Histone deacetylase (HDAC) inhibitors are successful for treatment of advanced cutaneous T-cell lymphoma but only show modest effect in solid tumors. Approaches for HDAC inhibitors to improve activity against solid tumors are necessary.Sulforhodamine B assay and flow cytometric analysis detected cell proliferation and cell-cycle progression, respectively. Protein expression was determined by Western blotting. Comet assay and DNA end-binding activity of Ku proteins detected DNA damage and DNA repair activity, respectively. siRNA technique was used for knockdown of specific cellular target.WJ25591 displayed inhibitory activity against HDAC1 and cell proliferation in human hormone-refractory prostate cancers PC-3 and DU-145. WJ25591 caused an arrest of cell-cycle at both G1- and G2-phase and increased protein expressions of p21 and cyclin E, followed by cell apoptosis. WJ25591-induced Bcl-2 down-regulation and activation of caspase-9, -8, and -3, suggesting apoptotic execution through both intrinsic and extrinsic apoptotic pathways. WJ25591 also significantly inhibited DNA repair activity but not directly induced DNA damage. Moreover, the proteasome inhibitor MG-132 dramatically sensitized WJ25591-induced cell apoptosis. The siRNA technique demonstrated that endoplasmic reticulum (ER) stress, in particular CHOP/GADD153 up-regulation, contributed to the synergistic effect.The data suggest that WJ25591 inhibited HDAC activity, leading to cell-cycle arrest and inhibition of DNA repair. Caspase cascades are subsequently triggered to execute cell apoptosis. MG-132 dramatically sensitizes WJ25591-mediated apoptosis, at least partly, through ER stress response. The data also reveal that combination of HDAC inhibitors and proteasome inhibitors may be a potential strategy against hormone-refractory prostate cancers.
Castration-resistant prostate cancer (CRPC) is refractory to hormone treatment and the therapeutic options are continuously advancing. This study aims to discover the anti-CRPC effects and underlying mechanisms of small-molecule compounds targeting topoisomerase (TOP) II and cellular components of DNA damage repair.Cell proliferation was determined in CRPC PC-3 and DU-145 cells using anchorage-dependent colony formation, sulforhodamine B assay and flow cytometric analysis of CFSE staining. Flow cytometric analyses of propidium iodide staining and JC-1 staining were used to examine the population of cell-cycle phases and mitochondrial membrane potential, respectively. Nuclear extraction was performed to detect the nuclear localization of cellular components in DNA repair pathways. Protein expressions were determined using Western blot analysis.A series of azathioxanthone-based derivatives were synthesized and examined for bioactivities in which WC-A13, WC-A14, WC-A15, and WC-A16 displayed potent anti-CRPC activities in both PC-3 and DU-145 cell models. These WC-A compounds selectively downregulated both TOP IIα and TOP IIβ but not TOP I protein expression. WC-A13, WC-A14, and WC-A15 were more potent than WC-A16 on TOP II inhibition, mitochondrial dysfunction, and induction of caspase cascades indicating the key role of amine-containing side chain of the compounds in determining anti-CRPC activities. Furthermore, WC-A compounds induced an increase of γH2AX and activated ATR-Chk1 and ATM-Chk2 signaling pathways. P21 protein expression was also upregulated by WC-A compounds in which WC-A16 showed the least activity. Notably, WC-A compounds exhibited different regulation on Rad51, a major protein in homologous recombination of DNA in double-stranded break repair. WC-A13, WC-A14, and WC-A15 inhibited, whereas WC-A16 induced, the nuclear translocation of Rad51.The data suggest that WC-A compounds exhibit anti-CRPC effects through the inhibition of TOP II activities, leading to mitochondrial stress-involved caspase activation and apoptosis. Moreover, WC-A13, WC-A14, and WC-A15 but not WC-A16 display inhibitory activities of Rad51-mediated DNA repair pathway which may increase apoptotic effect of CRPC cells.
