Intranucleosomal DNA Fragmentation Research Articles

Synthesis of Polyphenon-60 Functionalized Bimetallic Ag–Pt Nanostructures that Inhibit Proliferation of SiHa Cells

Bimetallic nanostructures exhibit unique catalytic and optical properties compared with monometallic nanostructures. However, the biological properties of bimetallic nanostructures have not been explored completely. In the present investigation, we developed an environmentally benign, cost-effective and facile one-pot synthetic route for bimetallic Ag–Pt nanostructure fabrication and functionalization. This synthetic route is an alternative to conventional chemical and physical methods. Polyphenon-60 was employed as reducing and functionalizing agents for the reduction of metal ions to Ag–Pt nanostructures at ambient temperature. Powder XRD results confirmed the crystalline phase of the Ag–Pt to be face-centered cubic. Transmission electron microscopy investigations of the Ag–Pt morphology suggested the formation of spherical and well dispersed Ag–Pt nanostructures 50–75 nm in size. The results of EDX analysis indicated that the prepared nanostructures were highly pure. An MTT colorimetric assay for measuring cytotoxic effects revealed that the Ag–Pt structures induced dose-dependent cell death in SiHa cells via apoptosis. The Ag–Pt nanostructures caused apoptotic cell death and intranucleosomal DNA fragmentation in SiHa cells. In the cell cycle, the percentage of cells in the G0/G1 and G2/M phases significantly increased in the treated SiHa cells, which suggests that the Ag–Pt structures induced cell cycle arrest in the G2/M phase. The polyphenon-60 functionalized Ag–Pt nanostructures can be applicable for cancer treatment.

Eco-friendly synthesis and characterization of platinum-copper alloy nanoparticles induce cell death in human cervical cancer cells

Bimetallic nanoparticles are considered next generation materials with synergistic optical, electrical and catalytic properties. They are composed of two different metals and exhibit superior performance compared with their monometallic nanoparticles. However, the effects of bimetallic nanoparticles on cells and organisms are unknown. Specifically, the effects of platinum-copper alloy nanoparticles (Pt@Cu) on human cervical cancer cell (SiHa) viability, cytological morphology and cell cycle are not yet well studied. In the present study, we have developed a green synthetic route to synthesize Pt@Cu using Polyphenon 60 as a reducing as well as a functionalizing agent. The synthesized Pt@Cu morphological and compositional properties were analyzed using high-resolution transmission electron microscopy and energy dispersive X-ray spectrometer, respectively. The electron microscopic images indicated the formation of spherical Pt@Cu aggregates, ∼30nm in size. The cell viability assay revealed that Pt@Cu inhibit the cell proliferation and enhanced nuclear morphological changes including cell shrinkage, intranucleosomal DNA fragmentation and chromatin condensation in SiHa cells. The cell cycle progression results suggest that Pt@Cu significantly increase the proportion of cells in the Sub G0 phase, which clearly indicates Pt@Cu induced Sub G0 phase arrest. Our finding suggests that bimetallic nanoparticles open up new opportunities for cancer therapeutics applications.

Green synthesis of bimetallic Au@Pt nanostructures and their application for proliferation inhibition and apoptosis induction in human cervical cancer cell.

Bimetallic Au@Pt nanostructures (Au@Pts) are potential candidates for optical, electrical, catalytic and biological applications. However, methods for the fabrication of Au@Pts using total tea polyphenols (TPPs), studies of the mechanism of action of Au@Pts on biological systems and studies on the application of Au@Pts in cancer diagnosis and therapy are sparse. In this study, we developed a simple, eco-friendly and low-cost method for the synthesis of Au@Pts to examine the cytotoxic effect of these Au@Pts on human cervical cancers in vitro. The gold and platinum ions were successfully reduced simultaneously using TPPs at room temperature. The prepared Au@Pts were characterized using UV-Vis spectrophotometery, X-ray diffractometery (XRD), energy-dispersive X-ray spectroscopy (EDS), and transmission electron microscopy (TEM). EDS and XRD confirmed the formation of the Au@Pt. Formation of Au@Pts with a size of 5-20 nm was confirmed using TEM. The cytotoxic properties of the Au@Pts were evaluated in human cervical cancer cells (SiHa). The cell viability results revealed that Au@Pts induce cell death in a dose- and time-dependent manner. The morphological features of the Au@Pt-exposed SiHa cells were observed and indicated cell death via cell shrinkage, intranucleosomal DNA fragmentation and chromatin condensation. During progression of the different phases of the cell cycle, the proportion of cells in the G2/M phase of the treated SiHa cells was significantly increased, which strongly confirmed that the Au@Pts induced apoptosis through the G2/M phase check points. Our findings demonstrate the activity of Au@Pts against cervical cancer cells and reveal strategies for the development of highly active bimetallic nanostructures for cancer therapeutics.

