Simultaneous Induction Of Apoptosis Research Articles

Tanshinone IIA Shows Higher Antiproliferative Activities than Sinapic Acid in 4 Cancer Cell Lines and Simultaneously Induces Apoptosis and Necroptosis in Human Lung Cancer A549 Cells

Tanshinone IIA (Tan IIA) and sinapic acid (SA) are 2 components separately isolated from 2 Asian medicinal plants, Hydnophytum formicarum Jack and Salvia miltiorrhiza Bunge. The antitumor activities of them were worth exploring, therefore, we examined their antitumor activities in A549, HCT116, HeLa, and Colo320 cancer cell lines by means of WST-1 assay. The results show that Tan IIA exerted far higher (IC50 from 1.0 ± 0.0 to 166.3 ± 24.0 µg/mL) antiproliferative activities than SA (IC50 from 2236.3 ± 484.1 to >10 000.0 µg/mL). Of the 4 cell lines, A549 cells were the most sensitive to Tan IIA; thus, we used Western blotting to explore the cytotoxic mechanisms of Tan IIA in A549 cells and found that they rely on simultaneous induction of apoptosis and necroptosis in the cells. Apoptosis was hallmarked by the induction of cleaved caspase-3 by Tan IIA and necroptosis by the necroptotic marker proteins cyclophilin A and high mobility group box 1 (HMGB1), as well as increased lactate dehydrogenase (LDH) activities. The necroptotic effect was confirmed by the necroptosis inhibitor necrostatin-1 (Nec-1), which eliminated these effects and restored cell survival rates. The levels of cyclophilin A decreased in response to the pan-caspase inhibitor z-VAD-fmk, and those of cleaved caspase-3 decreased in response to Nec-1. Conclusively, Tan IIA has the potential to prevent lung cancer and the mechanism seems to be apoptosis and necroptosis, of which the relationship is mutually interdependent. This is the first report of Tan IIA eliciting necroptosis in A549 cells. Tan IIA may be used for necroptosis-based cancer therapy, especially to overcome apoptosis resistance.

MiR-221 regulates proliferation, invasion, apoptosis and progression of prostate cancer cells by modulating E-cadherin/Wnt/β catenin axis

MicroRNA-221 (miR-221) is aberrantly expressed in various tumours including prostate carcinoma and can act as either tumour suppressor or oncogene depending on the tumour system and stage. Epithelial-mesenchymal transition (EMT) is a plastic transition in tumour progression characterized by decreased expression of epithelial markers and increased expression of mesenchymal markers and has been known to play a critical role in promoting metastases. In this study, we have demonstrated the functional role of miR-221 in regulating epithelial-mesenchymal transition in prostate cancer. Expression of miR-221 was found to be downregulated in androgen-independent prostate cancer cells and was found to be inversely correlated with EMT. Ectopic expression of miR-221 potentially inhibited invasion and migration of androgen-independent prostate cancers cells and inhibition of miR-221 expression rescued the anti-invasive effects of miR-221. Flow cytometric analysis further revealed that miR-221 could induce cell cycle arrest with simultaneous induction of apoptosis. Additionally, transfection with miR-221 mimic upregulated negative regulators of Wnt/β catenin pathway such as GSK-3β thereby inhibiting it and downregulated mesenchymal markers viz., N-cadherin and Vimentin with simultaneous upregulation of epithelial marker E-cadherin as revealed by real-time PCR and Western blot analysis. A study using xenograft model further confirmed the anti-tumorigenic and anti-metastatic role of miR-221 in prostate cancer. We believe that his study is the first report documenting the miR-221- Wnt/β catenin signaling-EMT regulatory circuit in prostate cancer.

Novel gene Merlot inhibits differentiation and promotes apoptosis of osteoclasts.

Extended osteoclast longevity is deeply involved in the pathogenesis of bone diseases such as osteoporosis and rheumatoid arthritis, though the mechanisms that determine osteoclast lifespan are not fully understood. Here we present findings indicating that the newly characterized gene Merlot, which encodes a highly conserved yet uncharacterized protein in vertebrates, is an important regulator for termination of osteoclastogenesis via induction of apoptosis. Mice lacking Merlot exhibited low bone mass due to increased osteoclast and bone resorption. Furthermore, osteoclast precursors overexpressing Merlot failed to differentiate into mature osteoclasts, while Merlot deficiency led to hyper-nucleation and prolonged survival of osteoclasts, accompanied by sustained nuclear localization of nuclear factor of activated T cell c1 (NFATc1) and derepression of glycogen synthase kinase-3β (GSK3β) activity, known to regulate NFATc1 activity and induce apoptosis. Merlot-deficient osteoclasts were found to represent suppression of caspase-3-mediated apoptosis and Merlot deficiency caused transcriptional downregulation of a proapoptotic cascade, including Bax, Bak, Noxa, and Bim, as well as the executor caspase members Casp-3, -6, and -7, and upregulation of anti-apoptotic Bcl2, resulting in a low apoptotic threshold. Thus, Merlot regulates osteoclast lifespan by inhibition of differentiation and simultaneous induction of apoptosis via regulation of the NFATc1-GSK3β axis.

