Selective Induction Of Cell Death Research Articles

Methods for Investigating Transient Receptor Potential Melastatin-2 (TRPM2): A Cation Channel Activated by ADP-Ribose and Involved in Cell Death.

Transient receptor potential melastatin-2 (TRPM2) is an emerging chemotherapeutic target due to its involvement in poly(ADP-ribose) metabolism and the ability to induce anticancer effects after antagonism of its functions. Normally functioning as a nonspecific cation channel that is activated by free ADP-ribose, TRPM2 is involved with many cellular processes, including the induction of cell death after oxidative stress. What is becoming clear is that antagonism of TRPM2 selectively induces anticancer effects in several types of cancer. We previously demonstrated decreased growth and proliferation, increased levels of DNA damage, and the selective induction of cell death in breast cancer and melanoma cells. Due to these effects, it appears that TRPM2 has a novel role in cancer cells. Further, this novel role appears to involve nuclear function, because our studies, as well as those from other independent groups, demonstrate a nuclear localization of TRPM2 in various types of cancers. Thus, as an emerging therapeutic target, it is important to describe research techniques that can be utilized to analyze TRPM2 function, determine its effects in cancerous and noncancerous cells, and provide molecular biological methods to inhibit or downregulate its function.

Selective Induction of Cell Death in Human M1 Macrophages by Smac Mimetics Is Mediated by cIAP-2 and RIPK-1/3 through the Activation of mTORC.

Inflammatory macrophages have been implicated in many diseases, including rheumatoid arthritis and inflammatory bowel disease. Therefore, targeting macrophage function and activation may represent a potential strategy to treat macrophage-associated diseases. We have previously shown that IFN-γ-induced differentiation of human M0 macrophages toward proinflammatory M1 state rendered them highly susceptible to the cytocidal effects of second mitochondria-derived activator of caspases mimetics (SMs), antagonist of the inhibitors of apoptosis proteins (IAPs), whereas M0 and anti-inflammatory M2c macrophages were resistant. In this study, we investigated the mechanism governing SM-induced cell death during differentiation into M1 macrophages and in polarized M1 macrophages. IFN-γ stimulation conferred on M0 macrophages the sensitivity to SM-induced cell death through the Jak/STAT, IFN regulatory factor-1, and mammalian target of rapamycin complex-1 (mTORC-1)/ribosomal protein S6 kinase pathways. Interestingly, mTORC-1 regulated SM-induced cell death independent of M1 differentiation. In contrast, SM-induced cell death in polarized M1 macrophages is regulated by the mTORC-2 pathway. Moreover, SM-induced cell death is regulated by cellular IAP (cIAP)-2, receptor-interacting protein kinase (RIPK)-1, and RIPK-3 degradation through mTORC activation during differentiation into M1 macrophages and in polarized M1 macrophages. In contrast to cancer cell lines, SM-induced cell death in M1 macrophages is independent of endogenously produced TNF-α, as well as the NF-κB pathway. Collectively, selective induction of cell death in human M1 macrophages by SMs may be mediated by cIAP-2, RIPK-1, and RIPK-3 degradation through mTORC activation. Moreover, blocking cIAP-1/2, mTORC, or IFN regulatory factor-1 may represent a promising therapeutic strategy to control M1-associated diseases.

Nanosilver reinforced Parmelia sulcata extract efficiently induces apoptosis and inhibits proliferative signalling in MCF-7 cells

The Lichen, Parmelia sulcata synthesizes various secondary metabolites, in which phenolic based compounds received much attention due to their importance in biomedical application. Especially the phenolic compound was effective against the cancer treatment. An effective administration of such plant natural product can represent a significant conventional management of cancer in terms of chemoprevention. The nanomedicines are group of agents that selectively interfere the cancer cells which leads to reduction of side effect thereby reducing the doses. Silver nanoparticles is a promising antitumor agent, however, the conventional production of silver nanoparticles have many drawbacks which led to increase in need of eco-friendly biological production methods. In this study, we made an attempt to synthesise a nano silver (Ps-AgNPs) from phenolic extract of lichen Parmelia sulcata extract. The Ps-AgNps was applied for anticancer activity using MCF-7 cells and the effect was characterised by western blotting method. The FTIR, XRD, UV and TEM results confirms the presence of silver nanoparticles in phenolic extract of lichen Parmelia sulcata. The cytotoxicity assay shows that the Ps-AgNPs is toxic against cancer cells (MCF-7) but not to normal cells (NIH3T3), which confirm the selective induction of cell death (apoptosis) against cancer cells. The Western blot analysis also clearly indicates the down regulation of inflammatory genes (TNF-alpha and IL-6) and cell cycle genes (PCNA and Cyclin-D1) thus promoting intrinsic apoptotic pathway. The results suggest that Ps-AgNPs can effectively kill cancer cells and can be used as an alternative therapeutic agent for cancer treatment.

