Leakage Of Cytochrome Research Articles

G-quadruplex-Based Artificial Transmembrane Channels Induce Cancer Cell Apoptosis by Perturbing Potassium Ion Homeostasis.

Transmembrane ion transport modality has received a widespread attention due to its apoptotic activation toward anticancer cell activities. In this study, G-quadruplex-based potassium-specific transmembrane channels have been developed to facilitate the intracellular K+ efflux, which perturbs the cellular ion homeostasis thereby inducing cancer cell apoptosis. Cholesterol-tag, a lipophilic anchor moiety, serves as a rudiment for the G-quadruplex immobilization onto the membrane, while G-quadruplex channel structure as a transport module permits ion binding and migration along the channels. A c-Myc sequence tagged with two-cholesterol is designed as a representative lipophilic G-quadruplex, which forms intramolecular parallel G-quadruplex with three stacks of G-quartets (Ch2-Para3). Fluorescence transport assay demonstrates Ch2-Para3 a high transport activity (EC50 = 10.9 × 10-6 m) and an ion selectivity (K+/Na+ selectivity ratio of 84). Ch2-Para3 mediated K+ efflux in cancer cells is revealed to purge cancer cells through K+ efflux-mediated cell apoptosis, which is confirmed by monitoring the changes in membrane potential of mitochondria, leakage of cytochrome c, reactive oxygen species yield, as well as activation of a family of caspases. The lipophilic G-quadruplex exhibits obvious antitumor activity in vivo without systemic toxicity. This study provides a functional scheme aimed at generating DNA-based selective artificial membrane channels for the purpose of regulating cellular processes and inducing cell apoptosis, which shows a great promising for anticancer therapy in the future.

Saikosaponin-A induces apoptosis of cervical cancer through mitochondria- and endoplasmic reticulum stress-dependent pathway in vitro and in vivo: involvement of PI3K/AKT signaling pathway

ABSTRACT Cervical cancer causes considerable mortality in women worldwide. Saikosaponin-A, a triterpenoid glycoside isolated from Bupleurum falcatum, has been proven to exert anti-cancer property. In this study, we evaluated the possibility of saikosaponin-A on cervical cancer in vitro and in vivo. The results showed that saikosaponin-A induced cell death and altered cellular morphology dose-dependently. Saikosaponin-A significantly induced apoptosis in HeLa cells, confirmed by Hoechst 33,342 staining and flow cytometry. Sequentially, saikosaponin-A triggered the mitochondrial-mediated apoptosis demonstrated by deficiency of MMP, induction of Bax/Bcl-2 ratio, leakage of cytochrome c to cytoplasm, and activation of caspase-3. Moreover, ER stress also participated in the apoptosis induced by saikosaponin-A in HeLa cells as indicated by the upregulation of GPR78, CHOP and caspase-12 expression. Furthermore, HeLa cells showed increased expressions of p-PI3K and p-AKT in response to saikosaponin-A treatment. Additionally, saikosaponin-A could inhibit HeLa tumor growth in nude mice and induce apoptosis, reflected by the induction of TUNEL and the expression of cytochrome c, caspase-3 and CHOP confirmed by immunohistochemistry. These findings at least to a certain extent suggested that saikosaponin-A triggered apoptosis through both mitochondrial pathway and ER stress pathway and inhibiting PI3K/Akt signaling, thereby contributing to against cervical cancer. This work provides a new understanding of saikosaponin-A on therapeutic application in treatment of cancer, which has the potential to be a promising candidate therapeutic agent for cervical cancer patients.

Apoptotic caspases suppress Mycobacterium bovis-induced IFN-β production in murine macrophage

Caspases are classified as inflammatory or apoptotic category. Inflammatory caspases participate in inflammasome activation, while apoptotic caspases mediate apoptotic activation. Previous studies have shown that apoptotic caspases prevent the production of IFN-β during apoptosis or virus infection. However, the relationship between apoptotic caspases and IFN-β production during intracellular bacterial infection is still unclear. Here, we investigated the role of apoptotic caspases in IFN-β production induced by Mycobacterium bovis (M. bovis) infection. M. bovis is an intracellular bacterium and belongs to the Mycobacterium tuberculosis complex. M. bovis infection can cause tuberculosis in animals and human beings. In the current study, we found that M. bovis infection triggered mitochondrial stress, which caused the leakage of cytochrome c into the cytoplasm, and in turn, activated the downstream caspase-9 and-3. Furthermore, our results showed that activation of apoptotic caspases reduced IFN-β production during M. bovis infection and vice versa. Confocal microscopy analysis revealed that apoptotic caspases prevented IFN-β production by decreasing p-IRF3 nuclear translocation. Our findings demonstrate that apoptotic caspases negatively regulate the production of IFN-β induced by an intracellular bacterial infection.

