Reevesioside F induces potent and efficient anti-proliferative and apoptotic activities through Na/K-ATPase α3 subunit-involved mitochondrial stress and amplification of caspase cascades

Reevesioside F, isolated from Reevesia formosana, induced anti-proliferative activity that was highly correlated with the expression of Na⁺/K⁺-ATPase α₃ subunit in several cell lines, including human leukemia HL-60 and Jurkat cells, and some other cell lines. Knockdown of α₃ subunit significantly inhibited cell apoptosis suggesting a crucial role of the α₃ subunit. Reevesioside F induced a rapid down-regulation of survivin protein, followed by release of cytochrome c from mitochondria and loss of mitochondrial membrane potential (ΔΨ_m). Further examination demonstrated the mitochondrial damage in leukemic cells through Mcl-1 down-regulation, Noxa up-regulation and an increase of the formation of truncated Bid, tBim and a 23-kDa cleaved Bcl-2 fragment. Furthermore, reevesioside F induced an increase of mitochondria-associated acetyl α-tubulin that may also contribute to apoptosis. The caspase cascade was profoundly activated by reevesioside F. Notably, the specific caspase-3 inhibitor z-DEVD-fmk significantly blunted reevesioside F-induced loss of ΔΨ_m and apoptosis, suggesting that caspase-3 activation may further amplify mitochondrial damage and apoptotic signaling cascade. In spite of being a cardiac glycoside, reevesioside F did not increase the intracellular Ca²⁺ levels. Moreover, CGP-37157 which blocked Na⁺/Ca²⁺ exchanger on plasma membrane and mitochondria did not modify reevesioside F-mediated effect. In summary, the data suggest that reevesioside F induces apoptosis through the down-regulation of survivin and Mcl-1, and the formation of pro-apoptotic fragments from Bcl-2 family members. The loss of ΔΨ_m and mitochondrial damage are responsible for the activation of caspases. Moreover, the amplification of caspase-3-mediated signaling pathway contributes largely to the execution of apoptosis in leukemic cells.     

17Alpha-estradiol arrests cell cycle progression at G2/M and induces apoptotic cell death in human acute leukemia Jurkat T cells

A pharmacological dose (2.5-10 μM) of 17α-estradiol (17α-E₂) exerted a cytotoxic effect on human leukemias Jurkat T and U937 cells, which was not suppressed by the estrogen receptor (ER) antagonist ICI 182,780. Along with cytotoxicity in Jurkat T cells, several apoptotic events including mitochondrial cytochrome c release, activation of caspase-9, -3, and -8, PARP degradation, and DNA fragmentation were induced. The cytotoxicity of 17α-E₂ was not blocked by the anti-Fas neutralizing antibody ZB-4. While undergoing apoptosis, there was a remarkable accumulation of G₂/M cells with the upregulatoin of cdc2 kinase activity, which was reflected in the Thr56 phosphorylation of Bcl-2. Dephosphorylation at Tyr15 and phosphorylation at Thr161 of cdc2, and significant increase in the cyclin B1 level were underlying factors for the cdc2 kinase activation. Whereas the 17α-E₂-induced apoptosis was completely abrogated by overexpression of Bcl-2 or by pretreatment with the pan-caspase inhibitor z-VAD-fmk, the accumulation of G₂/M cells significantly increased. The caspase-8 inhibitor z-IETD-fmk failed to influence 17α-E₂-mediated caspase-9 activation, but it markedly reduced caspase-3 activation and PARP degradation with the suppression of apoptosis, indicating the contribution of caspase-8; not as an upstream event of the mitochondrial cytochrome c release, but to caspase-3 activation. In the presence of hydroxyurea, which blocked the cell cycle progression at the G₁/S boundary, 17α-E₂ failed to induce the G₂/M arrest as well as apoptosis. These results demonstrate that the cytotoxicity of 17α-E₂ toward Jurkat T cells is attributable to apoptosis mainly induced in G₂/M-arrested cells, in an ER-independent manner, via a mitochondria-dependent caspase pathway regulated by Bcl-2.     

Diallyl disulfide induces apoptosis in human colon cancer cell line (COLO 205) through the induction of reactive oxygen species,endoplasmic reticulum stress,caspases casade and mitochondrial-dependent pathways

In this study, we investigated the effects of DADS on human colon cancer cell line COLO 205 on cell cycle arrest and apoptosis in vitro. After 24 h treatment of COLO 205 cells with DADS, the dose- and time-dependent decreases of viable cells were observed and the IC₅₀ was 22.47 μM. The decreased percentages of viable cells are associated with the production of ROS. Treatment of COLO 205 cells with DADS resulted in G2/M phase arrest and apoptosis occurrence through the mitochondrial-pathway (Bcl-2, Bcl-xL down-regulation and Bak, Bax up-regulation). DADS increased cyclin B, cdc25c-ser-216-9 and Wee1 but did not affect CDK1 protein and gene expression within 24 h of treatment. DADS-induced apoptosis was examined and confirmed by DAPI staining and DNA fragmentation assay. DADS promoted caspase-3, -8 and -9 activity and induced apoptosis were accompanied by increasing the levels of Fas, phospho-Ask1 and -JNK, p53 and decreasing the mitochondrial membrane potential which then led to release the cytochrome c, cleavage of pro-caspase-9 and -3. The COLO 205 cells were pre-treated with JNK inhibitor before leading to decrease the percentage of apoptosis which was induced by DADS. Inhibition of caspase-3 activation blocked DADS-induced apoptosis on COLO 205 cells.     