Detention of copper by sulfur nanoparticles inhibits the proliferation of A375 malignant melanoma and MCF-7 breast cancer cells

Selective induction of cell death or growth inhibition of cancer cells is the future of chemotherapy. Clinical trials have found that cancer tissues are enriched with copper. Based on this finding, many copper-containing compounds and complexes have been designed to “copper” cancer cells using copper as bait. However, recent studies have demonstrated that copper boosts tumor development, and copper deprivation from serum was shown to effectively inhibit the promotion of cancer. Mechanistically, copper is an essential cofactor for mitogen-activated protein kinase (MAPK)/extracellular activating kinase (ERK) kinase (MEK), a central molecule in the BRAF/MEK/ERK pathway. Therefore, depleting copper from cancer cells by directly sequestering copper has a wider field for research and potential for combination therapy. Based on the affinity between sulfur and copper, we therefore designed sulfur nanoparticles (Nano-S) that detain copper, achieving tumor growth restriction. We found that spherical Nano-S could effectively bind copper and form a tighter surficial structure. Moreover, this Nano-S detention of copper effectively inhibited the proliferation of A375 melanoma and MCF-7 breast cancer cells with minimum toxicity to normal cells. Mechanistic studies revealed that Nano-S triggered inactivation of the MEK-ERK pathway followed by inhibition of the proliferation of the A375 and MCF-7 cells. In addition, lower Nano-S concentrations and shorter exposure stimulated the expression of a copper transporter as compensation, which further increased the cellular uptake and anticancer activities of cisplatin. Collectively, our results highlight the potential of Nano-S as an anticancer agent or adjuvant through its detention of copper.

Cyclooxygenase-2 contributes to the selective induction of cell death by the endocannabinoid 2-arachidonoyl glycerol in hepatic stellate cells

The endogenous cannabinoid 2-arachidonoyl glycerol (2-AG) is an anti-fibrotic lipid mediator that induces apoptosis in hepatic stellate cells (HSCs), but not in hepatocytes. However, the exact molecular mechanisms of this selective induction of HSC death are still unresolved. Interestingly, the inducible isoform of cyclooxygenase, COX-2, can metabolize 2-AG to pro-apoptotic prostaglandin glycerol esters (PG-GEs). We analyzed the roles of COX-2 and endocannabinoid-derived PG-GEs in the differential susceptibility of primary activated HSCs and hepatocytes toward 2-AG-induced cell death. HSCs displayed significant COX-2 expression in contrast to hepatocytes. Similar to 2-AG, treatment of HSCs with PGD2-GE dose-dependently induced cell death independently from cannabinoid receptors that was accompanied by PARP- and caspase 3-cleavage. In contrast to 2-AG, PGD2-GE failed to induce significant ROS formation in HSCs, and depletion of membrane cholesterol did not rescue HSCs from PGD2-GE-induced apoptosis. These findings indicate differential engagement of initial intracellular signaling pathways by 2-AG and its COX-2-derived metabolite PGD2-GE, but similar final cell death pathways. Other PG-GEs, such as PGE2-or PGF2α-GE did not induce apoptosis in HSCs. Primary rat hepatocytes were mainly resistant against 2-AG- and PGD2-GE-induced apoptosis. HSCs, but not hepatocytes were able to metabolize 2-AG to PGD2-GE. As a proof of principle, HSCs from COX-2−/− mice lacked PDG2-GE production after 2-AG treatment. Accordingly, COX-2−/− HSCs were resistant against 2-AG-induced apoptosis. In conclusion, the divergent expression of COX-2 in HSCs and hepatocytes contributes to the different susceptibility of these cell types towards 2-AG-induced cell death due to the generation of pro-apoptotic PGD2-GE by COX-2 in HSCs. Modulation of COX-2-driven metabolization of 2-AG may provide a novel physiological concept allowing the specific targeting of HSCs in liver fibrosis.

