Identification of a novel antimicrobial peptide,scolopendin 1, derived from centipede Scolopendra subspinipes mutilans and its antifungal mechanism

In this study, a novel antimicrobial peptide, scolopendin 1, was identified from adult centipedes, Scolopendra subspinipes mutilans using RNA sequencing. Scolopendin 1 exerted an antimicrobial activity without inducing haemolysis of human erythrocytes. In order to understand the antifungal mechanism, a reactive oxygen species (ROS) assay was performed, which indicated that scolopendin 1 induced ROS accumulation in Candida albicans. Evaluation of fungal viability using N‐acetyl cysteine, a ROS scavenger, suggested that ROS are a major factor in scolopendin 1‐induced fungal cell death. Co‐staining of annexin V‐fluorescein isothiocyanate (FITC) and propidium iodide, and TUNEL and 4′,6‐diamidino‐2‐phenylindole (DAPI) assays confirmed that ROS‐induced fungal cell death is associated with apoptosis. To further investigate the mechanism that facilitates the progression of apoptosis, changes in intracellular Ca²⁺ concentration and mitochondrial dysfunction were examined. Ca²⁺, a signalling molecule in the apoptotic pathway, was increased in the cytosol and mitochondria, and ROS accumulation triggered mitochondrial depolarization and the release of cytochrome c, a pro‐apoptotic factor, from the mitochondria to the cytosol. Finally, the released cytochrome c activated intracellular caspase. The present study suggests that scolopendin 1 could emerge as a model molecule that targets the apoptotic pathway and provides a novel remedy.     

Resveratrol, a polyphenolic compound in red wine, protects against oxidized LDL-induced cytotoxicity in endothelial cells

BACKGROUND: Resveratrol, a polyphenolic constituent of red wine, has antioxidant effects. However, its protective effects against oxLDL-induced endothelial injury remained unclarified. METHODS: Primary human umbilical vein endothelial cell cultures (HUVECs) treated with oxLDL (200 microg/ml) were used to explore the protective effect of resveratrol. Cytotoxicity of oxLDL on HUVECs was studied by measuring lactate dehydrogenase (LDH) release, methylthiazol tetrazolium (MTT) and apoptotic cell death as characterized by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) stain. We also measured the production of reactive oxygen species (ROS) by using the fluorescnet probe 2', 7'-dichlorofluorescein acetoxymethyl ester (DCF-AM), and observed the activity of antioxidant enzymes. Furthermore, several apoptotic signaling pathway with increased cytosolic calcium, alteration of mitochondrial membrane potential, cytochrome c release and activation of caspase 3 were also investigated. RESULTS: Resveratrol attenuated oxLDL-induced cytotoxicity, apoptotic features, generation of ROS and intracellular calcium accumulation. OxLDL-induced mitochondria membrane potential collapase, cytochrome c release and activation of caspase 3 in HUVECs were also suppressed by resveratrol pretreatment. CONCLUSIONS: Red wine intake may protect against oxLDL-induced dysfunction of endothelial cells.     

Involvement of Mitochondrial and Reactive Oxygen Species in the Sonodynamic Toxicity of Chlorin e6 in Human Leukemia K562 Cells

It is well accepted that sonodynamic therapy (SDT) exerts cytotoxicity and anti-tumor activity in many human tumors through the induction of cell apoptosis. The aim of the work described here was to study the effect of chlorin e6 (Ce6)-mediated SDT on human chronic myelogenous leukemia K562 cells. Our results indicate that Ce6-mediated SDT can suppress the viability of K562 cells. SDT caused apoptosis as analyzed by annexin V-phycoerythrin/7-amino-actinomycin D staining as well as cleavage of caspase 3 and the polypeptide poly(ADP-ribose) polymerase. After SDT exposure, loss of mitochondrial membrane potential, translocation of Bax from cytoplasm to mitochondria and activation of caspase 9 indicated that the mitochondrial-related apoptotic pathway might be activated. This process was accompanied by rapid generation of reactive oxygen species (ROS). Scavenging of ROS significantly blocked caspase-3 expression and the killing effect of SDT on K562 cells. Stress-activated protein kinases c-jun NH2-terminal kinase (JNK) and the p38 mitogen-activated protein kinase were activated after SDT treatment. Together, these findings indicate that Ce6-mediated SDT triggers mitochondria- and caspase-dependent apoptosis; oxidative injury may play a vital role in apoptotic signaling cascades.     

