Elp1 facilitates RAD51-mediated homologous recombination repair via translational regulation.

BACKGROUND: RAD51-dependent homologous recombination (HR) is one of the most important pathways for repairing DNA double-strand breaks (DSBs), and its regulation is crucial to maintain genome integrity. Elp1 gene encodes IKAP/ELP1, a core subunit of the Elongator complex, which has been implicated in translational regulation. However, how ELP1 contributes to genome maintenance is unclear. METHODS: To investigate the function of Elp1, Elp1-deficient mouse embryonic fibroblasts (MEFs) were generated. Metaphase chromosome spreading, immunofluorescence, and comet assays were used to access chromosome abnormalities and DSB formation. Functional roles of Elp1 in MEFs were evaluated by cell viability, colony forming capacity, and apoptosis assays. HR-dependent DNA repair was assessed by reporter assay, immunofluorescence, and western blot. Polysome profiling was used to evaluate translational efficiency. Differentially expressed proteins and signaling pathways were identified using a label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics approach. RESULTS: Here, we report that Elp1 depletion enhanced genomic instability, manifested as chromosome breakage and genotoxic stress-induced genomic DNA fragmentation upon ionizing radiation (IR) exposure. Elp1-deficient cells were hypersensitive to DNA damage and exhibited impaired cell proliferation and defective HR repair. Moreover, Elp1 depletion reduced the formation of IR-induced RAD51 foci and decreased RAD51 protein levels. Polysome profiling analysis revealed that ELP1 regulated RAD51 expression by promoting its translation in response to DNA damage. Notably, the requirement for ELP1 in DSB repair could be partially rescued in Elp1-deficient cells by reintroducing RAD51, suggesting that Elp1-mediated HR-directed repair of DSBs is RAD51-dependent. Finally, using proteome analyses, we identified several proteins involved in cancer pathways and DNA damage responses as being differentially expressed upon Elp1 depletion. CONCLUSIONS: Our study uncovered a molecular mechanism underlying Elp1-mediated regulation of HR activity and provides a novel link between translational regulation and genome stability.

Synthesis and biological evaluation of 2-amino-1-thiazolyl imidazoles as orally active anticancer agents.

Designed from a high throughput screened hit compound, novel 2-amino-1-thiazolyl imidazoles were synthesized and demonstrated cytotoxicity against human cancer cells. 1-(4-Phenylthiazol-2-yl)-4-(thiophen-2-yl)-1H-imidazol-2-amine (compound 2), a 2-amino-1-thiazolyl imidazole, inhibited tubulin polymerization, interacted with the colchicine-binding sites of tubulins, and caused cell cycle arrest at the G(2)/M phase in human gastric cancer cells. Disruption of the microtubule structure in cancer cells by compound 2 was also observed. Compound 2 concentration-dependently inhibited the proliferation of cancer cells in histocultured human gastric and colorectal tumors. Given orally, compound 2 prolonged the lifespans of leukemia mice intraperitoneally inoculated with the murine P388 leukemic cells. We report 2-amino-1-thiazolyl imidazoles as a novel class of orally active microtubule-destabilizing anticancer agents.

Antiangiogenic activities and cisplatin-combined antitumor activities of BPR0L075.

BACKGROUND: Antimitotic tubulin-binding BPR0L075 is structurally analogous to the vascular-disrupting combretastatin A-4. MATERIALS AND METHODS: In vitro/in vivo models of endothelial cells cultures, Matrigel plug assay, tumor-bearing nude mice, and murine leukemia cells-inoculated mice were utilized to evaluate BPR0L075 for antiangiogenic and antitumoral activity spectra. RESULTS: BPR0L075 concentration-dependently inhibited proliferation and migration of human umbilical vein endothelial cells (HUVECs), disrupted capillary tube formations of HUVECs and rat aorta endothelial cells, and suppressed in vivo VEGF-mediated angiogenesis in Matrigel plugs in mice. Besides inhibiting the colony growth of cancer cells, BPR0L075 suppressed growth of subcutaneously-xenografted human lung, colorectal, and cervical solid tumors in nude mice. Combination treatments of BPR0L075 plus cisplatin, compared to either agent alone, demonstrated a stronger growth inhibition against the tumor xenografts in nude mice and longer lifespan in the leukemia mice. CONCLUSION: BPR0L075 is an antitumoral and antiangiogenic agent and potentiates the anticancer activity of cisplatin.