Abstract 13320: Direct Cardiac Actions of Empagliflozin Improve Mitochondrial Oxidative Phosphorylation and Attenuate Pressure-overload Heart Failure

Introduction: The underlying mechanisms by which empagliflozin (EMPA) and other sodium glucose co-transporter 2 (SGLT2) inhibitors attenuate heart failure (HF) are still poorly understood. However, this protection does not appear to be fully explained by their antihyperglycemic or diuretic effects. Hypothesis: EMPA attenuates HF by direct effects on the heart to improve its metabolism and function. Methods: C57BL/6J mice (4-6 months) were subjected to transverse aortic constriction (TAC) or sham surgeries. Two weeks after TAC, EMPA (10 mg/kg/day) or vehicle was administered daily for 4 additional weeks. Cardiac function was assessed by echocardiography and cardiac substrate metabolism measured in isolated perfused hearts. Transmission electron microscopy was used to evaluate mitochondrial morphology and molecular docking analysis to predict potential cardiac targets of EMPA. Results: EMPA increased survival and attenuated adverse left ventricle remodeling and cardiac fibrosis after TAC. EMPA also attenuated left ventricular systolic dysfunction (ejection fraction 51.6 vs. 40.2% p<0.05; fraction shortening 28.8 vs 18.4% p<0.05). EMPA rescued impaired glucose and fatty acid oxidation in failing hearts, while reducing glycolysis. Molecular docking analysis and isolated perfused heart experiments indicated that EMPA can directly bind glucose transporters in the heart to reduce glycolysis, and enhance AMP-activated protein kinase. EMPA treatment enhanced mitochondrial biogenesis, restored mitochondria cristae integrity, increased expression of endogenous antioxidants, and reduced cellular apoptosis caused by leakage of cytochrome C from mitochondria into the cytosol. These beneficial cardiac effects of EMPA occurred despite no alterations in fasting blood glucose, body weight, or daily urine volume. Conclusions. Our study demonstrated that EMPA may directly bind glucose transporters and reduce excessive glycolysis in failing hearts. EMPA enhanced mitochondrial biogenesis, improved mitochondrial oxidative phosphorylation, and reduced mitochondria-mediated apoptosis, thereby attenuating cardiac dysfunction and progression of HF.

Biocompatibility assessment of single-walled carbon nanotubes using Saccharomyces cerevisiae as a model organism

BackgroundSingle-walled carbon nanotubes (SWCNTs) have many potential applications in various fields. Especially, the unique physicochemical properties make them as the prime candidates for applications in biomedical fields. However, biocompatibility of SWCNTs has been a major concern for their applications. In the study, biocompatibility of oxidized SWCNTs (O-SWCNTs) was assessed using Saccharomyces cerevisiae (S. cerevisiae) as a model organism.ResultsCell proliferation and viability were significantly changed after exposure to O-SWCNTs (188.2 and 376.4 mg/L) for 24 h. O-SWCNTs were internalized in cells and distributed in cytoplasm, vesicles, lysosomes and cell nucleus. The average O-SWCNTs contents in S. cerevisiae were ranged from 0.18 to 4.82 mg/g during the exposure from 0 to 24 h, and the maximum content was reached at 18 h after exposure. Both penetration and endocytosis were involved in the internalization of O-SWCNTs in S. cerevisiae, and endocytosis was the main pathway. Cellular structures and morphology were changed after exposure to O-SWCNTs, such as undulating appearance at the membrane, shrinking of the cytosol, increased numbers of lipid droplets and disruption of vacuoles. ROS and antioxidant enzymes activities were observably changed following exposure. For the treatment at 376.4 mg/L, 20.8% of the total cells was undergone apoptosis. Decrease of mitochondrial transmembrane potential and leakage of cytochrome c from mitochondria were observed after exposure. Moreover, expression levels of apoptosis-related genes were significantly increased.ConclusionsO-SWCNTs can internalize in S. cerevisiae cells via direct penetration and endocytosis, and distribute in cytoplasm, vesicles, lysosomes and cell nucleus. Besides, O-SWCNTs (188.2 and 376.4 mg/L) can induce apoptosis in S. cerevisiae cells, and oxidative stress is involved in activation of the mitochondria-dependent apoptotic pathway.

