Potential Apoptosis Inducers Research Articles

ChemInform Abstract: NaSH in the Construction of Thiophene Ring Fused with N‐Heterocycles: A Rapid and Inexpensive Synthesis of Novel Small Molecules as Potential Inducers of Apoptosis.

AbstractThe first examples of the use of NaSH to construct a thiophene ring fused with N‐heterocycles via a SnAr‐type reaction—cyclization strategies are given.

ChemInform Abstract: Ligand‐Free MCR for Linking Quinoxaline Framework with a Benzimidazole Nucleus: A New Strategy for the Identification of Novel Hybrid Molecules as Potential Inducers of Apoptosis.

AbstractBenzimidazole functionalized quinoxalines are prepared in a multi‐component‐reaction.

A Pd(ii)-catalyzed C–H activation approach to densely functionalized N-heteroaromatics related to neocryptolepine and their evaluation as potential inducers of apoptosis

Bioactive indoloquinolines are synthesized via Pd(ii)-catalyzed intramolecular oxidative C–H alkenylation as the key step.

NaSH in the construction of thiophene ring fused with N-heterocycles: A rapid and inexpensive synthesis of novel small molecules as potential inducers of apoptosis

A facile construction of a thiophene ring fused with N-heterocycles has been achieved via the reaction of NaSH with 2-chloro-3-alkynyl quinoxalines/pyrazines leading to novel 2-substituted thieno[2,3-b]pyrazine/quinoxaline derivatives as potential inducers of apoptosis. Some of them showed encouraging pharmacological properties when tested in zebrafish.

Ligand-free MCR for linking quinoxaline framework with a benzimidazole nucleus: a new strategy for the identification of novel hybrid molecules as potential inducers of apoptosis.

We report a true MCR involving the reaction of N-(prop-2-ynyl)quinoxalin-2-amine derivatives with 2-iodoanilines and tosyl azide in the presence of 10 mol% of CuI and Et3N in DMSO to afford the pre-designed hybrid molecules containing quinoxaline framework linked with a benzimidazole nucleus. The MCR proceeds in the absence of any ligand and/or lateral addition of the catalyst/base affording products within 30 min in good yields, some of which showed encouraging apoptosis inducing properties in zebrafish.

Ruthenium(II) complexes as apoptosis inducers by stabilizing c-myc G-quadruplex DNA

Two ruthenium(II) complexes, [Ru(L)2(p-tFMPIP)](ClO4)2 (L = bpy, 1; phen, 2; p-tFMPIP = 2-(4-(trifluoromethyphenyl)-1H-imidazo[4,5f][1,10] phenanthroline)), were prepared by microwave-assisted synthesis technology. The inhibitory activity evaluated by MTT assay shown that 2 can inhibit the growth of MDA-MB-231 cells with inhibitory activity (IC50) of 16.3 μM, which was related to the induction of apoptosis. Besides, 2 exhibit low toxicity against normal HAcat cells. The inhibitory growth activity of both complexes related to the induction of apoptosis was also confirmed. Furthermore, the studies on the interaction of both complexes with c-myc G4 DNA shown that 1 and 2 can stabilize the conformation of c-myc G4 DNA in groove binding mode, which has been rational explained by using DFT theoretical calculation methods. In a word, this type of ruthenium(II) complexes can act as potential apoptosis inducers with low toxicity in clinic by stabilizing c-myc G4 DNA.

Abstract 825: Id3: Tumor suppressor of squamous cell carcinoma.