Microtubule disrupting agent-mediated inhibition of cancer cell growth is associated with blockade of autophagic flux and simultaneous induction of apoptosis.

ObjectivesGiven that autophagy inhibition is a feasible way to enhance sensitivity of cancer cells towards chemotherapeutic agents, identifying potent autophagy inhibitor has obvious clinical relevance. Here, we investigated ability of TN‐16, a microtubule disrupting agent, on modulation of autophagic flux and its significance in promoting in vitro and in vivo cancer cell death.Materials and methodsThe effect of TN‐16 on cancer cell proliferation, cell division, autophagic process and apoptotic signalling was assessed by various biochemical (Western blot and SRB assay), morphological (TEM, SEM, confocal microscopy) and flowcytometric assays. In vivo anti‐tumour efficacy of TN‐16 was investigated in syngeneic mouse model of breast cancer.ResultsTN‐16 inhibited cancer cell proliferation by impairing late‐stage autophagy and induction of apoptosis. Inhibition of autophagic flux was demonstrated by accumulation of autophagy‐specific substrate p62 and lack of additional LC3‐II turnover in the presence of lysosomotropic agent. The effect was validated by confocal micrographs showing diminished autophagosome‐lysosome fusion. Further studies revealed that TN‐16–mediated inhibition of autophagic flux promotes apoptotic cell death. Consistent with in vitro data, results of our in vivo study revealed that TN‐16–mediated tumour growth suppression is associated with blockade of autophagic flux and enhanced apoptosis.ConclusionsOur data signify that TN‐16 is a potent autophagy flux inhibitor and might be suitable for (pre‐) clinical use as standard inhibitor of autophagy with anti‐cancer activity.

Immunotoxicity of titanium dioxide nanoparticles via simultaneous induction of apoptosis and multiple toll-like receptors signaling through ROS-dependent SAPK/JNK and p38 MAPK activation.

BackgroundTitanium dioxide nanoparticles (TiO2 NPs) represent a scientific breakthrough in the areas of biological and medicinal applications. Interaction of TiO2 NPs with components of innate immune system remains elusive.AimThis study explored in vitro immunotoxicity of murine macrophage RAW 264.7 to TiO2 NPs (20 nm, negative charge) and its underlying molecular mechanism by way of immunoredox profiling.Materials and methodsIn this study, chemically synthesized BSA-functionalized TiO2 NPs (20 nm, negative charge) were characterized and immunotoxicity was investigated on RAW 264.7 cells.ResultsWe found that reactive oxygen species levels significantly increased with increasing nitric oxide production, whereas depleting endogenous antioxidant super oxide dismutase as well as nuclear factor erythroid 2-related factor 2 (Nrf2) protein levels. Furthermore, NPs exposure increased the expression of apoptotic factors such as BAX, BIM, and PUMA with disruption of mitochondrial membrane potential (Δψm) that lead to decrease in immunocytes. Molecular immune profiling revealed the activation of multiple toll-like receptors (TLRs) 4/9/12/13 simultaneously with the phosphorylation of p-p38MAPK and p-SAPK/c-Jun N-terminal kinase (JNK) compared to untreated control.ConclusionCollectively, this study shows that the molecular nature of TiO2SA20(−) NP-induced immunotoxicity in RAW 264.7 macrophage is simultaneous induction of immunocyte apoptosis and multiple TLRs signaling through oxidative stress-dependent SAPK/JNK and p38 mitogen-associated protein kinase activation. This is the first study to address newer molecular mechanism of TiO2SA20(−) NP-induced immunotoxicity.