CGA-N12, a peptide derived from chromogranin A, promotes apoptosis of Candida tropicalis by attenuating mitochondrial functions.

CGA-N12 (the amino acid sequence from the 65th to the 76th residue of the N-terminus of chromagranin A) is an antifungal peptide derived from human chromogranin A (CGA). In our previous investigation, CGA-N12 was found to have specific anti-candidal activity, though the mechanism of action remained unclear. Here, we investigated the effects of CGA-N12 on mitochondria. We found that CGA-N12 induced an over-generation of intracellular reactive oxygen species and dissipation in mitochondrial membrane potential, in which the former plays key roles in the initiation of apoptosis and the latter is a sign of the cell apoptosis. Accordingly, we assessed the apoptosis features of Candida tropicalis cells after treatment with CGA-N12 and found the following: leakage of cytochrome c and uptake of calcium ions into mitochondria and the cytosol; metacaspase activation; and apoptotic phenotypes, such as chromatin condensation and DNA degradation. In conclusion, CGA-N12 is capable of inducing apoptosis in C. tropicalis cells through mitochondrial dysfunction and metacaspase activation. Antifungal peptide CGA-N12 from human CGA exhibits a novel apoptotic mechanism as an antifungal agent.

NR4A1-induced increase in the sensitivity of a human gastric cancer line to TNFα-mediated apoptosis is associated with the inhibition of JNK/Parkin-dependent mitophagy.

Tumor necrosis factor α (TNFα)-based immunotherapy is the vital host defense system against the progression of gastric cancer (GC) as a pro-inflammatory and pro-apoptotic cytokine. However, resistance limits its therapeutic efficiency. Therefore, an increasing number of studies are focusing on the development of drugs or methods with which to enhance the treatment efficacy of TNFα. Nuclear receptor subfamily 4 group A member 1 (NR4A1) has been shown to exert antitumor effects through several mechanisms, such as by inhibiting proliferation, as well as pro-apoptotic and potent pro-oxidant effects. In this study, we examined the effects and mechanisms of action of NR4A1 on the apoptosis of GC cells treated with TNFα, with particular focus on mitochondrial homeostasis. We found that TNFα treatment decreased NR4A1 expression. Moreover, the overexpression of NR4A1 in the presence of TNFα further increased GC cell apoptosis. Mechanistically, the overexpression of NR4A1 augmented caspase-9-dependent mitochondrial apoptosis, as evidenced by reduced mitochondrial membrane potential, reactive oxygen species (ROS) overproduction, mitochondrial permeability transition pore (mPTP) opening and the leakage of cytochrome c (Cyt-c) leakage. Moreover, NR4A1 overexpression also evoked mitochondrial energy disorder via the suppression of mitochondrial respiratory complex expression. Furthermore, we found that TNFα treatment activated Parkin-dependent mitophagy. Excessive Parkin-dependent mitophagy blocked mitochondrial apoptosis, undermining the toxic effects of TNFα on cells. However, NR4A1 overexpression suppressed Parkin-dependent mitophagy via the inhibition of c-Jun N-terminal kinase (JNK). Re-activation of the JNK/Parkin pathway abrogated the inhibitory effects of NR4A1 on mitophagy, eventually limiting cell apoptosis. Collectively, this study confirmed that NR4A1 sensitizes GC cells to TNFα-induced apoptosis through the inhibition of JNK/Parkin-dependent mitophagy.

Homocysteine Induces Apoptosis of Human Umbilical Vein Endothelial Cells via Mitochondrial Dysfunction and Endoplasmic Reticulum Stress.