Valproic acid inhibits ATP‐triggered Ca2+ release via a p38‐dependent mechanism in bEND.3 endothelial cells

Valproic acid (VA) is currently used to treat epilepsy and bipolar disorder. It has also been demonstrated to promote neuroprotection and neurogenesis. Although beneficial actions of VA on brain blood vessels have also been demonstrated, the effects of VA on brain endothelial cell (EC) Ca²⁺ signaling are hitherto unreported. In this report, we examined the effects of VA on agonist‐triggered Ca²⁺ signaling in mouse cortical bEND.3 EC. While VA (100 μm ) did not cause an acute inhibition of ATP‐triggered Ca²⁺ signaling, a 30‐min VA treatment strongly suppressed ATP‐triggered intracellular Ca²⁺ release; however, such treatment did not affect Ca²⁺ release triggered by cyclopiazonic acid, an inhibitor of SERCA Ca²⁺ pump, suggesting there was no reduction in Ca²⁺ store size. VA‐activated p38 signaling, and VA‐induced inhibition of ATP‐triggered Ca²⁺ release was prevented by SB203580, a p38 inhibitor, suggesting VA caused the inhibition by activating p38. Remarkably, VA treatment did not affect acetylcholine‐triggered Ca²⁺ release, suggesting VA may not inhibit inositol 1,4,5‐trisphosphate‐induced Ca²⁺ release per se, and may not act directly on Gq or phospholipase C. Taken together, our results suggest VA treatment, via a p38‐dependent mechanism, led to an inhibition of purinergic receptor‐effector coupling.     

N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine induces apoptosis through the activation of caspases-3 and -8 in human platelets. A role for endoplasmic reticulum stress

Summary. Background: Apoptosis or programmed cell death involves a number of biochemical events, including the activation of caspases, which lead to specific cell morphology changes and ultimately cell death. Traditionally, two apoptotic pathways have been described: the cell‐surface death receptor‐dependent extrinsic pathway and the mitochondria‐dependent intrinsic pathway. Alternatively, apoptosis has been reported to be induced by endoplasmic reticulum (ER) stress, which is mainly induced by a reduction in intraluminal free Ca²⁺ concentration ([Ca²⁺]_ER). Objectives: The present study aimed to investigate the development of apoptotic events after ER stress induced by N,N,N′,N′‐tetrakis(2‐pyridylmethyl)ethylenediamine (TPEN), an ER Ca²⁺ chelator, in human platelets. Methods: Changes in cytosolic free Ca²⁺ concentration, caspase activity and phosphatidylserine externalization were determined by fluorimetric techniques. Results: Our results indicate that TPEN reduces the amount of free Ca²⁺ releasable by the Ca²⁺‐mobilizing agonist thrombin. TPEN induced activation of caspase‐3, ‐8 and ‐9 and subsequent phosphatidylserine externalization. The ability of TPEN to induce phosphatidylserine externalization was smaller than that of thrombin. In addition, TPEN was able to induce phosphorylation of the eukaryotic initiation factor 2α (eIF2α). TPEN‐mediated caspase‐3 activation requires functional caspase‐8, but is independent of H₂O₂ generation. Activation of caspase‐3 and ‐8 by TPEN was prevented by salubrinal, an agent that prevents ER stress‐induced apoptosis. Conclusion: These findings provide experimental evidence for the existence of ER stress‐mediated apoptosis in human platelets, a process that might limit platelet life span upon prolonged stimulation with agonists.     