New Platinum(II) agent induces bimodal death of apoptosis and autophagy against A549 cancer cell

Agents with multiple modes of tumor cell death can be effective chemotherapeutic drugs. One example of a bimodal chemotherapeutic approach is an agent that can induce both apoptosis and autophagic death. Thus far, no clinical anticancer drug has been shown to simultaneously induce both these pathways. Mono-functional platinum complexes are potent anticancer drug candidates which act through mechanisms distinct from cisplatin. Here, we describe the synthesis and characterize of two mono-functional platinum complexes containing 8-substituted quinoline derivatives as ligands. In comparison to cisplatin, n-Mon-Pt-1 exhibited a greater in vitro cytotoxicity, was more effective in resistant cells and elicited a better anticancer effect. Mechanistic experiments indicate that n-Mon-Pt-1 mainly accumulates in mitochondria, and stimulates significant TrxR inhibition, ROS release and an ER stress response, ultimately resulting in a simultaneous induction of apoptosis and autophagy. Importantly, compared to cisplatin, n-Mon-Pt-1 exhibits lower acute toxicity and better anticancer activity in a murine tumor model.

Mitochondria-targeted platinum(II) complexes induce apoptosis-dependent autophagic cell death mediated by ER-stress in A549 cancer cells

Agents with multiple modes of tumor cell death can be effective chemotherapeutic drugs. One example of a bimodal chemotherapeutic approach is an agent that can induce both apoptosis and autophagic death. Thus far, no clinical anticancer drug has been shown to simultaneously induce both these pathways. Mono-functional platinum complexes are potent anticancer drug candidates which act through mechanisms distinct from cisplatin. Here, we describe the synthesis and characterize of two mono-functional platinum complexes containing 8-substituted quinoline derivatives as ligands, [PtL1Cl]Cl [L1 = (Z)-1-(pyridin-2-yl)-N-(quinolin-8-ylmethylene) methanamine] (Mon-Pt-1) and [PtL2Cl]Cl [L2 = (Z)-2-(pyridin-2-yl)-N-(quinolin-8-ylmethylene) ethanamine] (Mon-Pt-2). In comparison to cisplatin, Mon-Pt-2 exhibited a greater in vitro cytotoxicity, was more effective in resistant cells and elicited a better anticancer effect. Mechanistic experiments indicate that Mon-Pt-2 mainly accumulates in mitochondria, and stimulates significant TrxR inhibition ROS release and an ER stress response, mediated by mitochondrial dysfunction, ultimately resulting in a simultaneous induction of apoptosis and autophagy. Importantly, compared to cisplatin, Mon-Pt-2 exhibits lower acute toxicity and better anticancer activity in a murine tumor model. To the best of our knowledge, Mon-Pt-2 is the first mono-functional platinum complex inducing pro-death autophagy and apoptosis of cancer cells.

Receptor-specific TRAIL as a means to achieve targeted elimination of activated hepatic stellate cells

Activated hepatic stellate cells (HSCs) are known to play a central role in liver fibrosis and their elimination is a crucial step toward the resolution and reversion of liver fibrosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a molecule that may contribute to the apoptotic removal of activated HSC through binding to its dedicated receptors. In the present study, we investigated the potential application of recombinant receptor-specific TRAIL proteins in the efficient elimination of activated HSCs. Our finding revealed differential contribution of TRAIL receptors among HSCs populations with activated hepatic stellate cells expresses more TRAIL receptors DR5. In vitro treatment of activated HSCs with DR5-specific or wild-type TRAIL variants induced a significant reduction in viability and extracellular matrix production, whereas no significant decrease in viability was associated with the treatment of cells by DR4-specific TRAIL. Our analysis indicate the successful application of the DR5 receptor-specific TRAIL variant in the targeted elimination of activated HSCs via interference with collagen production and simultaneous induction of apoptosis via activation of the caspase pathway. DR5 receptor-specific TRAIL may thus represent a new therapeutic compound for the treatment of liver fibrosis.

Simultaneous induction of apoptosis and necroptosis by Tanshinone IIA in human hepatocellular carcinoma HepG2 cells