Homocysteine- (Hcy-) induced endothelial cell apoptosis has been suggested as a cause of Hcy-dependent vascular injury, while the proposed molecular pathways underlying this process are unclear. In this study, we investigated the adverse effects of Hcy on human umbilical vein endothelial cells (HUVEC) and the underlying mechanisms. Our results demonstrated that moderate-dose Hcy treatment induced HUVEC apoptosis in a time-dependent manner. Furthermore, prolonged Hcy treatment increased the expression of NOX4 and the production of intracellular ROS but decreased the ratio of Bcl-2/Bax and mitochondrial membrane potential (MMP), resulting in the leakage of cytochrome c and activation of caspase-3. Prolonged Hcy treatment also upregulated glucose-regulated protein 78 (GRP78), activated protein kinase RNA-like ER kinase (PERK), and induced the expression of C/EBP homologous protein (CHOP) and the phosphorylation of NF-κb. The inhibition of NOX4 decreased the production of ROS and alleviated the Hcy-induced HUVEC apoptosis and ER stress. Blocking the PERK pathway partly alleviated Hcy-induced HUVEC apoptosis and the activation of NF-κb. Taken together, our results suggest that Hcy-induced mitochondrial dysfunction crucially modulated apoptosis and contributed to the activation of ER stress in HUVEC. The excessive activation of the PERK pathway partly contributed to Hcy-induced HUVEC apoptosis and the phosphorylation of NF-κb.

Toxicological effects of multi-walled carbon nanotubes on Saccharomyces cerevisiae: The uptake kinetics and mechanisms and the toxic responses

Using Saccharomyces cerevisiae as an experimental model, the potential toxicological effects of oxidized multi-walled carbon nanotubes (MWCNTs) were investigated following exposure to 0–600mg/L for 24h. Results indicated that MWCNTs (>100mg/L) had adverse effects on the cell proliferation. MWCNTs were clearly visible in lysosome, vacuole, endosome, mitochondria, multivesicular body and localization in the perinuclear region. The uptake kinetics data demonstrated that the maximum MWCNTs content (209.61mg/g) was reached at 3h, and a steady state was reached after 18h. Based on the combined results of transmission electron microscope, endocytosis inhibition experiments and endocytosis-related genes (END3, END6, Sla2 and Rsp5) expression analysis, we elucidated MWCNTs uptake mechanism: (i) via a direct penetration of single MWCNTs; (ii) via endocytosis of single MWCNTs; and (iii) via endocytosis of MWCNTs aggregates. The percentage of apoptosis was significant increased at 600mg/L. The decrease of mitochondrial transmembrane potential and the leakage of cytochrome c shown dose-dependent manners. Interestingly, there was no significant increase of reactive oxygen species (ROS). The apoptosis-related genes (SOD1, SOD2, Yca1, Nma111 and Nuc1) were significant changed. These results obtained in our study demonstrated that oxidized MWCNTs induce Saccharomyces cerevisiae apoptosis via mitochondrial impairment pathway.

Protective Role of Hinokitiol Against H2O2-Induced Injury in Human Corneal Epithelium

ABSTRACTPurpose: We recently found that hinokitiol has anti-inflammatory activity in human corneal epithelial (HCE) cells. Herein, we investigated the protective role of hinokitiol against H2O2-induced injury in HCE cells and the mechanisms that underlie its action.Methods: HCE cells were incubated with different concentrations of hinokitiol or dimethylsulfoxide (DMSO), which served as a vehicle control, before H2O2 stimulus. The cell viability was evaluated using a cell counting kit-8 (CCK-8) assay. TUNEL, phosphorylated histone γH2A.X, cleaved caspase-3 expression analyses, and location of cytochrome c were conducted to detect cell injury and apoptosis. Reactive oxygen species (ROS), catalase (CAT), superoxide dismutase (SOD), methane dicarboxylic aldehyde (MDA), and total antioxidative capacity (T-AOC) were used to determine oxidative stress. Bcl-2 and Bax protein expressions were measured by western blotting.Results: Hinokitiol significantly improved the cell viability, decreased the apoptosis rate, inhibited DNA damage, and reduced cleaved caspase-3 expression and the leakage of cytochrome c from mimitochondrion to cytoplasm of HCE cells against the oxidative stress induced by H2O2. Generation of ROS and MDA and decreased activity of CAT, SOD, and T-AOC were also ameliorated by hinokitiol administration. Moreover, Bcl-2 expression was down-regulated while Bax was up-regulated by H2O2 stimulus, which were reversed by hinokitiol application.Conclusion: Hinokitiol protects HCE cells against H2O2-induced injury likely by its antioxidant activity and modulating the Bcl-2 signaling pathway.