Caspase-mediated apoptosis and caspase-independent cell death induced by irofulven in prostate cancer cells

Irofulven (hydroxymethylacylfulvene) is a novel antitumor drug, which acts by alkylating cellular macromolecular targets. The drug is a potent inducer of apoptosis in various types of tumor cells, whereas it is nonapoptotic in normal cells. This study defined molecular responses to irofulven involving mitochondrial dysfunction and leading to death of prostate tumor LNCaP-Pro5 cells. Irofulven caused early (2-5 hours) translocation of the proapoptotic Bax from cytosol to mitochondria followed by the dissipation of mitochondrial membrane potential and cytochrome c release at 4 to 12 hours. These effects preceded caspase activation and during the first 6 hours were not affected by caspase inhibitors. Processing of caspase-9 initiated the caspase cascade at approximately 6 hours and progressed over time. The activation of the caspase cascade provided a positive feedback loop that enhanced Bcl-2-independent translocation and cytochrome c release. General and specific caspase inhibitors abrogated irofulven-induced apoptotic DNA fragmentation with the following order of potency: pan-caspase > or = caspase-9 > caspase-8/6 > caspase-2 > caspase-3/7 > caspase-1/4. Abrogation of caspase-mediated DNA fragmentation failed to salvage irofulven-treated cells from growth inhibition and loss of viability, demonstrating a substantial contribution of a caspase-independent cell death. Monobromobimane, an inhibitor of alternative caspase-independent apoptotic pathway that is mediated by mitochondrial permeability transition, antagonized both apoptosis, measured as phosphatidylserine externalization, and cytotoxicity of irofulven. Collectively, the results indicate that irofulven-induced signaling is integrated at the level of mitochondrial dysfunction. The induction of both caspase-dependent and caspase-independent death pathways is consistent with pleiotropic effects of irofulven, which include targeting of cellular DNA and proteins.     

Advanced glycation end products‐induced chondrocyte apoptosis through mitochondrial dysfunction in cultured rabbit chondrocyte

Advanced glycation end products (AGEs) are an important mediator in osteoarthritis (OA) and cause apoptosis in articular chondrocytes. Mitochondrial function is involved in modulating apoptosis of articular chondrocytes. This study was performed to investigate the mechanism of AGEs‐induced chondrocyte apoptosis. The ratio of apoptotic cell and cell viability was surveyed by TUNEL, MTT,LDH release assay. The reactive oxygen species was determined by the fluorescent probe 2’, 7’‐dichlorofluorescein diacetate. The expression of caspase‐3 and cytochrome c was detected by Western blot. The mitochondrial membrane potential (?Ψm) was evaluated by rhodamine‐123 fluorescence. We found that AGEs induced apoptosis in primary rabbit chondrocytes, upregulation of ROS production, cytochrome c, and caspase‐3 levels. Simultaneously, AGEs decreases the levels of ?Ψm and ATP production; however, the antibody of AGEs (sRAGE) and antioxidant‐N‐acetylcys‐teine (NAC) significantly reversed AGEs‐induced the above damage thus to protect the cells from apoptosis. These observations suggested that the mechanism of AGEs‐induced chondrocyte apoptosis was primarily via ROS production and mitochondria‐mediated caspase‐3 activation.     

The apoptotic effect of hesperetin on human cervical cancer cells is mediated through cell cycle arrest,death receptor,and mitochondrial pathways