Tanshinone IIA (Tan IIA), a constituent of the traditional medicinal plant Salvia miltiorrhiza BUNGE, has been reported to possess anticancer activity through induction of apoptosis in many cancer cells. Surprisingly, the present study finds that Tan IIA simultaneously causes apoptosis and necroptosis in human hepatocellular carcinoma HepG2 cells. We further find that apoptosis can be converted to necroptosis by pan-caspase inhibitor Z-VAD-fmk, and the two death modes can be blocked by necroptotic inhibitor necrostatin-1. The underlying mechanisms are revealed by analysis of the signaling molecules using western blotting. In control cells, FLICE inhibitory protein in short form (FLIPS) is expressed in relatively high levels and binds to caspase 8 in ripoptosome, which supposedly sustains cell survival. However, in Tan IIA-treated cells, FLIPS is down-regulated and may thus cause homodimer formation of cleaved caspase 8, cleavage of receptor-interacting serine/threonine-protein kinases 1, 3 (RIP1, RIP3), and mixed-lineage kinase domain-like (MLKL), in turn leads to cell apoptosis. In parallel, Tan IIA causes necroptosis by forming a suggested necrosomal complex composed of RIP1/RIP3. Regarding the inhibitors, z-VAD-fmk diminishes the cleaved caspase 8, RIP1, RIP3, and MLKL induced by Tan IIA, and reconstructs the ripoptosome complex, which marks cells moving from apoptosis to necroptosis. Nec-1 recovers the Tan IIA down-regulated FLIPS, consequently causes FLIPS to form heterodimer with caspase 8 and thus block apoptosis. Meanwhile, cleaved forms of RIP1 and RIP3 were observed preventing necroptosis. Intriguingly, the cytotoxicity of tumor necrosis factor-related apoptosis-inducing ligand to HepG2 cells is enhanced by Tan IIA in a pilot study, which may be attributed to low FLIPS levels induced by Tan IIA. In short, Tan IIA simultaneously induces both Nec-1 inhibition and FLIPS regulation-mediated apoptosis/necroptosis, which has not been previously documented. Moreover, the involvement of the cleavage type of MLKL in executing necroptosis warrants further investigation.

Telomelysin shows potent antitumor activity through apoptotic and non‐apoptotic cell death in soft tissue sarcoma cells

This study investigated the pathway underlying the antitumor activity of telomelysin, a telomerase-dependent, replication-selective oncolytic adenovirus, in soft tissue sarcoma cells. Treatment with telomelysin alone resulted in simultaneous induction of apoptosis and autophagy, whereas cotreatment with telomelysin and 3-methyladenine significantly reduced cell viability and increased apoptosis and the cellular ATP level compared to treatment with telomelysin alone, indicating that telomelysin-mediated autophagy is a death-protective but not death-promoting process. Cotreatment with Z-Val-Ala-Asp-CH2F significantly increased cellular ATP depletion compared to telomelysin-alone treatment while inhibiting telomelysin-induced apoptosis and having no significant effect on cell viability, indicating that it promotes transition from apoptotic to necrotic cell death.

Cytotoxic effect of evodiamine in SGC-7901 human gastric adenocarcinoma cells via simultaneous induction of apoptosis and autophagy

Evodiamine, an alkaloid isolated from Evodia rutaecarpa, possesses potent anticancer activity. Although many reports have elucidated the cytotoxic effects of evodiamine in a variety of cancer cells, little is known about the mechanism of evodiamine-induced cytotoxic activity in gastric cancer cells. To date, no report has addressed the synchronized role of autophagy and apoptosis in evodiamine-induced cytotoxic activity. This study was conducted to investigate the synchronized role of autophagy and apoptosis in evodiamine-induced cytotoxic activity on SGC-7901 human gastric adenocarcinoma cells and further to elucidate the underlying molecular mechanisms. The MTT assay was used to examine the cytotoxicity of evodiamine against SGC-7901 gastric adenocarcinoma cells. The effects of evodiamine on the cell cycle and apoptosis were measured by flow cyto-metry and cellular morphology was observed under a phase contrast microscope. Acridine orange (AO) staining was used to detect autophagy. The expression levels of Bcl-2 and Bax were detected by Western blotting. The expression level of Beclin‑1 in SGC-7901 cells was monitored by reverse transcription-polymerase chain reaction (RT-PCR). Here, we found that evodiamine significantly inhibited the proliferation of SGC-7901 cells and induced G2/M phase cell cycle arrest. Furthermore, both autophagy and apoptosis were activated during the evodiamine-induced death of SGC-7901 cells. Evodiamine-induced autophagy is partially involved in the death of SGC-7901 cells which was confirmed by using the autophagy inhibitor 3-methyladenine (3-MA). Additionally, Beclin-1 is involved in evodiamine-induced autophagy and the pro-apoptotic mechanisms of evodiamine may be associated with down-regulation of Bcl-2 and up-regulation of Bax expression. The inhibitory effects on SGC-7901 cells were associated with apoptosis, autophagy and cell cycle arrest at the G2/M phase in a dose-dependent manner. These results suggest that evodiamine is an effective natural compound for the treatment of gastric cancer and may represent a candidate for invivo studies of monotherapies or combined antitumor therapies.