Abstract Skin cancer, the most common type of cancer with more than 1 million cases diagnosed in the US every year, occurs at least once to about half of Americans 65 years or older. The three main types of skin cancer—Basal Cell Carcinoma (BCC), Squamous Cell Carcinoma (SCC), and melanoma have mortality rates that are estimated to be 0.1%, 4.3% and 10% respectively in 2011. The Inhibitor of DNA Binding/Differentiation 3 (Id3) protein, a helix-loop-helix (HLH), dominant negative transcription factor, is one of four Id proteins found in mammals (Id1-4). The Id protein family plays a role in many cellular processes, including differentiation, proliferation, apoptosis, and angiogenesis. Previous studies in the Rosenthal lab have shown that Id3 over-expression in immortalized human keratinocytes induces apoptosis in part by activation of a Bax-caspase-9-caspase-3-dependent mitochondrial pathway via upregulation of bax promoter activity. To further elucidate the roles of Id3 in apoptosis, proliferation, and tumorigenesis, our studies focused on effects of induced Id3 expression on tumor growth in vivo. To investigate the role of Id3 in SCC, a tetracycline (Tet)-regulated expression (T-REx™) system was utilized to induce Id3 expression in A431 cells, a human malignant cervical SCC cell line. Immunoblot and RT-PCR analysis of A431-TRex-Id3 cells verified Id3 expression in the presence of Tet. When Id3 was induced by Tet in these cells in vitro, caspase-3 and -9 were activated. Cells were stably transfected with GFP to monitor tumor growth over thirty days by Maestro® in vivo imaging. Effects of Id3 on tumorigenesis in vivo was examined by subcutaneous injection of these cells onto athymic nude mice, half of which were fed with a doxycycline (Dox; a tetracycline derivative)-containing feed, leading to induced Id3 expression in the tumor, and half with a regular diet. Subcutaneously-injected A431 cells formed significantly (50%) smaller tumors upon Id3 induction. Immunofluorescent staining of tumor sections from Dox-fed mice revealed a 1.5-fold increase in active caspase-3 but no significant change in Ki-67, a proliferation marker. To investigate the mechanism of tumor shrinkage caused by Id3, microarray analysis was performed, and four genes were found to be significantly up- or down-regulated by Id3. Id3 deletion mutants were further created to study the domain(s) responsible for the decrease in tumor size. Mutation analysis showed that Id3 induces Max dimerization protein 3 (MXD3) protein expression through its HLH domain. Other candidate genes remain to be tested. The role of Id3 in SCC cell killing by cisplatin, a commonly used chemotherapeutic drug for SCC, is also currently under investigation. Our results together indicate that Id3 is a potential apoptosis inducer and therefore a therapeutic target in treating SCC. Citation Format: You-Shin Chen, Cynthia Rosenthal, Dean Rosenthal. Id3: Tumor suppressor of squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 825. doi:10.1158/1538-7445.AM2013-825

Cellular imaging assay for early evaluation of an apoptosis inducer

The objective of this study was to propose a feasibility of a cellular imaging assay as an alternative to the conventional cytotoxicity assay, such as MTS assay, for apoptosis monitoring. As an apoptosis monitoring parameter, affinity interaction between phosphatidylserine (PS) and annexin V was chosen. First, the specific binding affinity between annexin V and PS in phospholipid bilayers consisting of various molar (0-15%) composition of PS was measured using a surface plasmon resonance biosensor. As PS composition increased, the binding level of annexin V increased proportionally. Second, various concentrations (0.1-10 μM) of staurosporine were used as to induce apoptosis and introduced to MCF-7 breast carcinoma cells. The cellular fluorescence images from annexin V-FITC conjugate were obtained by confocal microscopy, and their fluorescence intensities were quantified by image scanning. Dose-apoptosis (or cell death) relationships were very similar to those from MTS and FACS assays. In summary, our cellular imaging method could serve as a quicker and simpler alternative to MTS (end point assay) and FACS (flow cytometry) to screen potential apoptosis inducers.

Indoloquinolizidine Derivatives as Novel and Potent Apoptosis Inducers and Cell‐Cycle Blockers

A collection of approximately 11 000 natural-product derived and inspired compounds was screened for potential apoptosis inducers in the human tumour cell lines HepG2 (liver), HeLa (cervix) and MCF-7 (breast) by means of MTT and ATP-luminescence assays, automated cell counting, caspase 3/7 assay as well as by fluorescence activated cell sorting (FACS) analysis. A group of seven indoloquinolizidine derivatives was identified that exhibited IC(50) values for cell proliferation as low as 2 mumol L(-1), with no major necrosis of cells detectable. At the same time, an increase in the rate of apoptosis of up to 600 % relative to the reference level was observed. FACS analysis indicated that these effects are related to an arrest of cells in the G(2)M phase of the cell cycle.