Synthesis and biological activities of 2-amino-1-arylidenamino imidazoles as orally active anticancer agents.

2-Amino-1-arylidenaminoimidazoles, a novel class of orally (po) active microtubule-destabilizing anticancer agents, were synthesized. The compounds were designed from a hit compound identified in a drug discovery platform by using cancer cell-based high throughput screening assay. Selective synthesized compounds exerted cell cytotoxicity against human cancer cells. The underlying mechanisms for the anticancer activity were demonstrated as interacting with the tubulins and inhibiting microtubule assembly, leading to proliferation inhibition and apoptosis induction in the human tumor cells. Furthermore, two compounds showed in vivo anticancer activities in both po and intravenously (iv) administered routes and prolonged the life spans of murine leukemic P388 cells-inoculated mice. These new po active antimitotic anticancer agents are to be further examined in preclinical studies and developed for clinical uses.

Anti-inflammatory mechanisms of phenanthroindolizidine alkaloids.

The molecular mechanisms for the anti-inflammatory activity of phenanthroindolizidine alkaloids were examined in an in vitro system mimicking acute inflammation by studying the suppression of lipopolysaccharide (LPS)/interferon-gamma (IFNgamma)-induced nitric oxide production in RAW264.7 cells. Two of the phenanthroindolizidine alkaloids, NSTP0G01 (tylophorine) and NSTP0G07 (ficuseptine-A), exhibited potent suppression of nitric oxide production and did not show significant cytotoxicity to the LPS/IFNgamma-stimulated RAW264.7 cells, in contrast to their respective cytotoxic effects on cancer cells. Tylophorine was studied further to investigate the responsible mechanisms. It was found to inhibit the induced protein levels of tumor necrosis factor-alpha, inducible nitric-oxide synthase (iNOS), and cyclooxygenase (COX)-II. It also inhibited the activation of murine iNOS and COX-II promoter activity. However, of the two common responsive elements of iNOS and COX-II promoters, nuclear factor-kappaB (NF-kappaB) and adaptor protein (AP)1, only AP1 activation was inhibited by tylophorine in the LPS/IFNgamma-stimulated RAW264.7 cells. Further studies showed that the tylophorine enhanced the phosphorylation of Akt and thus decreased the expression and phosphorylation levels of c-Jun protein, thereby causing the subsequent inhibition of AP1 activity. Furthermore, the tylophorine was able to block mitogen-activated protein/extracellular signal-regulated kinase kinase 1 activity and its downstream signaling activation of NF-kappaB and AP1. Thus, NSTP0G01 exerts its anti-inflammatory effects by inhibiting expression of the proinflammatory factors and related signaling pathways.

Benzothiazolium compounds: novel classes of inhibitors that suppress the nitric oxide production in RAW264.7 cells stimulated by LPS/IFNgamma.

A series of benzothiazolium compounds were identified as novel classes of inhibitors of nitric oxide production in a cell culture system. They exhibited approximately 1600 folds potency with IC(50) at approximately 50nM to several microM as compared to IC(50) 88.4microM of l-NMMA, a known inhibitor of nitric oxide synthase. The mechanistic studies suggest that decreased iNOS protein synthesis and mRNA transcription, at least in part, were related to the inhibitory activity of effective benzothiazolium compounds. The correlation of in vivo and in vitro activities using mouse paw edema model was also demonstrated.

Concise synthesis and structure-activity relationships of combretastatin A-4 analogues, 1-aroylindoles and 3-aroylindoles, as novel classes of potent antitubulin agents.