Hesperetin, a flavonoid from citrus fruits, has several bioactivities such as anti‐inflammatory, antihypertensive, antiatherogenic effects. However, studies elucidating the role and the mechanism(s) of action of hesperetin in cervical cancer are sparse. In this study, we investigated the mechanism of the antiproliferative and apoptotic actions exerted by hesperetin on human cervical cancer SiHa cells. The viability of SiHa cells was evaluated using the MTT assay, apoptosis by acridine orange/ethidium bromide, propidium iodide, TUNEL assay, and Annexin V‐Cy3, cell cycle distribution and mitochondrial transmembrane potential using flow cytometry, and apoptotic marker genes using quantitative real‐time PCR. The treatment of SiHa cells with hesperetin (IC_50, 650 μm ) showed a marked concentration‐ and time‐dependent inhibition of proliferation and induced the G2/M phase in a dose‐dependent manner after 24 h. There was an attenuation of mitochondrial membrane potential with increased expression of caspase‐3, caspase‐8, caspase‐9, p53, Bax, and Fas death receptor and its adaptor protein Fas‐associated death domain–containing protein (FADD), indicating the participation of both death receptor– and mitochondria‐related mechanisms. Furthermore, hesperetin‐induced apoptosis was confirmed by TUNEL and Annexin V‐Cy3. This study shows that hesperetin exhibits a potential anticancer activity against human cervical cancer cell lines in vitro through the reduction in cell viability and the induction of apoptosis. Altogether, these data sustain our contention that hesperetin has anticancer properties and merits further investigation as a potential therapeutic agent.     

Alpha-lipoic acid attenuates hemorrhagic shock-induced apoptotic signaling and vascular hyperpermeability

Hemorrhagic shock (HS) is associated with the disruption of endothelial cell barrier leading to vascular hyperpermeability. Previous studies from our laboratory implicate reactive oxygen species (ROS) and the intrinsic apoptotic signaling cascades as mediators of vascular hyperpermeability after HS. Here we report the protective effects of alpha-lipoic acid, a natural antioxidant with antiapoptotic properties, against vascular hyperpermeability after HS. Hemorrhagic shock was induced in Sprague-Dawley rats by withdrawing blood to reduce the MAP to 40 mmHg for 60 min followed by resuscitation for 60 min. The rats were given fluorescein isothiocyanate-albumin (50 mg/kg) i.v., and the mesenteric postcapillary venules were examined for change in hyperpermeability using intravital microscopy. Mitochondrial ROS formation and change in mitochondrial transmembrane potential were measured using dihydrorhodamine 123 and the cationic dye JC-1, respectively. The mitochondrial release of cytochrome c and activation of caspase 3 were measured using enzyme-linked immunosorbent assay and fluorometric assay, respectively. Hemorrhagic shock resulted in vascular hyperpermeability and mitochondrial ROS formation. The activation of mitochondrial intrinsic apoptotic signaling pathway was evidenced from mitochondrial depolarization, an increase in cytochrome c release, and activation of caspase 3. alpha-Lipoic acid (100 mg/kg) given before the shock period attenuated vascular hyperpermeability, mitochondrial ROS formation, mitochondrial depolarization, cytochrome c release, and activation of caspase 3 (P < 0.05). Together, these results demonstrate that alpha-lipoic acid provides protection against vascular hyperpermeability by modulating the mitochondrial "intrinsic" apoptotic signaling.     

Caspase activation is required for gemcitabine activity in multiple myeloma cell lines

The objective of this study was to determine potential mechanisms of apoptotic activity of gemcitabine, a pyrimidine nucleoside analogue, in the MM1.S multiple myeloma (MM) cell line. A MM cell line that is sensitive to glucocorticoids (MM1.S) was used for this study. Immunoblotting analysis, cell cycle assays, and annexin V staining were performed to determine whether gemcitabine induced apoptosis in this model. Furthermore, we attempted to delineate the apoptotic pathway by measuring caspase-8 and -9 activity using fluorometric assays. Loss of mitochondrial membrane potential was measured by flow cytometry. Gemcitabine treatment caused apoptosis in MM cell lines as measured by an increase in DNA cleavage, an increase in annexin V binding, a decrease in the mitochondrial membrane potential, and activation of caspase activity. Furthermore, cleavage of the caspase substrate poly(ADP-ribose) polymerase and caspase-3 activation were documented as early as 8 h after treatment with gemcitabine. Caspase-8 and -9 were activated by gemcitabine treatment in this cell line, suggesting several mechanisms of action including death receptor pathway and mitochondrial damage. The addition of interleukin 6 to MM1.S cells treated with gemcitabine offered no protection against gemcitabine-induced cell death. Gemcitabine induced apoptosis in the MM1.S cell line, and its activity required caspase activation. There is a suggestion that mitochondrial integrity is being affected with gemcitabine in this system. Gemcitabine acts independently of interleukin 6, suggesting potential important therapeutic implications in MM patients.     