Polycyclic aromatic hydrocarbon-contaminated soils: bioaugmentation of autochthonous bacteria and toxicological assessment of the bioremediation process by means of Vicia faba L.

Two bacterial strains, Achromobacter sp. (ACH01) and Sphingomonas sp. (SPH01), were isolated from a heavily polycyclic aromatic hydrocarbon (PAH)-contaminated soil (5431.3 ± 102.3ppm) for their capacity to use a mixture of anthracene, pyrene, phenanthrene and fluorene as sole carbon sources for growth and for the capacity to produce biosurfactants. The two strains were exploited for bioaugmentation in a biopile pilot plant to increase the bioavailability and the degradation of the residual PAH contamination (99.5 ± 7.1ppm) reached after 9months of treatment. The denaturing gel gradient electrophoresis (DGGE) profile of the microbial ecology of the soil during the experimentation showed that the bioaugmentation approach was successful in terms of permanence of the two strains in the soil in treatment. The bioaugmentation of the two bacterial isolates positively correlated with the PAH depletion that reached 7.9 ± 2ppm value in 2months of treatment. The PAH depletion was assessed by the loss of the phyto-genotoxicity of soil elutriates on the model plant Vicia faba L., toxicological assessment adopted also to determine the minimum length of the decontamination process for obtaining both the depletion of the PAH contamination and the detoxification of the soil at the end of the process. The intermediate phases of the bioremediation process were the most significant in terms of toxicity, inducing genotoxic effects and selective DNA fragmentation in the stem cell niche of the root tip. The selective DNA fragmentation can be related to the selective induction of cell death of mutant stem cells that can compromise offsprings.

Targeting of the Tumor Necrosis Factor Receptor Superfamily for Cancer Immunotherapy

The tumor necrosis factor (TNF) ligand and cognate TNF receptor superfamilies constitute an important regulatory axis that is pivotal for immune homeostasis and correct execution of immune responses. TNF ligands and receptors are involved in diverse biological processes ranging from the selective induction of cell death in potentially dangerous and superfluous cells to providing costimulatory signals that help mount an effective immune response. This diverse and important regulatory role in immunity has sparked great interest in the development of TNFL/TNFR-targeted cancer immunotherapeutics. In this review, I will discuss the biology of the most prominent proapoptotic and co-stimulatory TNF ligands and review their current status in cancer immunotherapy.

The novel endocannabinoid noladin ether holds putative anti-fibrotic properties by selectively inducing cell death in hepatic stellate cells

Background: We could previously demonstrate that the endogenous cannabinoid 2-arachidonoyl glycerol (2-AG) is an antifibrotic lipid mediator that blocks proliferation and induces apoptotic cell death in hepatic stellate cells (HSCs), but not in hepatocytes. Recently a novel endocannabinoid termed noladin ether (NE, 2-arachidonyl glyceryl ether) has been identified, which represents a more stable entity against enzymatic hydrolysis than 2-AG. Aims: To test the effects of NE on hepatic cell populations. Methods: Primary hepatocytes and HSCs were isolated from healthy rat or mouse liver by collagenase perfusion. Cell death was analyzed by LDH assay and western blot for caspase 3- and PARP-cleavage. Glutathione (GSH) was depleted by DL-buthionine-(S,R)-sulfoximine (BSO) treatment. Antioxidant resistance was enhanced by Trolox application. ROS formation was detected by DCFDA fluorescence. Membrane cholesterol was depleted by methyl-beta-cyclodextrin (MCD). Results: NE dose-dependently induced cell death in activated primary HSCs after 2–4h, starting from 2µM. In contrast to 2-AG, which induced late apoptosis in HSCs, NE induced hallmarks of necrosis, lack of caspase-3 or PARP cleavage. ROS formation in HSCs was significantly and even more increased by NE than by 2-AG. The antioxidant Trolox significantly decreased NE-induced death, thus indicating an ROS-dependent cell death mechanism. Although the cannabinoid receptors CB1, CB2 and TRPV1 were expressed in HSCs, pharmacological blockade failed to block NE-mediated cell death, indicating a cannabinoid-receptor independent mechanism. Interestingly, membrane cholesterol depletion with MCD completely inhibited NE-induced cell death. Similarly to 2-AG, NE did not induce cell death in hepatocytes. The resistance toward NE in hepatocytes was due to high levels of antioxidants such as GSH, since GSH depletion with BSO significantly increased NE-induced cell death in hepatocytes. Intriguingly, adenoviral overexpression of the putative endocannabinoid degradation enzyme fatty acid amide hydrolase (FAAH) in HSCs did not rescue from NE-induced cell death, underlining the stability of NE against enzymatic hydrolysis. Conclusion: NE effectively caused cell death in activated HSCs, but not in hepatocytes. NE-induced cell death was mediated through membrane cholesterol-rich lipid rafts and pronounced ROS formation, but independently from cannabinoid receptors. The selective induction of cell death in HSCs and its high stability against enzymatic hydrolysis proposes NE as a potential endogenous antifibrogenic mediator.