A putative link between phagocytosis-induced apoptosis and hemocyanin-derived phenoloxidase activation

Apoptosis and phagocytosis are crucial processes required for developmental morphogenesis, pathogen deterrence and immunomodulation in metazoans. We present data showing that amebocytes of the chelicerate, Limulus polyphemus, undergo phagocytosis-induced cell death after ingesting spores of the fungus, Beauveria bassiana, in vitro. The observed biochemical and morphological modifications associated with dying amebocytes are congruent with the hallmarks of apoptosis, including: extracellularisation of phosphatidylserine, intranucleosomal DNA fragmentation and an increase in caspase 3/7-like activities. Previous studies have demonstrated that phosphatidylserine is a putative endogenous activator of hemocyanin-derived phenoloxidase, inducing conformational changes that permit phenolic substrate access to the active site. Here, we observed extracellular hemocyanin-derived phenoloxidase activity levels increase in the presence of apoptotic amebocytes. Enzyme activity induced by phosphatidylserine or apoptotic amebocytes was reduced completely upon incubation with the phosphatidylserine binding protein, annexin V. We propose that phosphatidylserine redistributed to the outer plasma membrane of amebocytes undergoing phagocytosis-induced apoptosis could interact with hemocyanin, thus facilitating its conversion into a phenoloxidase-like enzyme, during immune challenge.

Naringin induces death receptor and mitochondria-mediated apoptosis in human cervical cancer (SiHa) cells

Cervical cancer is the second most common female cancer worldwide, and it remains a challenge to manage preinvasive and invasive lesions. Fruit-based cancer prevention entities, such as flavonoid and their derivatives, have demonstrated a marked ability to inhibit preclinical models of epithelial cancer cell growth and tumor formation. Here, we extend the role of naringin-mediated chemoprevention to that of cervical carcinogenesis. The present study sought to investigate the therapeutic potential effect of naringin on apoptosis in human cervical SiHa cancer cells. Viability of SiHa cells was evaluated by the MTT assay, apoptosis and mitochondrial transmembrane potential by flow cytometry, and pro-apoptotic related genes by Real-time quantitative PCR. Naringin showed a 50% inhibition of SiHa human cervical cancer cells at a concentration of 750μM. SiHa cells exhibited apoptotic cell death, intranucleosomal DNA fragmentation, morphological changes and decline in the mitochondrial transmembrane potential. In addition, administration of naringin increased the expression of caspases, p53 and Bax, Fas death receptor and its adaptor protein FADD. These results suggest that the induction of apoptosis by naringin is through both death-receptor and mitochondrial pathways. Taken together, our results suggest that naringin might be an effective agent to treat human cervical cancer.

