A Novel Histone Deacetylase Inhibitor Exhibits Antitumor Activity via Apoptosis Induction, F-Actin Disruption and Gene Acetylation in Lung Cancer

BACKGROUND: Lung cancer is the leading cause of cancer mortality worldwide, yet the therapeutic strategy for advanced non-small cell lung cancer (NSCLC) is limitedly effective. In addition, validated histone deacetylase (HDAC) inhibitors for the treatment of solid tumors remain to be developed. Here, we propose a novel HDAC inhibitor, OSU-HDAC-44, as a chemotherapeutic drug for NSCLC. METHODOLOGY/PRINCIPAL FINDINGS: The cytotoxicity effect of OSU-HDAC-44 was examined in three human NSCLC cell lines including A549 (p53 wild-type), H1299 (p53 null), and CL1-1 (p53 mutant). The antiproliferative mechanisms of OSU-HDAC-44 were investigated by flow cytometric cell cycle analysis, apoptosis assays and genome-wide chromatin-immunoprecipitation-on-chip (ChIP-on-chip) analysis. Mice with established A549 tumor xenograft were treated with OSU-HDAC-44 or vehicle control and were used to evaluate effects on tumor growth, cytokinesis inhibition and apoptosis. OSU-HDAC-44 was a pan-HDAC inhibitor and exhibits 3-4 times more effectiveness than suberoylanilide hydroxamic acid (SAHA) in suppressing cell viability in various NSCLC cell lines. Upon OSU-HDAC-44 treatment, cytokinesis was inhibited and subsequently led to mitochondria-mediated apoptosis. The cytokinesis inhibition resulted from OSU-HDAC-44-mediated degradation of mitosis and cytokinesis regulators Auroroa B and survivin. The deregulation of F-actin dynamics induced by OSU-HDAC-44 was associated with reduction in RhoA activity resulting from srGAP1 induction. ChIP-on-chip analysis revealed that OSU-HDAC-44 induced chromatin loosening and facilitated transcription of genes involved in crucial signaling pathways such as apoptosis, axon guidance and protein ubiquitination. Finally, OSU-HDAC-44 efficiently inhibited A549 xenograft tumor growth and induced acetylation of histone and non-histone proteins and apoptosis in vivo. CONCLUSIONS/SIGNIFICANCE: OSU-HDAC-44 significantly suppresses tumor growth via induction of cytokinesis defect and intrinsic apoptosis in preclinical models of NSCLC. Our data provide compelling evidence that OSU-HDAC-44 is a potent HDAC targeted inhibitor and can be tested for NSCLC chemotherapy

Mitochondrial Apoptosis and FAK Signaling Disruption by a Novel Histone Deacetylase Inhibitor, HTPB, in Antitumor and Antimetastatic Mouse Models

BACKGROUND: Compound targeting histone deacetylase (HDAC) represents a new era in molecular cancer therapeutics. However, effective HDAC inhibitors for the treatment of solid tumors remain to be developed. METHODOLOGY/PRINCIPAL FINDINGS: Here, we propose a novel HDAC inhibitor, N-Hydroxy-4-(4-phenylbutyryl-amino) benzamide (HTPB), as a potential chemotherapeutic drug for solid tumors. The HDAC inhibition of HTPB was confirmed using HDAC activity assay. The antiproliferative and anti-migratory mechanisms of HTPB were investigated by cell proliferation, flow cytometry, DNA ladder, caspase activity, Rho activity, F-actin polymerization, and gelatin-zymography for matrix metalloproteinases (MMPs). Mice with tumor xenograft and experimental metastasis model were used to evaluate effects on tumor growth and metastasis. Our results indicated that HTPB was a pan-HDAC inhibitor in suppressing cell viability specifically of lung cancer cells but not of the normal lung cells. Upon HTPB treatment, cell cycle arrest was induced and subsequently led to mitochondria-mediated apoptosis. HTPB disrupted F-actin dynamics via downregulating RhoA activity. Moreover, HTPB inhibited activity of MMP2 and MMP9, reduced integrin-β1/focal adhesion complex formation and decreased pericellular poly-fibronectin assemblies. Finally, intraperitoneal injection or oral administration of HTPB efficiently inhibited A549 xenograft tumor growth in vivo without side effects. HTPB delayed lung metastasis of 4T1 mouse breast cancer cells. Acetylation of histone and non-histone proteins, induction of apoptotic-related proteins and de-phosphorylation of focal adhesion kinase were confirmed in treated mice. CONCLUSIONS/SIGNIFICANCE: These results suggested that intrinsic apoptotic pathway may involve in anti-tumor growth effects of HTPB in lung cancer cells. HTPB significantly suppresses tumor metastasis partly through inhibition of integrin-β1/FAK/MMP/RhoA/F-actin pathways. We have provided convincing preclinical evidence that HTPB is a potent HDAC targeted inhibitor and is thus a promising candidate for lung cancer chemotherapy