MSFTZ, a flavanone derivative, induces human hepatoma cell apoptosis via a reactive oxygen species- and caspase-dependent mitochondrial pathway

Hepatocellular carcinoma (HCC) is the most common malignancy of the liver. It is unfortunate that HCCs are highly refractory to conventional chemotherapy, radiation therapy, and even immunotherapy. Thus, novel therapeutic targets need to be sought for the successful treatment of HCCs. We now report that (+/-)-(3aRS,4SR)-2-(2-chloro-4-methylsulfonylphenyl)-4'-chloro-3alpha,4-diethoxy-flavane[4,3-d]-D1,9b-1,2,3-thiadiazoline (MSFTZ), a synthesized flavanone derivative, induced growth arrest and apoptosis of HCCs both in vitro and in vivo. MSFTZ induced a time- and dose-dependent increase in HCC apoptosis through caspase-3 activation and poly(ADP-ribose) polymerase-1 cleavage. Activation of caspase-9 induced by MSFTZ suggested that MSFTZ-induced signaling was mediated through a mitochondrial death pathway. In addition, we observed an elevation of reactive oxygen species (ROS) and a consequent loss of mitochondrial membrane potential, further suggesting that MSFTZ-induced death signaling was mediated through a mitochondrial oxygen stress pathway. These events were associated with a decrease and increase in Bcl-2 and Bax expression, respectively, as well as phosphorylation of mitogen-activated protein kinase (MAPK) and activation of p53-MDM2 pathway. However, the antioxidant N-acetylcysteine opposed MSFTZ-mediated mitochondrial dysfunction, caspase activation, Bcl-2/Bax modulation, and apoptosis, supporting the role of ROS in the apoptotic process. We were surprised that we failed to observe the protective effect of N-acetylcysteine against MSFTZ-induced MAPK activation. Furthermore, MSFTZ had an antitumor effect in vivo by 34.8 to 78.7% reduction of tumor size in SMMC-7721-xenografted nude mice. We conclude that MSFTZ induces HCC cell apoptosis both in vivo and in vitro via caspase- and ROS-dependent mitochondrial pathway. In addition, MSFTZ has potential as a novel therapeutic agent for the treatment of HCC.     

Sulforaphane generates reactive oxygen species leading to mitochondrial perturbation for apoptosis in human leukemia U937 cells

Sulforaphane, an isothiocyanate found in cruciferous vegetables, has been shown to possess growth-inhibiting and apoptosis-inducing activities in cancer cell lines in vitro. In order to further explore the critical events leading to apoptosis in sulforaphane-treated U937 human leukemia cells, the following effects of sulforaphane on components of the mitochondrial apoptotic pathway were examined: generation of reactive oxygen species (ROS), alteration of the mitochondrial membrane potential (MMP), and the expression changes of Bcl-2 family proteins. The cytotoxic effect of sulforaphane was mediated by its induction of apoptosis as characterized by the occurrence of DNA ladders, apoptotic bodies and chromosome condensation in U937 cells. The sulforaphane-induced apoptosis in U937 cells correlated with the generation of intracellular ROS, collapse of MMP, activation of caspase-3, and down-regulation of anti-apoptotic Bcl-2 expression. The quenching of ROS generation with antioxidant N-acetyl-l-cysteine conferred significant protection against sulforaphane-elicited ROS generation, disruption of the MMP, caspase-3 activation and apoptosis. In conclusion, the present study reveals that the cellular ROS generation plays a pivotal role in the initiation of sulforaphane-triggered apoptotic death in U937 cells.     