Gamma-Ray Susceptibility of Immature and Mature Hippocampal Cultured Cells

Ionizing radiation suppresses neurogenesis in the mammalian brain. This in vitro study compared the detrimental effect of acute gamma-irradiation on immature hippocampal cells with mature cells. Both rat immature (0.5 day in vitro (DIV)) and mature hippocampal cells (14 DIV) were irradiated with 0-4 Gy gamma-rays. Cell viability was analyzed by using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. DNA fragmentation study was performed by extracting intracellular DNA. Morphological features of apoptosis were characterized by 4',6-diamidine-2'-phenylindole, dihydrochloride (DAPI) staining. MTT assay revealed that the survival rate of immature hippocampal cells declined in a dose-dependent manner within the range of irradiation applied, but was not changed in mature cells. Intranucleosomal DNA fragmentation in a ladder like pattern was dose-dependently increased in immature cells, but not in mature cells. The number of apoptotic nuclei in immature cells increased significantly in a dose-dependent manner within the range of irradiation applied. Active caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP) expressions in immature hippocampal cells at 6 hr after 2 Gy exposure were markedly higher than control levels. The significantly greater radiosensitivity of immature hippocampal cells than that of the mature cells, indicates that the susceptibility of such hippocampal cells depends on their maturation. In addition, gamma-irradiation may induce caspase-dependent apoptosis in immature hippocampal cells.

Induction of Ca 2+ signal mediated apoptosis and alteration of IP3R1 and SERCA1 expression levels by stress hormone in differentiating C2C12 myoblasts

Glucocorticoid (GC) are stress hormones, whose cytotoxicity has been shown in various cells. The imbalance of calcium homeostasis is believed to be associated with the dexamethasone (DEX, a synthetic GC)-induced apoptosis. Here we show that in C2C12 myoblasts, DEX markedly up-regulated the expression of inositol 1,4,5-triphosphate receptor 1 (IP3R1) and down-regulated the expression of SERCA1 (sarcoendoplasmic reticulum Ca 2+-ATPase 1), leading to calcium overload. Furthermore, the imbalance of calcium homeostasis increased the level of BAX, decreased the level of Bcl-2, induced cytochrome c release and activated caspase-3, leading to intranucleosomal DNA fragmentation and plasma membrane damage, eventually resulting in cell apoptosis. Taken together, by using C2C12 myoblasts as a model system, we demonstrated a novel mechanism for stress hormone-induced apoptosis: it is dependent on the induction of intracellular calcium overload via the alterations of IP3R1 and SERCA1 expressions.

Intratympanic Treatment of Acute Inner Ear Disorders with AM-111: A New Cell-Permeable JNK Ligand

Background and Aim: Acute inner ear disorders like sudden deafness, acoustic trauma, tinnitus, and others are thought to initiate complex biochemical processes in the sensory hair cells that can lead to apoptosis. Apoptosis is an active process that is characterized by chromatin condensations, intracellular fragmentation, and intranucleosomal DNA fragmentation. The molecular mechanism behind it is the JNK (c-Jun N-terminal kinase) group of mitogen-activated protein kinases (MAPK), also known as stress-activated protein kinases. In different animal models, the intratympanic administration of AM-111, a cell-permeable JNK ligand also known as D-JNKI, has been shown to prevent hearing loss after acute acoustic trauma. Functional and morphological analysis of the treated ears revealed excellent otoprotective properties that could be obtained even when AM-111 was administered hours after the noise exposure. Blocking the signal pathway with D-JNKI-1 is therefore a promising way to protect the morphological integrity and physiological function of the inner ear.

Immunomodulatory role of piperine in cadmium induced thymic atrophy and splenomegaly in mice

Cadmium being a potent immunotoxicant, affects both humoral and cell mediated immunity. In rodents, it is primarily characterized by marked thymic atrophy and splenomegaly. Cadmium induces apoptosis in mice and suppresses the immune functions. Piperine, major alkaloid of Piper longum Linn. and Piper nigrum Linn. with a long history of medicinal value, has shown anti-apoptotic activity in vitro. Thus, to delineate its role in vivo, piperine (2.5 mg/kg/day, oral, 7 days) treated Balb/C mice were administered Cd as CdCl 2 (1.8 mg/kg, i.p., once, 4th day). The various biochemical indexes of cell damage such as cytotoxicity (MTT assay), oxidative stress (glutathione, reactive oxygen species), apoptosis (mitochondrial membrane potential, caspase-3 activity, phosphatidylserine externalization, apoptotic DNA, intranucleosomal DNA fragmentation) along with lymphocyte phenotyping, cell proliferative response and cytokine secretion (IL-2 and IFNγ) were assessed in thymic and splenic single cell suspensions. Lowering of body weight gain and cellularity and a loss in cell viability seen in Cd group, were abrogated by piperine treatment. Similarly, oxidative stress and apoptotic markers altered by Cd were also modulated by this alkaloid. In addition, a pronounced inhibition of cell proliferative response, alterations in T- and B-cell phenotypes, cytokine release and morphological changes were restored to normalcy. The present in vivo data corroborating with our previous in vitro findings, provide confirmatory evidence of the immuno-protective efficacy of piperine.