Synthesis and molecular structure of a zinc complex of the vitamin K3 analogue phthiocol

The complex [Zn(phthiocol)2(H2O)2]; 1, where phthiocol is 2-hydroxy-3-methyl-1,4-naphthoquinone, has been synthesized and characterized by elemental analysis, FT-IR, 1H NMR, UV–vis spectroscopy, thermogravimetric (TG) analysis, electrochemical and single crystal X-ray diffraction studies. The νCO stretch shifts to lower frequencies upon complexation of phthiocol to Zn2+. 1H NMR spectra show an upfield shift of the benzenoid ring protons in 1. There is a bathochromic shift of the LMCT band in the UV–vis spectra of 1. Single crystal X-ray structure of 1 show distorted octahedral geometry around Zn2+. Two phthiocol ligands are in plane with the metal, while water molecules are trans to this plane. Coordination of deprotonated phthiocol ligands is ‘trans, trans’ to Zn2+. Intra as well as intermolecular interactions are observed in 1. Molecules of 1 show three dimensional network through CH⋯O and OH⋯O interactions. Additional anodic peaks are observed in cyclic voltammogram of phthiocol ligand due to oxidation of reduced species formed during reduction. One-electron reduction of 1 is shown to be reversible and DFT studies define this redox event as ligand-centered

Rapid efficient synthesis and characterization of silver, gold, and bimetallic nanoparticles from the medicinal plant Plumbago zeylanica and their application in biofilm control