1,25-Dihydroxyvitamin D3, recombinant human transforming growth factor-beta 1, and recombinant human bone morphogenetic protein-2 induce in vitro differentiation of canine osteosarcoma cells.

Effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), recombinant human transforming growth factor (rhTGF)-beta 1 and recombinant human bone morphogenetic protein (rhBMP)-2 on differentiation in four different canine osteosarcoma cell lines (POS53B, 53C, 53D and 14A) were examined using markers specifically expressed by phenotypic osteoblasts. 1,25(OH)2D3 increased alkaline phosphatase (ALP) activity in one cell line, osteocalcin production in two lines and type I collagen production in three lines. RhTGF-beta 1 increased ALP activity in one clonal cell, osteocalcin production in one clonal cell and type I collagen production in two clonal cells. RhBMP-2 increased ALP activity in all clonal cells, osteocalcin production in two clonal cells and type I collagen production in three clonal cells. Thus, these agents induced differentiation in osteosarcoma cells at different efficacies. Electron microscopic study revealed that these agents increased cellular activity in all cell lines with no evidence of degeneration of cell organelle by drug cytotoxicity. In some cultures treated with either 1,25(OH)2D3 or rhBMP-2, apoptotic cells were observed. Based on the change in markers, rhBMP-2 and 1,25(OH)2D3 seemed to be more effective than rhTGF-beta 1. These agents are potential inducers of apoptosis.

Variable expression of apoptotic phenotype in keratinocytes treated with ultraviolet radiation, ceramide, or suspended in semisolid methylcellulose.

Apoptosis is a form of cellular suicide and is activated in various cells, including keratinocytes, in response to physiological and pathological stimuli. A current hypothesis holds that apoptotic cells in a concerted manner express characteristic phenotypic features comprising cytoplasmic budding, pyknosis, chromatin condensation, karyorhexis and internucleosomal DNA fragmentation. In this study we investigated the effects of different potential inducers of apoptosis on cultured human keratinocytes. Viability was determined with vital dyes (ethidium bromide, trypan blue), cell morphology was investigated with electron microscopy, and nuclear and DNA integrity was assessed with TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) or DNA gel electrophoresis. Irradiation with 50 mJ/cm2 ultraviolet B (UVB), treatment with C8 ceramide, or suspension in a semisolid medium caused apoptosis which was ultrastructurally different from necrotic induced by very high (300 mJ/cm2) doses of UVB. However, the phenotype of dying cells did not exhibit all typical features of apoptosis and cell morphology depended on the method used to induce apoptosis. Cells irradiated with ultraviolet B or treated with C8 ceramide developed large and small budding and DNA nicks but not chromatin condensation or classical karyorhexis. In UVB-irradiated cells a novel form of karyorhexis was observed manifested by formation of a few very small chromatin fragments in nuclear periphery. Cells suspended in methylcellulose developed DNA nicks and pyknosis, but not budding or karyorhexis. In neither case could the typical internucleosomal DNA fragments be detected by gel electrophoresis. These morphological results indicate that in keratinocytes induction of effect or death mechanisms is not concerted but depends on the stimulus inducing apoptosis.

Induction of apoptosis in human HaCaT keratinocytes.