The synthesis and study of the structure-activity relationships of two new classes of synthetic antitubulin compounds based on 1-aroylindole and 3-aroylindole skeletons are described. Lead compounds 3, 10, and 14 displayed potent cytotoxicities with IC50 = 0.9-26 nM against human NUGC3 stomach, MKN45 stomach, MESSA uterine, A549 lung, and MCF-7 breast carcinoma cell lines. The inhibition of proliferation correlated with in vitro polymerization inhibitory activities. Structure-activity relationships revealed that 6-methoxy substitution of 3-aroylindoles and 5-methoxy substitution of 1-aroylindoles contribute to a significant extent for maximal activity by mimicking the para substitution of the methoxy group to the carbonyl group in the case of aminobenzophenones. Addition of a methyl group at the C-2 position on the indole ring exerts an increased potency. The 3,4,5-trimethoxybenzoyl moiety was necessary for better activity but not essential and can be replaced by 3,5-dimethoxybenzoyl and 3,4,5-trimethoxybenzyl moieties. We conclude that 1- and 3-aroylindoles constitute an interesting new class of antitubulin agents with the potential to be clinically developed for cancer treatment.

Synthesis and in vitro cytotoxicity of 5-substituted 2-cyanoimino-4-imidazodinone and 2-cyanoimino-4-pyrimidinone derivatives.

A series of 5-substituted 2-cyanoimino-4-imidazodinone and 2-cyanoimino-4-pyrimidinone derivatives were synthesized and their anticancer cytotoxicity were evaluated in in vitro assay. It was found that the bulky aryl functionality in the 5-position of the 2-cyanoimino-4-imidazolidinone compounds was essential for the cytotoxicity of these heterocyclic compounds. Some of the derivatives exhibited modest cytotoxicity against a variety of cancer cell lines. One of the derivatives, [1-[6-(4-chlorophenoxy)hexyl]-5-oxo-4-phenyl-3-(4-pyridyl)tetrahydro-1H-2-imidazolyliden]aminomethanenitrile (Compound 11), exhibited the most potent cytotoxic activity with IC(50) in the nanomolar range. The cytotoxicity of these derivatives was selection with no apparent toxic effect toward normal fibroblasts.

Synthesis and biological evaluation of N-heterocyclic indolyl glyoxylamides as orally active anticancer agents.

A series of N-heterocyclic indolyl glyoxylamides were synthesized and evaluated for in vitro and in vivo anticancer activities. They exhibited a broad spectrum of anticancer activity not only in murine leukemic cancer cells but also in human gastric, breast, and uterus cancer cells as well as their multidrug resistant sublines with a wide range of IC(50) values. They also induced apoptosis and caused DNA fragmentation in human gastric cancer cells. Among the compounds studied, 7 showed the most potent activity of growth inhibition (IC(50) = 17-1711 nM) in several human cancer cells. Given orally, compounds 7 and 13 dose-dependently prolonged the survival of animals inoculated with P388 leukemic cancer cells. N-Heterocyclic indolyl glyoxylamides may be useful as orally active chemotherapeutic agents against cancer and refractory cancerous diseases of multidrug resistance phenotype.

Synthesis and anticancer evaluation of vitamin K(3) analogues.

Novel vitamin K(3) analogues were synthesized and evaluated for their anticancer activity. Compound 6, 9, 10, 11, 14, and (+/-)15 demonstrated a strong inhibitory activity against the tumor cells of A-549, Hep G2, MCF7, MES-SA, MES-SA/Dx5, MKN45, SW-480, and TW-039. Compound (+/-)15 displayed potent tumor cell cytotoxicity, and compound 14 selectively affected MCF7, even though it did not influence normal cells Detroit551 and WI-38. Compound (+/-)15 inhibited MES-SA and MES-SA/Dx5, and this specific result shows that compound (+/-)15 may become a good anticancer drug candidate.

Synthesis and structure-activity relationship of 2-aminobenzophenone derivatives as antimitotic agents.

A new type of inhibitor of tubulin polymerization was discovered on the basis of the combretastatin molecular skeleton. The lead compounds in this series, compounds 6 and 7, strongly inhibited tubulin polymerization in vitro and significantly arrested cells at the G(2)/M phase. Compounds 6 and 7 yielded 50- to 100-fold lower IC(50) values than did combretastatin A-4 against Colo 205, NUGC3, and HA22T human cancer cell lines as well as similar or greater growth inhibitory activities than did combretastain A-4 against DLD-1, HR, MCF-7, DU145, HONE-1, and MES-SA/DX5 human cancer cell lines. Structure-activity relationship information revealed that introduction of an amino group at the ortho position of the benzophenone ring plays an integral role for increased growth inhibition.