3-Bromopyruvate and sodium citrate induce apoptosis in human gastric cancer cell line MGC-803 by inhibiting glycolysis and promoting mitochondria-regulated apoptosis pathway

Cancer cells are mainly dependent on glycolysis to generate adenosine triphosphate (ATP) and intermediates required for cell growth and proliferation. Thus, inhibition of glycolysis might be of therapeutic value in antitumor treatment. Our previously studies had found that both 3-bromopyruvate (BP) and sodium citrate (SCT) can inhibit tumor growth and proliferation in vitro and in vivo. However, the mechanism involved in the BP and SCT mediated antitumor activity is not entirely clear. In this work, it is demonstrated that BP inhibits the enzyme hexokinase (HK) activity and SCT suppresses the phosphofructokinase (PFK) activity respectively, both the two agents decrease viability, ATP generation and lactate content in the human gastric cancer cell line MGC-803. These effects are directly correlated with blockage of glycolysis. Furthermore, BP and SCT can induce the characteristic manifestations of mitochondria-regulated apoptosis, such as down-regulation of anti-apoptosis proteins Bcl-2 and Survivin, up-regulation of pro-apoptosis protein Bax, activation of caspase-3, as well as leakage of cytochrome c (Cyt-c). In summary, our results provided evidences that BP and SCT inhibit the MGC-803 cells growth and proliferation might be correlated with inhibiting glycolysis and promoting mitochondria-regulated apoptosis.

Bortezomib-mediated downregulation of S-phase kinase protein-2 (SKP2) causes apoptotic cell death in chronic myelogenous leukemia cells.

BackgroundProteasome inhibitors are attractive cancer therapeutic agents because they can regulate apoptosis-related proteins. Bortezomib also known as Velcade®, a proteasome inhibitor that has been approved by the food and drug administration for treatment of patients with multiple myeloma, and many clinical trials are ongoing to examine to the efficacy of bortezomib for the treatment of other malignancies. Bortezomib has been shown to induce apoptosis and inhibit cell growth of many cancer cells. In current study, we determine whether bortezomib induces cell death/apoptosis in CML.MethodsCell viability was measured using MTT assays. Apoptosis was measured by annexin V/PI dual staining and DNA fragmentation assays. Immunoblotting was performed to examine the expression of proteins. Colony assays were performed using methylcellulose.ResultsTreatment of CML cells with bortezomib results in downregulation of S-phase kinase protein 2 (SKP2) and concomitant stabilization of the expression of p27Kip1. Furthermore, knockdown of SKP2 with small interference RNA specific for SKP2 caused accumulation of p27Kip1. CML cells exposed to bortezomib leads to conformational changes in Bax protein, resulting in loss of mitochondrial membrane potential and leakage of cytochrome c to the cytosol. In the cytosol, cytochrome c causes sequential activation of caspase-9, caspase-3, PARP cleavage and apoptosis. Pretreatment of CML cells with a universal inhibitor of caspases, z-VAD-fmk, prevents bortezomib-mediated apoptosis. Our data also demonstrated that bortezomib treatment of CML downregulates the expression of inhibitor of apoptosis proteins. Finally, inhibition of proteasome pathways by bortezomib suppresses colony formation ability of CML cells.ConclusionsAltogether, these findings suggest that bortezomib suppresses the cell proliferation via induction of apoptosis in CML cells by downregulation of SKP2 with concomitant accumulation of p27Kip1, suggesting that proteasomal pathway may form novel therapeutic targets for better management of CML.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0823-y) contains supplementary material, which is available to authorized users.

Sox6 suppression induces RA-dependent apoptosis mediated by BMP-4 expression during neuronal differentiation in P19 cells.