Although cell death by apoptosis has been recognized as an important control mechanism in the maintenance of tissue homeostasis and in the elimination of cells with damaged DNA, information on the induction and characteristics of apoptosis in keratinocytes is rather scarce. Apoptotic mechanisms may play an important role in normal and disturbed homeostasis of the skin. In the present study, we therefore investigated the effects of several potential inducers of apoptosis in the human keratinocyte cell line HaCaT. Apoptosis was assessed with respect to morphological changes by light and electron microscopic examinations and to DNA integrity by a specific ELISA. UVB irradiation induced the morphology and internucleosomal DNA fragmentation characteristic of apoptosis in a dose- and time-dependent manner. Interferon-gamma caused DNA cleavage at the linker regions without producing morphological features consistent with apoptotic cell death. In contrast, treatment with dithranol and NP-40 resulted in necrotic alterations in the keratinocytes. Treatment with the calcium ionophore A23187 caused morphological changes which were similar to the characteristics of 'nonapoptotic programmed cell death'. Dexamethasone, tumor necrosis factor-alpha, transforming growth factor-beta, TPA, retinoic acid, the podophyllin derivative etoposide, the thromboxane A2 analogue U46619, cycloheximide, and the nitric oxide donors sodium nitroprusside and S-nitroso-glutathione, which are all known to induce apoptosis in other cell types, did not affect HaCaT keratinocytes. These results demonstrate that apoptosis can be induced in keratinocytes in vitro but the apoptosis differs from that in other cell types, such as haematopoietic cells, with regard to the type of inducer and/or the sensitivity of the target cells. Since keratinocytes are affected by numerous external and internal stimuli, they might posses several protective mechanisms to prevent apoptosis and to ensure the structural integrity of the outermost barrier of the body.

Dolichyl Phosphate, a Potent Inducer of Apoptosis in Rat Glioma C6 Cells

Exposure of rat glioma C6 cells to dolichyl phosphate resulted in cell shrinkage followed by nuclear fragmentation and internucleosomal cleavage of genomic DNA, yielding ladder patterns of oligonucleosomal fragments, all characteristics of apoptosis, This phenomenon occurred in a dose and time dependent manner. Dolichol and prenol failed to induce apoptosis. The inhibitors of N-glycosylation, tunicamycin and swainsonine had no apparent effect on dolichyl phosphate-induced apoptosis. Apoptotic changes were also observed in HL-60 cells, SIRC cells and HeLa cells. Thus, dolichyl phosphate functions as a potential apoptosis inducer as well as an essential carrier lipid in the biosynthesis of N-linked glycoprotein.

Induction of apoptosis in human HaCaT keratinocytes

Although cell death by apoptosis has been recognized as an important control mechanism in the maintenance of tissue homeostasis and in the elimination of cells with damaged DNA, information on the induction and characteristics of apoptosis in keratinocytes is rather scarce. Apoptotic mechanisms may play an important role in normal and disturbed homeostasis of the skin. In the present study, we therefore investigated the effects of several potential inducers of apoptosis in the human keratinocyte cell line HaCaT. Apoptosis was assessed with respect to morphological changes by light and electron microscopic examinations and to DNA integrity by a specific ELISA. UVB irradiation induced the morphology and internucleosomal DNA fragmentation characteristic of apoptosis in a dose- and time-dependent manner. Interferon-γ caused DNA cleavage at the linker regions without producing morphological features consistent with apoptotic cell death. In contrast, treatment with dithranol and NP-40 resulted in necrotic alterations in the keratinocytes. Treatment with the calcium ionophore A23187 caused morphological changes which were similar to the characteristics of ‘nonapoptotic programmed cell death’. Dexamethasone, tumor necrosis factor-α, transforming growth factor-β, TPA, retinoic acid, the podophyllin derivative etoposide, the thromboxane A2 analogue U46619, cycloheximide, and the nitric oxide donors sodium nitroprusside and S-nitroso-glutathione, which are all known to induce apoptosis in other cell types, did not affect HaCaT keratinocytes. These results demonstrate that apoptosis can be induced in keratinocytes in vitro but the apoptosis differs from that in other cell types, such as haematopoietic cells, with regard to the type of inducer and/or the sensitivity of the target cells. Since keratinocytes are affected by numerous external and internal stimuli, they might posses several protective mechanisms to prevent apoptosis and to ensure the structural integrity of the outermost barrier of the body.