The endocannabinoid N-arachidonoyl dopamine (NADA) selectively induces oxidative stress-mediated cell death in hepatic stellate cells but not in hepatocytes

The endocannabinoid system is a crucial regulator of hepatic fibrogenesis. We have previously shown that the endocannabinoid anandamide (AEA) is a lipid mediator that blocks proliferation and induces death in hepatic stellate cells (HSCs), the main fibrogenic cell type in the liver, but not in hepatocytes. However, the effects of other endocannabinoids such as N-arachidonoyl dopamine (NADA) have not yet been investigated. The NADA-synthesizing enzyme tyrosine hydroxylase was mainly expressed in sympathetic neurons in portal tracts. Its expression pattern stayed unchanged in normal or fibrotic liver. NADA dose dependently induced cell death in culture-activated primary murine or human HSCs after 2-4 h, starting from 5 μM. Despite caspase 3 cleavage, NADA-mediated cell death showed typical features of necrosis, including ATP depletion. Although the cannabinoid receptors CB1, CB2, or transient receptor potential cation channel subfamily V, member 1 were expressed in HSCs, their pharmacological or genetic blockade failed to inhibit NADA-mediated death, indicating a cannabinoid-receptor-independent mechanism. Interestingly, membrane cholesterol depletion with methyl-β-cyclodextrin inhibited AEA- but not NADA-induced death. NADA significantly induced reactive oxygen species formation in HSCs. The antioxidant glutathione (GSH) significantly decreased NADA-induced cell death. Similar to AEA, primary hepatocytes were highly resistant against NADA-induced death. Resistance to NADA in hepatocytes was due to high levels of GSH, since GSH depletion significantly increased NADA-induced death. Moreover, high expression of the AEA-degrading enzyme fatty acid amide hydrolase (FAAH) in hepatocytes also conferred resistance towards NADA-induced death, since pharmacological or genetic FAAH inhibition significantly augmented hepatocyte death. Thus the selective induction of cell death in HSCs proposes NADA as a novel antifibrogenic mediator.

Selective Induction of Cell Death in Melanoma Cell Lines through Targeting of Mcl-1 and A1

Melanoma is an often fatal form of skin cancer which is remarkably resistant against radio- and chemotherapy. Even new strategies that target RAS/RAF signaling and display unprecedented efficacy are characterized by resistance mechanisms. The targeting of survival pathways would be an attractive alternative strategy, if tumor-specific cell death can be achieved. Bcl-2 proteins play a central role in regulating survival of tumor cells. In this study, we systematically investigated the relevance of antiapoptotic Bcl-2 proteins, i.e., Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and A1, in melanoma cell lines and non-malignant cells using RNAi. We found that melanoma cells required the presence of specific antiapoptotic Bcl-2 proteins: Inhibition of Mcl-1 and A1 strongly induced cell death in some melanoma cell lines, whereas non-malignant cells, i.e., primary human fibroblasts or keratinocytes were not affected. This specific sensitivity of melanoma cells was further enhanced by the combined inhibition of Mcl-1 and A1 and resulted in 60% to 80% cell death in all melanoma cell lines tested. This treatment was successfully combined with chemotherapy, which killed a substantial proportion of cells that survived Mcl-1 and A1 inhibition. Together, these results identify antiapoptotic proteins on which specifically melanoma cells rely on and, thus, provide a basis for the development of new Bcl-2 protein-targeting therapies.