Sox6 is a transcription factor that induces neuronal differentiation in P19 cells; its suppression not only inhibits neuronal differentiation but also induces retinoic acid (RA)-dependent apoptosis of P19 cells. In the present study, we found that Sox6 suppression-induced apoptosis was mediated by activation of caspase 9 and 3. Moreover, we noted a weak leakage of cytochrome c into the cytoplasm from the mitochondria, indicating that apoptosis occurs through a mitochondrial pathway in Sox6-suppressed P19 (P19[anti-Sox6]) cells. Sox6 suppression in the presence of RA also induced the expression and secretion of bone morphogenetic protein 4 (BMP-4). Addition of an anti-BMP-4 antibody for neutralization increased cell viability and led to RA-dependent death of P19[anti-Sox6] cells. Our results indicate that Sox6 suppression induces RA-dependent cell death of P19 cells, mediated by BMP-4 expression and secretion. Normally, high Sox6 expression leads to RA-mediated neuronal differentiation in P19 cells; however, Sox6 deficiency induces production and secretion of BMP-4, which mediates selective cell death. Our findings suggest that Sox6 contributes to cell survival by suppressing BMP-4 transcription during neuronal differentiation.

PK11195-chitosan-graft-polyethylenimine-modified SPION as a mitochondria-targeting gene carrier

Superparamagnetic iron oxide nanoparticle (SPION) holds great potential as a gene delivery system due to its unique properties, such as good biocompatibility and non-invasive targeting ability. In this study, we modified SPION with chitosan-graft-PEI (CHI-g-PEI) and PK11195, to fabricate a mitochondria-targeting gene carrier, PK-CP-SPION. PK-CP-SPION manifested prominent physicochemical properties for magnetic guided gene delivery, and it could effectively condense and protect DNA at proper weight ratios. The in vitro cytotoxicity of PK-CP-SPIONs was mild. Under an external magnetic field, the transfection efficiency of PK-CP-SPIONs was comparable to PEI 25 K with shorter transfection time. PK11195 facilitated the specific accumulation of PK-CP-SPIONs in mitochondria, leading to the leakage of cytochrome c, the dissipation of mitochondrial membrane potential and subsequently the activation of mitochondria apoptosis pathway. These results indicated that with further development, PK-CP-SPIONs could serve as a multifunctional nanoplatform for magnetic targeting gene delivery and mitochondria-targeting therapy, leading enhanced therapeutic effect towards tumor cells.

Abstract 258: Nanoparticle-mediated Targeting of a Chemical Inhibitor of Drp1 to the Mitochondria Induces Cardioprotection From Myocardial Ischemia-reperfusion Injury

Background: Mitochondria (MITO) injury including MITO permeability transition pore (mPTP) opening plays a major role in the mechanism of ischemia-reperfusion (IR) injury. Intravenous administration of an inhibitor of mPTP opening, cyclosporine A, can reduce IR injury in animals and patients with acute myocardial infarction (MI), however; the power of cardioprotection by cyclosporine A is insufficient. We tested the hypothesis that nanoparticle-mediated targeting of Mdivi1, a chemical inhibitor of Drp1, to MITO enhances cardioprotection from IR injury. Methods and Results: We formulated poly(lactic acid/glycolic acid) (PLGA) nanoparticles containing Mdivi1 (Mdivi1-NP) or FITC (FITC-NP). In neonatal rat cardiomyocytes, PLGA nanoparticles accumulated in MITO after the addition of hydrogen peroxide (H2O2) that represents oxidative stress during IR (Fig.A). Treatment with Mdivi1-NP reduced H2O2-induced MITO division and cardiomyocyte death (Fig.B). This Mdivi1-NP cardioprotective effect was not seen over adenovirus harboring Drp1 shRNA transduced cardiomyocyte. In an in vivo and ex vivo murine model, treatment with Mdivi1-NP at the time of reperfusion reduced infarct size more effectively than Mdivi1 alone (Fig.C). Interestingly, Mdivi1-NP inhibited the leakage of cytochrome c to cytosol and MITO swelling, and reduced IR injury in both wildtype and cyclophilin D (a key regulatory molecule for mPTP opening)-KO mice (Fig.D). Conclusions: Mdivi1-NP enhanced cardioprotection against IR injury through mechanisms independent of mPTP opening. Mdivi1-NP can be a novel cardioprotective strategy in acute MI.

The chemopotential effect of Annona muricata leaves against azoxymethane-induced colonic aberrant crypt foci in rats and the apoptotic effect of Acetogenin Annomuricin E in HT-29 cells: a bioassay-guided approach.