A two-stage poly(ethylenimine)-mediated cytotoxicity: implications for gene transfer/therapy.

Poly(ethylenimine) (PEI) is a cationic macromolecule commonly used in gene transfer/therapy protocols with high transfection efficiency both in vitro and in vivo. PEI is also cytotoxic, but the molecular basis of its cytotoxicity is poorly understood. Here, we have demonstrated that branched (25 kDa) and linear (750 kDa) PEI can both induce membrane damage and initiate apoptosis in three clinically relevant human cell lines (Jurkat T cells, umbilical vein endothelial cells, and THLE3 hepatocyte-like cells). We have defined Phase I toxicity as early necrotic-like changes (30 min) resulting from compromised membrane integrity, assessed by considerable lactate dehydrogenase release and phosphatidylserine translocation from the inner plasma membrane to the outer cell surface. Phase II cytotoxicity (24 h) was due to activation of a "mitochondrially mediated apoptotic program," resulting from PEI-induced channel formation in the outer mitochondrial membrane. This led to the release of proapoptotic cytochrome c, subsequent activation of caspase 3, and alteration in mitochondrial membrane potential as a result of caspase translocation into the mitochondria. The reported observations have important implications for the design and execution of gene therapy protocols as well for controlling intracellular distribution of drugs with cationic-based polymer-delivery systems.     

Dependence of reactive oxygen species and FLICE inhibitory protein on lipofectamine-induced apoptosis in human lung epithelial cells

Cationic liposomes such as lipofectamine (LF) are widely used as nonviral gene delivery vectors; however, their clinical application is limited by their cytotoxicity. These agents have been shown to induce apoptosis as the primary mode of cell death, but their mechanism of action is not well understood. The present study investigated the mechanism of LF-induced apoptosis and examined the role of reactive oxygen species (ROS) in this process. We found that LF induced apoptosis of human epithelial H460 cells through a mechanism that involves caspase activation and ROS generation. Inhibition of caspase activity by pan-caspase inhibitor (z-VAD-fmk) or by specific caspase-8 inhibitor (z-IETD-fmk) or caspase-9 inhibitor (z-LEHD-fmk) inhibited the apoptotic effect of LF. Overexpression of FLICE-inhibitory protein (FLIP) or B-cell lymphoma-2, which are known inhibitors of the extrinsic and intrinsic death pathways, respectively, similarly inhibited apoptosis by LF. Induction of apoptosis by LF was shown to require ROS generation because its inhibition by ROS scavengers or by ectopic expression of antioxidant enzyme superoxide dismutase and glutathione peroxidase strongly inhibited the apoptotic effect of LF. Electron spin resonance studies showed that LF induced multiple ROS; however, superoxide was found to be the primary ROS responsible for LF-induced apoptosis. The mechanism by which ROS mediate the apoptotic effect of LF involves down-regulation of FLIP through the ubiquitination pathway. In demonstrating the role of FLIP and ROS in LF death signaling, we document a novel mechanism of apoptosis regulation that may be exploited to decrease cytotoxicity and increase gene transfection efficiency of cationic liposomes.     