Apoptosis induced by 2-acetyl-3-(6-methoxybenzothiazo)-2-yl-amino-acrylonitrile in human leukemia cells involves ROS-mitochondrial mediated death signaling and activation of p38 MAPK

Benzothiazoles are multitarget agents with broad spectrum of biological activity. 2-Acetyl-3-(6-methoxybenzothiazo)-2-yl-amino-acrylonitrile (AMBAN) is a new synthetically prepared derivative, which in our previous study showed cytotoxic effects towards tumor cells. The aim of the present study was to examine the antiproliferative and apoptosis inducing activities of AMBAN towards human leukemia HL60 and U937 cells. Further, the molecular mechanism involved in AMBAN-induced apoptosis was investigated. Benzothiazole inhibited the growth and induced programmed cell death of HL60 and U937 cells. In addition, AMBAN elevated the level of reactive oxygen species, decreased the mitochondrial membrane potential, activated caspases 9 and 3, induced the cytochrome c release and PARP cleavage and led to intranucleosomal DNA fragmentation. Further, p38 MAPK was associated with the apoptotic activity of AMBAN. It can be concluded that AMBAN-induced apoptosis in HL60 and U937 cells through mitochondrial/caspase 9/caspase 3-dependent pathway.

The role of apoptosis in Listeria monocytogenes neural infection: Listeriolysin O interaction with neuroblastoma Neuro-2a cells

Listeria monocytogenes is the etiological agent of meningitis that affects individuals at high risk such as pregnant women, neonates, the elderly and immunocompromised individuals. Infection by this intracellular pathogen can be lethal if not diagnosed and treated. Mouse neuroblastoma Neuro-2a cells, a neuron-like cell line, were infected with L. monocytogenes. In this study apoptotic changes of neuroblastoma Neuro-2a cells infected with strains of Listeria producing different listeriolysin O levels are investigated by cytotoxicity assay, cellular viability assay, DAPI staining, intranucleosomal DNA fragmentation test, and monoclonal antibodies against ss-DNA. Results show that after internalization, the bacteria induced morphological, functional and genetic changes in the cells characteristic of apoptosis, which was dose-and time-dependent on listeriolysin O. Neuroblastoma Neuro-2a cells represent an interesting model cell line to further the understanding of Listeria pathogenesis within the central nervous system.

Role of oxidative stress and apoptosis in cadmium induced thymic atrophy and splenomegaly in mice

Cadmium immunotoxicity in rodents is primarily characterized by marked thymic damage and splenomegaly. To understand the toxicity of Cd on lymphoid cells in vivo, a single dose of Cd as CdCl 2 (1.8 mg/kg, i.p.) was administered to male BALB/c mice and cytotoxicity (MTT assay), oxidative stress indicators (glutathione, reactive oxygen species) and apoptotic markers (mitochondrial membrane potential, caspase-3 activity, phosphatidylserine externalization, apoptotic DNA, intranucleosomal DNA fragmentation) were assessed in thymic and splenic single cell suspensions, at various time intervals. Lowering of body weight gain and cellularity and a loss in cell viability was seen in the Cd treated mice. The earliest significant increase in ROS at 18 h, followed by mitochondrial membrane depolarization, caspase-3 activation and GSH depletion at 24 h in spleen and later at 48 h in thymus, strongly implicate the possible involvement of ROS. A pronounced inhibition of cell proliferative response at 48 h and 72 h may also be linked to Cd induced apoptosis. The morphological alterations including thymic cortical cell depletion and an increase in red pulp with diminished white pulp in spleen were observed at 48 h and beyond. The splenic cells appeared more susceptible than thymus cells to the adverse effects of Cd. The present study, therefore, demonstrates potentiation of oxidative stress followed by mitochondrial-caspase dependent apoptotic pathway. This may, in part, be responsible for causing suppression of cell proliferative response, thymic atrophy and splenomegaly.