Reciprocal Regulation of The Survival and Apoptosis of Th17 and Th1 Cells in The Colon

The immediate early response gene X-1 (IEX-1) is a stress-inducible gene involved in the regulation of cell growth, apoptosis and inflammation. Acute colitis was induced by treatment of IEX-1 knockout (KO) and wild type (WT) control mice with dextran sulfate sodium (DSS), whereas chronic colitis was induced in Rag-/- mice by adoptive transfer of CD4(+) CD45RB(hi) T cells isolated from the two strains of mice. The diseases and responses of lamina propria lymphocytes were analyzed in the mice. IEX-1 KO mice produced IL-17 in the colon significantly greater than WT control mice following DSS treatment owing to better survival and differentiation of both IL-17-secreting γδ T cells and Th17 cells. The altered level of IL-17 production contributed critically to the reduced colon inflammation in IEX-1 KO mice, and administration of neutralizing anti-IL-17 antibody increased susceptibility of the animal to the disease. Strikingly, in contrast to the better survival of T cells producing IL-17, lack of IEX-1 enhanced apoptosis in proinflammatory T cells producing interferon gamma (IFN-γ). Enhanced apoptosis in Th1 cells and better survival of Th17 cells may both result in a delayed onset of colitis in Rag-/- mice receiving pathogenic CD4(+) CD45RB(hi) T cells isolated from IEX-1 KO animals compared to those mice transferred with WT counterparts The present study demonstrates the refractoriness of IEX-1 knockout (KO) mice to DSS-induced colitis and diminished pathogenesis of IEX-1-deficient CD4(+) CD45RB(hi) T cells. These data demonstrate that IEX-1 reciprocally regulates T-cell survival and apoptosis in a subset-dependent fashion. Inhibition of IEX-1 may thus offer novel strategies for colitis treatment by simultaneous induction of apoptosis in proinflammatory Th1 cells while promoting the survival and differentiation of a protective T-cell subset.

New inhibitors of protein kinase CK2, analogues of benzimidazole and benzotriazole

Derivatives of 4,5,6,7-tetrabromobenzotriazole (TBBt) and 4,5,6,7-tetrabromobenzimidazole (TBBi) with IC50 in the low micromolar range and with high selectivity belong to the most promising inhibitors of protein kinase CK2 (casein kinase 2). Treatment of various cell lines with TBBt, TBBi or 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) affected cell viability with simultaneous induction of apoptosis. The inhibitory activity of newly synthesized hydroxyalkyl derivatives of TBBi and TBBt depends on the length of the alkyl chain. The hydroxypropyl substituted derivatives show higher or similar inhibitory activity than the parent compounds when tested with human protein kinase CK2. To test the distribution of this class of compounds in mammals, [14C] TBBi was synthesized.

Hyperthermic preconditioning protects pig islet grafts from early inflammation but enhances rejection in immunocompetent mice.

The induction of heat shock proteins (HSP) protects isolated islet cells against the cytotoxicity of inflammatory mediators in vitro. Very little information is available about the effect of HSP overexpression on function of preconditioned islet grafts. The present study investigated the function of heat-exposed pig islets after transplantation into immunocompetent mice in comparison with in vitro resistance against inflammatory mediators. Pig islets were preconditioned at 43 degrees C or sham treated prior to subcapsular transplantation into diabetic C57/Bl6j mice. Nondiabetic mice simultaneously receiving preconditioned and control islets were subjected to bilateral nephrectomy for determination of pig insulin. Resistance against H2O2, NO, human Il-1beta, IFN-gamma, or TNF-alpha was assessed by trypan blue exclusion and insulin determination. Heat-induced protein expression was confirmed by Western blot analysis. Graft preconditioning increased resistance against H2O2, NO, or cytokines (p < 0.05) but decreased survival in nondiabetic mice (p < 0.05) and function in diabetic mice (p < 0.01). Upregulation of caspase-3 activity as well as Bax, Fas, FasL, and DFF expression (p < 0.05) indicated simultaneous induction of apoptosis. The coexpression of HSP and proapoptotic proteins reveals the dual character of the stress response simultaneously starting mechanisms for protection and apoptosis. In vitro assays seem to reflect only insufficiently the situation of islets after transplantation.