Platelet senescence and phosphatidylserine exposure

BACKGROUND: The exposure of phosphatidylserine occurs during platelet (PLT) activation and during in vitro storage. Phosphatidylserine exposure also occurs during apoptosis after the release of mitochondrial cytochrome c. We have examined the role of cytochrome c release, mitochondrial membrane potential (ΔΨm), and cyclophilin D (CypD) in phosphatidylserine exposure due to activation and storage. STUDY DESIGN AND METHODS: The exposure of phosphatidylserine and the loss of ΔΨm were determined in a flow cytometer using fluorescein isothiocyanate–lactadherin and JC‐1, a lipophilic cationic reporter dye. The role of CypD was determined with cyclosporin A and CypD‐deficient murine PLTs. Cytochrome c‐induced caspase‐3 and Rho‐associated kinase I (ROCK1) activation were determined by immunoblotting and using their inhibitors. RESULTS: Collagen‐ and thrombin‐induced exposure of phosphatidylserine was accompanied by a decrease in ΔΨm. Cyclosporin A inhibited the phosphatidylserine exposure and the loss of ΔΨm. CypD^?/? mice had decreased loss of ΔΨm and impaired phosphatidylserine exposure. Collagen and thrombin did not induce the release of cytochrome c nor the activation of caspase‐3 and ROCK1. In contrast, in PLTs stored for more than 5 days, the phosphatidylserine exposure was associated with cytochrome c–induced caspase‐3 and ROCK1 activation. ABT737, a BH3 mimetic that induces mitochondrial pathway of apoptosis, induced cytochrome c release and activation of caspase‐3 and ROCK1 and phosphatidylserine exposure independent of CypD. CONCLUSION: These results show that in stored PLTs cytochrome c release and the subsequent activation of caspase‐3 and ROCK1 mediate phosphatidylserine exposure and it is distinct from activation‐induced phosphatidylserine exposure.     

Flow Cytometric Analysis of Ca2+-Induced Membrane Permeability Transition of Isolated Rat Liver Mitochondria

The membrane permeability transition (MPT) of mitochondria plays an important role in the mechanism of apoptotic cell death in various cells. Classic type MPT is induced by Ca²⁺ in the presence of inorganic phosphate and respiratory substrate, and is characterized by various events including generation of reactive oxygen species (ROS), membrane depolarization, swelling, release of Ca²⁺ and high sensitivity to cyclosporine A. However, the sequence of these events and the effect of antioxidants on their events remain obscure. Flow cytometry is a convenient method to investigate the order of events among various functions occurring in MPT using a limited amount of mitochondria (200 µl of 0.02 mg protein/ml) without contamination by other organelles. Flow cytometric analysis revealed that Ca²⁺ sequentially induced ROS generation, depolarization, swelling and Ca²⁺ release in mitochondria by a cyclosporine A-inhibitable mechanism. These results were supported by the finding that Ca²⁺-induced MPT was inhibited by antioxidants, such as glutathione and N-acetylcysteine. It was also revealed that various inhibitors of Ca²⁺-induced phospholipase A₂ suppressed all of the events associated with Ca²⁺-induced MPT. These results suggested that ROS generation and phospholipase A₂ activation by Ca²⁺ underlie the mechanism of the initiation of MPT.     

Antitumor effect of N-succinyl-chitosan nanoparticles on K562 cells

Polymer nanoparticles have become attractive for its prominent property recent years. In this paper, antitumor effects of N-succinyl-chitosan nanoparticles (NSCNP), a nanoparticles derivated from chitosan, were evaluated in K562 cells, including cell viability comparison, cell morphology analysis, DNA fragmentation detection, cell surface potential and mitochondrial membrane potential (MMP) measurement, intracellular ROS and Ca²⁺ concentration evaluation. Our results revealed NSCNP could inhibit the proliferation of K562 with an IC₅₀ of 14.26 μg/ml (24 h); decrease the zeta potential; disrupt the mitochondrial membrane potential; increase ROS generation and Ca²⁺ concentration. Cytomorphology study and DNA fragment analysis reveal characteristics of apoptosis and necrosis, indicating that the antitumor effect of NSCNP achieved by necrosis and apoptosis induction in K562 cells.     