// Mei-Ling Chan 1 , Chia-Chun Yu 1 , Jui-Ling Hsu 1 , Wohn-Jenn Leu 1 , She-Hung Chan 1 , Lih-Ching Hsu 1 , Shih-Ping Liu 2, 3 , Polina M. Ivantcova 4 , Özdemir Dogan 5 , Stefan Bräse 6, 7 , Konstantin V. Kudryavtsev 4, 8 and Jih-Hwa Guh 1 1 School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan 2 Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan 3 Department of Urology, National Taiwan University Hospital, Taipei, Taiwan 4 Department of Medicinal Chemistry, Faculty of Chemistry, Lomonosov Moscow State University, Moscow, Russian Federation 5 Department of Chemistry, Middle East Technical University, Ankara, Turkey 6 Institute of Organic Chemistry, Karlsruhe Institute of Technology, Karlsruhe, Germany 7 Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany 8 Institute of Physiologically Active Compounds, Russian Academy of Sciences, Chernogolovka, Moscow region, Russian Federation Correspondence to: Konstantin V. Kudryavtsev, email: kudr@med.chem.msu.ru Jih-Hwa Guh, email: jhguh@ntu.edu.tw Keywords: β-dipeptide, c-Myc, PI3K/Akt, mTOR, hormone-refractory prostate cancer Received: October 21, 2016 Accepted: May 08, 2017 Published: May 20, 2017 ABSTRACT The use of peptides that target cancer cells and induce anticancer activities through various mechanisms is developing as a potential anticancer strategy. KUD983, an enantiomerically pure β-dipeptide derivative, displays potent activity against hormone-refractory prostate cancer (HRPC) PC-3 and DU145 cells with submicromolar IC 50 . KUD983 induced G1 arrest of the cell cycle and subsequent apoptosis associated with down-regulation of several related proteins including cyclin D1, cyclin E and Cdk4, and the de-phosphorylation of RB. The levels of nuclear and total c-Myc protein, which could increase the expression of both cyclin D1 and cyclin E, were profoundly inhibited by KUD983. Furthermore, it inhibited PI3K/Akt and mTOR/p70S6K/4E-BP1 pathways, the key signaling in multiple cellular functions. The transient transfection of constitutively active myristylated Akt (myr-Akt) cDNA significantly rescued KUD983-induced caspase activation but did not blunt the inhibition of mTOR/p70S6K/4E-BP1 signaling cascade suggesting the presence of both Akt-dependent and -independent pathways. Moreover, KUD983-induced effect was enhanced with the down-regulation of anti-apoptotic Bcl-2 members (e.g., Bcl-2, and Mcl-1) and IAP family members (e.g., survivin). Notably, KUD983 induced autophagic cell death using confocal microscopic examination, tracking the level of conversion of LC3-I to LC3-II and flow cytometric detection of acidic vesicular organelles-positive cells. In conclusion, the data suggest that KUD983 is an anticancer β-dipeptide against HRPCs through the inhibition of cell proliferation and induction of apoptotic and autophagic cell death. The suppression of signaling pathways regulated by c-Myc, PI3K/Akt and mTOR/p70S6K/4E-BP1 and the collaboration with down-regulation of Mcl-1 and survivin may explain KUD983-induced anti-HRPC mechanism.
Chalcones are responsible for biological activity throughout fruits, vegetables, and medicinal plants in preventing and treating a variety of inflammation-related diseases. However, their structure-activity relationship (SAR) in inhibiting inflammasome activation has not been explored. We synthesized numerous chalcones and determined their SAR on lipopolysaccharide (LPS)-primed ATP-induced NLRP3 inflammasome activation. 11Cha1 displayed good inhibitory activity on release reaction of caspase-1, IL-1β, and IL-18. It significantly inhibited LPS-induced phosphorylation and proteolytic degradation of IĸB-α and nuclear translocation of NF-ĸB, but had little effect on mitogen-activated protein kinases (MAPKs) activities. Furthermore, 11Cha1 blocked LPS-induced up-regulation of NLRP3, pro-caspase-1, ASC, IL-18, and IL-1β, indicating the suppression on priming step of inflammasome activation. ASC dimerization and oligomerization are considered to be direct evidence for inflammasome activation. 11Cha1 profoundly inhibited ATP-induced formation of ASC dimers, trimers, and oligomers, and the assembly of ASC, pro-caspase-1, and NLRP3 in inflammasome formation. Decrease of intracellular K+ levels is the common cellular activity elicited by all NLRP3 inflammasome activators. 11Cha1 substantially diminished ATP-mediated K+ efflux, confirming the anti-NLRP3 inflammasome activity of 11Cha1. In summary, the SAR of chalcone derivatives in anti-inflammasome activities was examined. Besides, 11Cha1 inhibited both priming and activation steps of NLRP3 inflammasome activation. It inhibited NF-ĸB activation and subsequently suppressed the up-regulation of NLRP3 inflammasome components including NLRP3, ASC, pro-caspase-1, pro-IL-18, and pro-IL-1β. Next, 11Cha1 blocked ATP-mediated K+ efflux and suppressed the assembly and activation of NLRP3 inflammasome, leading to the inhibition of caspase-1 activation and proteolytic cleavage, maturation, and secretion of IL-1β and IL-18.
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