C18:3-GM1a induces apoptosis in Neuro2a cells: enzymatic remodeling of fatty acyl chains of glycosphingolipids

GM1a [Gal beta1-3GalNAc beta1-4(NeuAc alpha2-3)Gal beta1-4Glc beta1-1Cer] is known to support and protect neuronal functions. However, we report that alpha-linolenic acid-containing GM1a (C18:3-GM1a), which was prepared using the reverse hydrolysis reaction of sphingolipid ceramide N-deacylase, induced apoptosis in neuronal cells. Intranucleosomal DNA fragmentation, chromatin condensation, and caspase activation, all typical features of apoptosis, were observed when mouse neuroblastoma Neuro2a cells were cultured with C18:3-GM1a but not GM1a containing stearic acid (C18:0) or oleic acid (C18:1). The phenotype of Neuro2a cells induced by C18:3-GM1a was similar to that evoked by lyso-GM1a. However, lyso-GM1a caused a complete disruption of lipid microdomains of Neuro2a cells and hemolysis of sheep erythrocytes, whereas C18:3-GM1a did neither. C18:3-GM1a, but not lyso-GM1a, was found to be abundant in lipid microdomains after the removal of loosely bound GM1a by BSA. The activation of stress-activated protein kinase/c-Jun N-terminal kinase in Neuro2a cells was observed with lyso-GM1a but not C18:3-GM1a. These results indicate that the mechanism of apoptosis induced by C18:3-GM1a is distinct from that caused by lyso-GM1a. This study also clearly shows that fatty acid composition of gangliosides significantly affected their pharmacological activities when added to the cell cultures and suggests why naturally occurring gangliosides do not possess polyunsaturated fatty acids as a major constituent.

Novel Peptide Treated Macrophage Induces Apoptosis in Tumor Cell Line P815

In vitro macrophages treated with novel peptides have been shown to develop enhanced tumoricidal activity against tumor target cell (P815), though the exact mechanism is not known. In the present study, we have investigated the mechanism involved in the tumor cell cytotoxicity mediated by novel peptides treated macrophage and involvement of possible effectors molecule. Peritoneal exudated macrophages treated with LPS, peptides, and LPS plus peptides and when cocultured with tumor cell P815 caused tumor cell death by induction of apoptosis. The results of our experiment reveal a specific pattern of intranucleosomal DNA fragmentation detected by agarose gel electrophoresis and also with microscopic examination of the cells revealed nuclear alteration characteristic of apoptosis. Viability studies showed that most of the cells undergoing apoptosis were found to be non-viable even after 24 h coculture. Macrophage induced apoptosis in tumor target cells even in the absence of cell to cell contact through diffusible effectors molecule. The study thus shows that the novel peptide treated macrophage can kill tumor cell P815 by extracellular release of effectors molecule NO (nitric oxide) that act by inducing apoptosis in a target cell-specific manner.

Apoptosis and cell cycle disturbances induced by coumarin and 7-hydroxycoumarin on human lung carcinoma cell lines

Coumarin and 7-hydroxycoumarin have anti-tumour actions in vitro and in vivo. There are no previous reports on the cytostatic and apoptotic actions of coumarin and 7-hydroxycoumarin in non-small cell lung carcinoma (NSCLC) cell lines. Here we report on: (1) the inhibition of cell proliferation, (2) the phase in which cell cycle arrest occurs, and (3) the induction of apoptosis. Inhibition of cell proliferation was determined by 3 H -thymidine incorporation. The effects on cell cycle phases were determined at 100 μg/ml of coumarin or 7-hydroxycoumarin using propidium iodide and flow cytometry. Higher concentrations were used to study apoptosis, detected by: (1) morphological cell changes, (2) sub G 1 peak detection and (3) Annexin-V assay. Peripheral blood mononuclear cells (PBMC) stimulated with phytohemagglutinin were used as controls. The actions of these compounds depended on drug concentrations and on histological cell type. Coumarin and 7-hydroxycoumarin inhibited cell growth by inducing cell cycle arrest in the G 1 phase in all the lung carcinoma cell lines. Apoptosis required large concentrations of the coumarin compounds and was observed in adenocarcinomas. Apoptosis was not associated with intra-nucleosomal DNA fragmentation. Apoptosis was not observed in squamous lung carcinoma cell lines, but an increase in G 1 cell cycle arrest was detected. In PBMC, only large concentrations of the coumarin compounds elicited a cystostatic action. Coumarins in combination with other anti-neoplastic drugs might increase the effectiveness of NSCLC treatments.