Costunolide,an active sesquiterpene lactone,induced apoptosis via ROS-mediated ER stress and JNK pathway in human U2OS cells

Costunolide, an active sesquiterpene lactone, is derived from many herbal medicines and it exhibits a broad spectrum of bioactivities such as anti-inflammatory, potential anti-tumor activity. Herein we assessed the anti-cancer effects of costunolide on U2OS cells and explored the underlying molecular mechanisms. The experiment data show that Costunolide exhibited significant anti-tumor activity by apoptosis related assays including Annexin V-FITC/PI flow cytometric analysis and 4,6-diamino-2-phenyl indole (DAPI) staining morphological analysis. Furthermore, we found Costunolide induced the loss of mitochondrial transmembrane potential, down-regulated Bcl-2/Bax ratio, encouraged Cyt-c release and caspase activation. All those effects are contributed by reactive oxygen species (ROS) generation and ER stress-induced mitochondrial dysfunction which are also responsible for c-Jun N-terminal kinase (JNK) activation. After the treatment of JNK inhibitor SP600125, it obviously reversed costunolide-induced apoptosis. Given N-acetyl-l-cysteine (NAC) effectively blocked the activation of JNK. Taken together, our results demonstrate that costunolide induces apoptosis in human U2OS cells through ROS generation and p38 MAPK/JNK activation.     

Epoxyeicosatrienoic acids attenuate reactive oxygen species level, mitochondrial dysfunction, caspase activation, and apoptosis in carcinoma cells treated with arsenic trioxide

Epoxyeicosatrienoic acids (EETs) and the cytochrome P450 epoxygenase CYP2J2 promote tumorogenesis in vivo and in vitro via direct stimulation of tumor cell growth and inhibition of tumor cell apoptosis. Herein, we describe a novel mechanism of inhibition of tumor cell apoptosis by EETs. In Tca-8113 cancer cells, the antileukemia drug arsenic trioxide (ATO) led to the generation of reactive oxygen species (ROS), impaired mitochondrial function, and induced apoptosis. 11,12-EET pretreatment increased expression of the antioxidant enzymes superoxide dismutase and catalase and inhibited ATO-induced apoptosis. 11,12-EET also prevented the ATO-induced activation of p38 mitogen-activated protein kinase, c-Jun NH(2)-terminal kinase, caspase-3, and caspase-9. Therefore, 11,12-EET-pretreatment attenuated the ROS generation, loss of mitochondrial function, and caspase activation observed after ATO treatment. Moreover, the CYP2J2-specific inhibitor compound 26 enhanced arsenic cytotoxicity to a clinically relevant concentration of ATO (1-2 μM). Both the thiol-containing antioxidant, N-acetyl-cysteine, and 11,12-EET reversed the synergistic effect of the two agents. Taken together, these data indicate that 11,12-EET inhibits apoptosis induced by ATO through a mechanism that involves induction of antioxidant proteins and attenuation of ROS-mediated mitochondrial dysfunction.     

5-Aminoimidazole-4-carboxamide riboside sensitizes TRAIL- and TNF{alpha}-induced cytotoxicity in colon cancer cells through AMP-activated protein kinase signaling

Death receptor-mediated tumor cell death, either alone or in combination with other anticancer drugs, is considered as a new strategy for anticancer therapy. In this study, we have investigated the effects and molecular mechanisms of 5-aminoimidazole-4-carboxamide riboside [AICAR; a pharmacologic activator of AMP-activated protein kinase (AMPK)] in sensitizing tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)- and TNFalpha-induced apoptosis of human colon cancer HCT116 cells. The cytotoxic action of AICAR requires AMPK activation and may occur at various stages of apoptotic pathways. AICAR cotreatment with either TRAIL or TNFalpha enhances activities of caspase-8, caspase-9, and caspase-3; down-regulates the antiapoptotic protein Bcl-2; increases the cleavage of Bid and results in the decrease of mitochondrial membrane potential; potentiates activation of p38 and c-Jun NH(2)-terminal kinase; and inhibits nuclear factor-kappaB activity. In addition, this sensitized cell apoptosis was neither observed in p53-null HCT116 cells nor affected by the cotreatment with mevalonate. In summary, we have developed a novel strategy of combining AICAR with TRAIL for the treatment of colon cancer cells. The sensitization effect of AICAR in cell apoptosis was mediated through AMPK pathway, requires p53 activity, and involves mitochondria-dependent apoptotic cascades, p38 and c-Jun NH(2)-terminal kinase.