Gayatri R Salunke,1 Sougata Ghosh,1 RJ Santosh Kumar,2 Samiksha Khade,1 Priya Vashisth,3 Trupti Kale,4 Snehal Chopade,5 Vikas Pruthi,3 Gopal Kundu,4 Jayesh R Bellare,6 Balu A Chopade1,5 1Institute of Bioinformatics and Biotechnology, University of Pune, 2National Chemical Laboratory, Pune, 3Department of Biotechnology, Indian Institute of Technology, Roorkee, 4National Centre for Cell Science, Pune University Complex, 5Department of Microbiology, University of Pune, Pune, 6Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai, India Background: Nanoparticles (NPs) have gained significance in medical fields due to their high surface-area-to-volume ratio. In this study, we synthesized NPs from a medicinally important plant – Plumbago zeylanica. Materials and methods: Aqueous root extract of P. zeylanica (PZRE) was analyzed for the presence of flavonoids, sugars, and organic acids using high-performance thin-layer chromatography (HPTLC), gas chromatography-time of flight-mass spectrometry (GC-TOF-MS), and biochemical methods. The silver NPs (AgNPs), gold NPs (AuNPs), and bimetallic NPs (AgAuNPs) were synthesized from root extract and characterized using ultraviolet-visible spectra, X-ray diffraction (XRD), energy-dispersive spectrometry (EDS), transmission electron microscopy (TEM), and dynamic light scattering (DLS). The effects of these NPs on Acinetobacter baumannii, Staphylococcus aureus, and Escherichia coli biofilms were studied using quantitative biofilm inhibition and disruption assays, as well as using fluorescence, ­scanning electron microscopy, and atomic force microscopy. Results: PZRE showed the presence of phenolics, such as plumbagin, and flavonoids, in addition to citric acid, sucrose, glucose, fructose, and starch, using HPTLC, GC-TOF-MS, and quantitative analysis. Bioreduction of silver nitrate (AgNO3) and chloroauric acid (HAuCl4) were confirmed at absorbances of 440 nm (AgNPs), 570 nm (AuNPs), and 540 nm (AgAuNPs), respectively. The maximum rate of synthesis at 50°C was achieved with 5 mM AgNO3 within 4.5 hours for AgNPs; and with 0.7 mM HAuCl4 within 5 hours for AuNPs. The synthesis of AgAuNPs, which completed within 90 minutes with 0.7 mM AgNO3 and HAuCl4, was found to be the fastest. Fourier-transform infrared spectroscopy confirmed bioreduction, while EDS and XRD patterns confirmed purity and the crystalline nature of the NPs, respectively. TEM micrographs and DLS showed about 60 nm monodispersed Ag nanospheres, 20–30 nm Au nanospheres adhering to form Au nanotriangles, and about 90 nm hexagonal blunt-ended AgAuNPs. These NPs also showed antimicrobial and antibiofilm activity against E. coli, A. baumannii, S. aureus, and a mixed culture of A. baumannii and S. aureus. AgNPs inhibited biofilm in the range of 96%–99% and AgAuNPs from 93% to 98% in single-culture biofilms. AuNPs also showed biofilm inhibition, with the highest of 98% in S. aureus. AgNPs also showed good biofilm disruption, with the highest of 88% in A. baumannii. Conclusion: This is the first report on rapid and efficient synthesis of AgNPs, AuNPs and AgAuNPs from P. zeylanica and their effect on quantitative inhibition and disruption of bacterial biofilms. Keywords: P. zeylanica, AgNPs, AuNPs, AgAuNPs, biofilm inhibition and disruption, GC-TOF-M

Function of Integrin-Linked Kinase in Modulating the Stemness of IL-6–Abundant Breast Cancer Cells by Regulating γ-Secretase–Mediated Notch1 Activation in Caveolae

Interleukin-6 (IL-6) and Notch signaling are important regulators of breast cancer stem cells (CSCs), which drive the malignant phenotype through self-renewal, differentiation, and development of therapeutic resistance. We investigated the role of integrin-linked kinase (ILK) in regulating IL-6–driven Notch1 activation and the ability to target breast CSCs through ILK inhibition. Ectopic expression/short hairpin RNA-mediated knockdown of ILK, pharmacological inhibition of ILK with the small molecule T315, Western blot analysis, immunofluorescence, and luciferase reporter assays were used to evaluate the regulation of IL-6–driven Notch1 activation by ILK in IL-6–producing triple-negative breast cancer cell lines (MDA-MB-231, SUM-159) and in MCF-7 and MCF-7IL-6 cells. The effects of ILK on γ-secretase complex assembly and cellular localization were determined by immunofluorescence, Western blots of membrane fractions, and immunoprecipitation. In vivo effects of T315-induced ILK inhibition on CSCs in SUM-159 xenograft models were assessed by mammosphere assays, flow cytometry, and tumorigenicity assays. Results show that the genetic knockdown or pharmacological inhibition of ILK suppressed Notch1 activation and the abundance of the γ-secretase components presenilin-1, nicastrin, and presenilin enhancer 2 at the posttranscriptional level via inhibition of caveolin-1-dependent membrane assembly of the γ-secretase complex. Accordingly, knockdown of ILK inhibited breast CSC-like properties in vitro and the breast CSC subpopulation in vivo in xenograft tumor models. Based on these findings, we propose a novel function of ILK in regulating γ-secretase–mediated Notch1 activation, which suggests the targeting of ILK as a therapeutic approach to suppress IL-6–induced breast CSCs