<0.05 using the Student's t-test. LacZ siRNA control sequence (7) were ordered from Bioneer Corporation. All siRNA sequences were transfected (at 100 nM final Immunoblotting. Protein isolation and immunoblotting were concentration) into PC-3 cells using Lipofectamine™ RNAiMAX performed as previously described (5). At the end of drug Transfection Reagent (Invitrogen, Life Technologies, Grand Island, treatments, PC-3 cells were washed with ice-cold PBS and NY, USA) according to the manufacturer’s instructions. Knockdown harvested using Pierce lysis buffer supplemented with protease and efficiency was assessed by immunoblotting. Data representative of phosphatase inhibitor cocktails (Pierce, Rockford, IL, USA). Protein three independent experiments (triplicates in each) are presented as assay was performed on cell lysates using protein dye concentrate the mean±SEM. Differences were considered statistically significant (BioRad, Hercules, CA, USA) and 40 μg of each sample were at p<0.05 using Student's t-test. boiled in sodium dodecyl sulfate sample buffer (BioRad) before being resolved
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