In Vitro Cytotoxic Effects of Stabilizing Sugars within Human Intravenous Immunoglobulin Preparations against the Human Macrophage THP-1 Cell-line

Intravenous immunoglobulin (IVIg) is a safe and effective therapy for the treatment of primary and secondary humoral immune deficiencies and autoimmune disorders. Both minor and more serious side effects may occur following IVIg administration in approximately 1-15% of infusions and stabilizing sugars found in IVIg preparations may contribute some of these. In this report, we aimed to determine the cytotoxic effects of IVIg as compared with four stabilizing sugars (glucose, sucrose, maltose and D-sorbitol) found in IVIg preparations on human monocyte-macrophages. The human THP-1 macrophage cell-line was used as a model to determine the effects of stabilizing sugars and IVIg preparations on cell viability and growth. The sugars differentially affected the viability of THP-1 cells. In experiments using doses of the sugars commonly found in IVIg preparations, cell viability and proliferation was unaffected when compared with doses of IVIg typically administered to patients (5 mg/ml). However, in an LDH-release cell lysis assay that measures changes in cell permeability, glucose (50 mg/ml) induced significant release of LDH as compared with complete IVIg (5 mg/ml, p<0.0001). Intranucleosomal DNA fragmentation was not detected at therapeutically relevant doses of IVIg. This suggested that THP-1 cell death was not due to apoptosis. We conclude that osmotic stress mediated by the sugars at high doses promoted THP-1 cell death. We propose that IVIg per se is not cytotoxic to the autonomously growing human THP-1 cell-line but rather, the stabilizing sugars used in the preparations are the cytotoxic factors. This observation was evident when preparations of IVIg were used at high concentrations but not at levels one would associate with clinically relevant doses of IVIg.

Apoptosis of Neuro2a cells induced by lysosphingolipids with naturally occurring stereochemical configurations

Lysosphingolipids, which lack the fatty acid moiety of sphingolipids, are known to be accumulated in some variants of sphingolipid storage diseases. Here, we report that lysosphingolipids with naturally occurring stereochemical configurations induce apoptosis in mouse neuroblastoma Neuro2a cells. The intracellular dehydrogenase activity and [3H]thymidine incorporation of Neuro2a cells were strongly suppressed by the addition of lysosphingolipids in a dose-dependent manner, whereas the parental sphingolipids had no effect. Intra-nucleosomal DNA fragmentation, chromatin condensation, and phosphatidylserine externalization, which are typical features of apoptosis, were observed when the cells were cultured with 40–80 μM of lysosphingolipids for 24–48 h in the presence of 5% fetal calf serum. Activation of caspase-3-like enzyme occurred after addition of lysosphingolipids followed by incubation at 37 °C for 24 h. The addition of an inhibitor of caspases, ZVAD-fmk, to the Neuro2a cell culture completely inhibited the elevation of caspase-3 activity but not the DNA fragmentation. These results may indicate that a caspase-3 independent signaling pathway is involved in the lysosphingolipid-induced apoptosis and suggest that accumulation of lysosphingolipids, but not parental sphingolipids, triggers the apoptotic cascade in neuronal cells of patients with sphingolipidoses. —Sueyoshi, N., T. Maehara, and M. Ito. Apoptosis of Neuro2a cells induced by lysosphingolipids with naturally occurring stereochemical configurations.

Cycloprodigiosin hydrochloride, a H+/Cl- symporter, induces apoptosis in human colon cancer cell lines in vitro.