Annona muricata has been used in folk medicine for the treatment of cancer and tumors. This study evaluated the chemopreventive properties of an ethyl acetate extract of A. muricata leaves (EEAML) on azoxymethane-induced colonic aberrant crypt foci (ACF) in rats. Moreover, the cytotoxic compound of EEAML (Annomuricin E) was isolated, and its apoptosis-inducing effect was investigated against HT-29 colon cancer cell line using a bioassay-guided approach. This experiment was performed on five groups of rats: negative control, cancer control, EEAML (250 mg/kg), EEAML (500 mg/kg) and positive control (5-fluorouracil). Methylene blue staining of colorectal specimens showed that application of EEAML at both doses significantly reduced the colonic ACF formation compared with the cancer control group. Immunohistochemistry analysis showed the down-regulation of PCNA and Bcl-2 proteins and the up-regulation of Bax protein after administration of EEAML compared with the cancer control group. In addition, an increase in the levels of enzymatic antioxidants and a decrease in the malondialdehyde level of the colon tissue homogenates were observed, suggesting the suppression of lipid peroxidation. Annomuricin E inhibited the growth of HT-29 cells with an IC50 value of 1.62 ± 0.24 μg/ml after 48 h. The cytotoxic effect of annomuricin E was further substantiated by G1 cell cycle arrest and early apoptosis induction in HT-29 cells. Annomuricin E triggered mitochondria-initiated events, including the dissipation of the mitochondrial membrane potential and the leakage of cytochrome c from the mitochondria. Prior to these events, annomuricin E activated caspase 3/7 and caspase 9. Upstream, annomuricin E induced a time-dependent upregulation of Bax and downregulation of Bcl-2 at the mRNA and protein levels. In conclusion, these findings substantiate the usage of A. muricata leaves in ethnomedicine against cancer and highlight annomuricin E as one of the contributing compounds in the anticancer activity of A. muricata leaves.

Nitro-Oleic Acid Attenuates OGD/R-Triggered Apoptosis in Renal Tubular Cells via Inhibition of Bax Mitochondrial Translocation in a PPAR-γ-Dependent Manner

Background: Nitroalkene derivatives of oleic acid (OA-NO₂) serve as high-affinity ligand for PPAR-γ, which regulates apoptosis, oxidation and inflammation and plays a central role in ischemia-reperfusion injury. In the present study, we elucidated the protective mechanisms of OA-NO₂ against renal ischemia-reperfusion injury. Methods: HK-2 cells were subjected to oxygen and glucose deprivation followed by re-oxygenation (OGD/R) to mimic renal ischemia-reperfusion injury. Cell apoptosis was analyzed by flow cytometry. Bax mitochondrial translocation, cytochrome c and apoptosis-inducing factor (AIF) cytosolic leakage and Akt/Gsk 3β phosphorylation were evaluated by Western blotting. Bax activation was visualized by immunocytochemistry. GW9662 and siRNA transfection were employed to examine the involvement of PPAR-γ. Results: OGD/R injury promoted mitochondrial translocation and activation of Bax, leakage of cytochrome c and AIF, subsequent caspase-3 activation, and eventually cell apoptosis. Pre-incubation with OA-NO₂ (1.25 µM, 45min) inhibited Bax activation and blocked apoptotic cascade, while the protective effects were negated by GW9662 or PPAR-γ siRNA. Moreover, OA-NO₂ restored Akt and Gsk 3β phosphorylation in a PPAR-γ-dependent way. Conclusion: These findings suggest that OA-NO₂ attenuates OGD/R-induced apoptosis by inhibiting Bax translocation and activation and the subsequent mitochondria-dependent apoptotic cascade in a PPAR-γ dependent manner.

On a sugary-relationship between caspases and lamins

On a sugary-relationship between caspases and lamins

Different apoptotic effects of [Pt(O,O'-acac)(γ-acac)(DMS)] and cisplatin on normal and cancerous human epithelial breast cells in primary culture.