Cox–2 contributes to the selective induction of cell death by the endocannabinoid 2-arachidonoyl glycerol in hepatic stellate cells through generation of prostaglandin-gycerol esters

Aims: We have shown previously that the endogenous cannabinoid 2-arachidonoyl glycerol (2-AG) is a lipid mediator that blocks proliferation and induces apoptosis in hepatic stellate cells (HSCs), but not in hepatocytes. However, the exact molecular mechanisms of this selective induction of HSC death still need to be clarified. AIM: To analyze a possible role of COX–2 in the differential cell death of primary HSCs and hepatocytes induced by 2-AG metabilization to pro-apoptotic prostaglandin D2 glycerol ester (PGD2-GE). Methods: Primary rat hepatocytes and rat HSCs were isolated from healthy rat liver by collagenase perfusion. 2-AG- or PGD2-GE-induced cell death was analyzed by LDH assay and western blot for caspase 3- and PARP-cleavage. Reactive oxygen species (ROS) formation was monitored by DCFDA fluorescence. COX–2 expression was determined by western blotting. Results: HSCs displayed significant COX–2 protein expression, whereas hepatocytes did not express notable levels of COX–2. Treatment of primary culture-activated rat HSCs with 2-AG dose-dependently induced cell death (70% after 24h at 10µM) and was accompanied by PARP- and caspase 3-cleavage. Similarly, PGD2-GE also induced apoptotic cell death dose-dependently in primary HSCs. In contrast, 2-AG did not induce cell death in primary rat hepatocytes at concentrations of up to 100uM, but PGD2-GE lead to marked cell death starting at 25µM. Both, 2-AG- and PGD2-GE induced ROS formation in HSCs, indicating common ROS-dependent pathways to induce cell death. Conclusion: HSCs and hepatocytes differentially express COX–2, leading to differential metabolization of the endocannabinoid 2-AG. The generation of PGD2-GE by COX–2 in HSCs possibly contributes to the different susceptibility of hepatocytes and HSCs towards 2-AG-induced cell death.

The role of the mitochondria in mediating cytotoxicity of anti-cancer therapies

Optimal cytotoxic anticancer therapy, at the cellular level, requires effective and selective induction of cell death to achieve a net reduction of biomass of malignant tissues. Standard cytotoxic chemotherapeutics have been developed based on the observations that mitotically active cancer cells are more susceptible than quiescent normal cells to chromosomal, microtubular or metabolic poisons. More recent development of molecularly targeted drugs for cancer focuses on exploiting biological differentials between normal and transformed cells for selective eradication of cancers. The common thread of "standard" and "novel" cytotoxic drugs is their ability to activate the apoptosis-inducing machinery mediated by mitochondria, also known as the intrinsic death signaling cascade. The aim of this article is to provide an overview of the role of the mitochondria, an energy-generating organelle essential for life, in mediating death when properly activated by cytotoxic stresses.

Molecular Pathogenesis of Alcohol‐Induced Hepatic Fibrosis

Alcohol abuse is a main cause of liver fibrosis and cirrhosis in the western world. Although the major mechanisms of fibrogenesis are independent of the origin of liver injury, alcoholic liver fibrosis features distinctive characteristics, including the pronounced inflammatory response of immune cells due to elevated gut-derived endotoxin plasma levels, increased formation of reactive oxygen species (ROS), ethanol-induced pericentral hepatic hypoxia or formation of cell-toxic and pro-fibrogenic ethanol metabolites (e.g., acetaldehyde or lipid oxidation products). These factors are together responsible for increased hepatocellular cell death and activation of hepatic stellate cells (HSCs), the key cell type of liver fibrogenesis. To date, removing the causative agent is the most effective intervention to prevent the manifestation of liver cirrhosis. A novel experimental approach in fibrosis therapy is the selective induction of cell death in HSCs. Substances such as gliotoxin, anandamide or antibody against tissue inhibitor of metalloproteinase (TIMP)-1 can selectively induce cell death in activated HSCs. These new results in basic science are encouraging for the search of new antifibrotic treatment.