Recently, we reported that cycloprodigiosin hydrochloride (cPrG.HCl), a novel H+/Cl- symporter, induces acidification of the cytosol and leads to apoptosis on rat and human liver cancer cells. In the present study, the effects of cPrG.HCl, a H+/Cl- symporter, were examined in colon cancer cell lines in vitro. In the MTT assay, cPrG.HCl inhibited the growth of two colon cancer cell lines (WiDr and SW480) in a dose- and time-dependent manner. The cPrG.HCl treatment of both types of cells induced apoptosis as confirmed by the appearance of a sub-G1 population and intranucleosomal DNA fragmentation. In addition, cPrG.HCl lowered pHi (below pH 6.8) respectively. Therefore, these results suggest that cPrG.HCl may be useful for the treatment of colon cancer cells.

Nitric Oxide Activates Apoptosis Signal - Regulating Kinase 1 (ASK 1) Forming S-Nitrosothiols with Reactive Thioredoxin SH-Groups. Nitric Oxide Synthase (NOS) Induction Mediated Inhibition of Poly-(ADP-RIBOSE)-Polymerase (PARP) and Protein Kinase C (PKC) Activities as well as Intranucleosomal DNA Fragmentation (IDF) Increase in the HEP-2 Cells.

INTRODUCTION. ASK 1 was recently identified as mitogen activated protein (MAP) kinase kinase kinase and activates p53, Jun-N-terminal kinase and stress activated protein kinases through MAP kinase kinase cascade phosphorylation. These factors cause the induction of apoptosis. Mammalian thioredoxin was established to be the direct inhibitor of ASK 1. It has been reported that superoxide activates ASK 1 forming sulfinic ions with thioredoxin SHgroups and making ASK 1 free. We assumed that nitric oxide (NO) may interact with thioredoxin SH-groups forming S-nitrosothiols. It was found that NO mediates apoptosis through p53 activation and mitochondrial dysfunction. The aim of this work was to study if nitric oxide activates ASK 1 forming S-nitrosothiols with reactive thioredoxin SH-groups and also the effects of the induction of NOS activity in the HEP-2 cells on PARP and PKC activities as well as IDF.

Cycloprodigiosin hydrochloride, a new H(+)/Cl(-) symporter, induces apoptosis in human and rat hepatocellular cancer cell lines in vitro and inhibits the growth of hepatocellular carcinoma xenografts in nude mice.

The effects of cycloprodigiosin hydrochloride (cPrG-HCl), a new H(+)/Cl(-) symporter, were examined in liver cancer cell lines in vitro and in vivo. In the in vitro MTT assay, cPrG-HCl inhibited the growth of 6 liver cancer cell lines (Huh-7, HCC-M, HCC-T, dRLh-84, and H-35, hepatocellular carcinoma; HepG2, hepatoblastoma) in a dose- and time-dependent manner. The 50% inhibitory concentrations (IC(50)) at 72 hours' treatment for liver cancer cell lines were 276 to 592 nmol/L, while that for isolated normal rat hepatocyte was 8.4 micromol/L. The cPrG-HCl treatment of Huh-7 cells induced apoptosis as confirmed by the appearance of a subG(1) population, intranucleosomal DNA fragmentation, and chromatin condensation. cPrG-HCl raised the pH of acidic organelles and lowered pHi (below pH 6.8). In addition, the apoptosis in Huh-7 cells induced by cPrG-HCl was strongly suppressed when the cells were cultured with imidazole, a cell-permeable base. In the in vivo assay, nude mice bearing subcutaneous xenografted Huh-7 cells received 2 weeks of treatment with cPrG-HCl (1 or 10 mg/kg/d) subcutaneously. This treatment significantly inhibited tumor growth compared with the control after 8 days. The control mice were treated with 1% dimethylsulfoxide (DMSO) in saline (vehicle). A histopathological examination using the terminal deoxynucleotidyl transferase mediated dUTP biotin nick end labeling (TUNEL) method showed apoptosis in the treated tumor cells. No pathological changes were observed in any organs, and the serum alanine transaminase levels remained within normal limits. These results suggest that cPrG-HCl may be useful for the treatment of hepatocellular carcinoma.