The aim of this study was to determine whether [platinum (Pt)(O,O'-acetylacetonate (acac))(γ-acac)(dimethylsulphide (DMS))] is differentially cytotoxic in normal and cancer cells, and to measure comparative levels of cytotoxicity compared with cisplatin in the same cells. We performed experiments on cancerous and normal epithelial breast cells in primary culture obtained from the same patients. The apoptotic effects [Pt(O,O'-acac)(γ-acac)(DMS)] and cisplatin in cancerous and normal breast cells were compared. Cancer cells were more sensitive to [Pt(O,O'-acac)(γ-acac)(DMS)] (IC50 = 5.22 ± 1.2 μmol·L(-1)) than normal cells (IC50 = 116.9 ± 8.8 μmol·L(-1)). However, the difference was less strong when cisplatin was used (IC50 = 96.0 ± 6.9 and 61.9 ± 6.1 μmol·L(-1) for cancer and normal cells respectively). Both compounds caused reactive oxygen species (ROS) production with different mechanisms: [Pt(O,O'-acac)(γ-acac)(DMS)] quickly activated NAD(P)H oxidase while cisplatin caused a slower formation of mitochondrial ROS. Cisplatin and [Pt(O,O'-acac)(γ-acac)(DMS)] caused activation of caspases, proteolysis of PARP and modulation of Bcl-2, Bax and Bid. [Pt(O,O'-acac)(γ-acac)(DMS)] also caused leakage of cytochrome c from the mitochondria. Overall, these processes proceeded more quickly in cells treated with [Pt(O,O'-acac)(γ-acac)(DMS)] compared with cisplatin. [Pt(O,O'-acac)(γ-acac)(DMS)] effects were faster and quantitatively greater in cancer than in normal cells. [Pt(O,O'-acac)(γ-acac)(DMS)] caused a fast decrease of mitochondrial membrane potential, especially in cancer cells. [Pt(O,O'-acac)(γ-acac)(DMS)] was specific to breast cancer cells in primary culture, and this observation makes this compound potentially more interesting than cisplatin.

Synergistic Cytotoxicity of Sorafenib with Busulfan and Nucleoside Analogs in Human FMS-like Tyrosine Kinase 3 Internal Tandem Duplications–Positive Acute Myeloid Leukemia Cells

Clofarabine (Clo), fludarabine (Flu), and busulfan (Bu) are used in pretransplantation conditioning therapy for patients with myeloid leukemia. To further improve their efficacy in FMS-like tyrosine kinase 3 internal tandem duplications (FLT3-ITD)-positive acute myeloid leukemia (AML), we investigated their synergism with sorafenib (Sor). Exposure of FLT3-ITD–positive MV-4-11 and MOLM 13 cells to Bu+Clo+Flu+Sor resulted in synergistic cytotoxicity; no such synergism was observed in the FLT3-wild type THP-1 and KBM3/Bu2506 cell lines. The drug synergism in MV-4-11 cells could be attributed to activation of DNA damage response, histone 3 modifications, inhibition of prosurvival kinases, and activation of apoptosis. Further, the phosphorylation of kinases, including FLT3, MAPK kinase (MEK), and AKT, was inhibited. The FLT3-ITD substrate STAT5 and its target gene PIM 2 product decreased when cells were exposed to Sor alone, Bu+Clo+Flu, and Bu+Clo+Flu+Sor. The level of the proapoptotic protein p53 upregulated modulator of apoptosis (PUMA) increased, whereas the level of prosurvival protein MCL-1 decreased when cells were exposed to Bu+Clo+Flu+Sor. The interactions of PUMA with MCL-1 and/or BCL-2 were enhanced when cells were exposed to Bu+Clo+Flu or Bu+Clo+Flu+Sor. The changes in the level of these proteins, which are involved in mitochondrial control of apoptosis, correlate with changes in mitochondrial membrane potential. Bu+Clo+Flu+Sor decreased mitochondrial membrane potential by 60% and caused leakage of cytochrome c, second mitochondria-derived activator of caspases (SMAC)/direct IAP Binding protein with low pI (DIABLO), and AIF from the mitochondria to the cytoplasm, caspase activation, and cell death, suggesting the activation of apoptosis. Analogous, synergistic cytotoxicity in response to Bu, Clo, Flu, and Sor was observed in mononuclear cells isolated from FLT3-ITD–positive AML patients. Although our previous studies were aimed at standardizing the conditioning regimen, the new findings suggest that patients with abnormal expression of FLT3 might further benefit from individualizing treatment through the addition of Sor to Bu+Clo+Flu, thereby providing personalized pretransplantation therapy.