TRAIL sensitizes for ionizing irradiation-induced apoptosis through an entirely Bax-dependent mitochondrial cell death pathway

The death ligand TRAIL has been suggested as a suitable biological agent for the selective induction of cell death in cancer cells. Moreover, TRAIL synergizes with DNA-damaging therapies such as chemotherapeutic drugs or ionizing irradiation (IR). Here, we show that synergy of TRAIL and IR, that is, crosssensitization between TRAIL and IR for induction of apoptosis, entirely depends on Bax proficiency in human DU145 and HCT116 carcinoma cells. DU145 prostate carcinoma cells that have lost Bax protein expression due to mutation fail to activate caspase-3 and -9 when exposed to TRAIL and IR. In contrast, TRAIL sensitized for IR-induced apoptosis and vice versa upon reconstitution of Bax expression. Notably, both DU145 and HCT116 still express significant levels of the multidomain proapoptotic Bcl-2 homolog Bak. This indicates that Bak is not sufficient to mediate crosssensitization and synergism between IR and TRAIL. These data clearly establish distinct roles for Bax and Bak in linking the TRAIL death receptor pathway to the mitochondrial apoptosis signaling cascade upon DNA damage by IR.

Differential sensitivity of normal and H-ras oncogene-transformed ratkidney epithelial cells to okadaic acid-induced apoptosis

H-ras oncogenes have been identified in greater than 50% of the most common forms of human neoplasia. Ras-related proteins have been postulated to mediate signal transduction pathways involving mitogen-activated protein (MAP) kinases and nuclear responses that may be involved in the induction of apoptosis. We examined whether expression of H-ras oncogene conferred resistance or susceptibility to the morphologic effects of the protein phosphatase inhibitor, okadaic acid, using a tumorigenic H-ras-transformed normal rat kidney epithelial cell line, NRK-H/6.1. We also examined whether okadaic acid induced apoptosis correlated with a differential effect on kinase activity in H-Ras-transformed cells as compared to the nontransformed NRK-52E cells. Treatment with varions concentrations of okadaic acid produced rapid and extensive morphologic changes characteristic of apoptosis in both cell types. Equimolar okadaic acid concentrations for 2 or 4 hr resulted in cell detachment and loss of membrane integrity (as measured by propidium iodide uptake) in 74% (0.5 μM) and 78% (1.0 μM) of the H-Ras-transformed cells as compared to 8 and 25%, respectively, in the non-transformed cells. Furthermore, a higher basal level of kinase activity was observed in the H-Ras-transformed cells as compared to the nontransformed cells. Okadaic acid-induced apoptosis correlated with activation of members of the MAP kinase family, raf-1 and protein kinase C (PKC). These studies show that H-ras oncogene expression imparts selective susceptibility to cell death induced by phosphatase inhibition. The observed increase in susceptibility to okadaic acid-induced apoptosis appears to involve the modulation of raf-1, PKC, and MAP kinase activities. These findings may be significant in the elucidation of mechanisms for selective induction of cell death in tumor cells expressing H-ras oncogene.

Selective induction of cell death in cancer cells by gallic acid.

Gallic acid (3,4,5-trihydroxybenzoic acid) is a naturally occurring plant phenol obtained by the hydrolysis of tannins and is know to show some pharmacological activities. In screening anti-cancer agents in traditional Chinese medicines, gallic acid was found to show cytotoxicity against all cancer cells that we examined in this study (IC50s: 4.8-13.2 micrograms/ml). Gallic acid was found to show cytotoxicity against primary cultured rat hepatocytes and macrophages, and lesser cytotoxicity against fibroblasts and endothelial cells. Cell death in dRLh-84 cells occurred within 6h after gallic acid treatment at a concentration of more than 20 micrograms/ml. A study of structurally related compounds suggested that the cytotoxicity shown by gallic acid was not a common feature in phenolic compounds, but was a fairly specific characteristic of gallic acid. That is, three adjacent phenolic hydroxyl groups of gallic acid were responsible for the cytotoxicity, and the carboxyl group was not responsible, but seemed to be implicated in distinguishing between normal cells and cancer cells.