rBTI induces apoptosis in human solid tumor cell lines by loss in mitochondrial transmembrane potential and caspase activation

The molecular mechanisms and the possible effects of a recombinant buckwheat trypsin inhibitor (rBTI) on the induction of apoptosis in the human solid tumor cells (EC9706, HepG2 and HeLa) were investigated. An MTT assay showed that rBTI could specifically inhibit the growth of solid tumor cells in a dose- and time-dependent manner. Analysis by flow cytometry indicated that the apoptosis of several tumor cells increased after treatment with rBTI in range of 6.25–50 μg/ml. DNA electrophoresis analysis showed the ‘DNA ladder’, typical of apoptosis. rBTI-induced apoptosis was shown to involve Bax and Bak up-regulation, Bcl-2 and Bcl-xl down-regulation, release of cytochrome c from the mitochondria to the cytosol, activation of caspase-3 and -9 and disruption of the mitochondrial transmembrane potential (Δψ_m). The z-DEVD-fmk caspase-3 inhibitor significantly inhibited rBTI-induced apoptosis. We concluded that rBTI can induce the apoptosis in several types of human solid tumor cells and promotes apoptosis through the mitochondrial apoptotic pathway.     

Effect of GADD45a on olaquindox-induced apoptosis in human hepatoma G2 cells: Involvement of mitochondrial dysfunction

Olaquindox, a quinoxaline 1, 4-dioxide derivative, has been widely used as a feed additive for promoting animal growth in China. The aim of present study was to investigate the effect of grow arrest and DNA damage 45 alpha (GADD45a) on olaquindox-induced apoptosis in HepG2 cells. The result showed that olaquindox induced the decrease of cell viability in a dose dependent manner. Compared to the control group, olaquindox treatment at 400 and 800 μg/mL increased the expression level of GADD45a protein and reactive oxygen species (ROS) production, decreased mitochondrial membrane potential (MMP), and subsequently increased the expression of Bax while decreased the expression of Bcl-2, leading to the release of cytochrome c (Cyt c). However, knockdown of GADD45a enhanced olaquindox-induced ROS production, disrupted MMP and subsequently caused Cyt c release, then further increased olaquindox- induced cell apoptosis by increasing the activities of caspase-9, caspase-3, and poly (ADP-ribose) polymerase (PARP). In conclusion, the results revealed that GADD45a played a critical role in olaquindox-induced apoptosis in HepG2 cells, which may embrace the regulatory ability on the mitochondrial apoptosis pathway.     

Antitumor effects of emodin on LS1034 human colon cancer cells in vitro and in vivo: Roles of apoptotic cell death and LS1034 tumor xenografts model

Emodin, an active natural anthraquinone derivative, is found in the roots and rhizomes of numerous Chinese medicinal herbs and exhibits anticancer effects on many types of human cancer cell lines. The aim of this study investigated that emodin induced apoptosis of human colon cancer cells (LS1034) in vitro and inhibited tumor nude mice xenografts bearing LS1034 in vivo. In in vitro study, emodin induced cell morphological changes, decreased the percentage of viability, induced G2/M phase arrest and increased ROS and Ca²⁺ productions as well as loss of mitochondrial membrane potential (ΔΨ_m) in LS1034 cells. Emodin-triggered apoptosis was also confirmed by DAPI staining and these effects are concentration-dependent. Western blot analysis indicated that the protein levels of cytochrome c, caspase-9 and the ratio of Bax/Bcl-2 were increased in LS1034 cells after emodin exposure. Emodin induced the productions of ROS and Ca²⁺ release, and altered anti- and pro-apoptotic proteins, leading to mitochondrial dysfunction and activations of caspase-9 and caspase-3 for causing cell apoptosis. In in vivo study, emodin effectively suppressed tumor growth in tumor nude mice xenografts bearing LS1034. Overall, the potent in vitro and in vivo antitumor activities of emodin suggest that it might be developed for treatment of colon cancer in the future.     

Anti-benzopyrene-7,8-diol-9,10-epoxide induces apoptosis via mitochondrial pathway in human bronchiolar epithelium cells independent of the mitochondria permeability transition pore

Anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) is a ubiquitous environmental pollutant contained in tobacco smoke, automobile exhausts and barbecued foods. The carcinogenicity of BPDE on animals has been well characterized, whereas its apoptotic effect is not well defined. A recent study has shown that BPDE-mediated apoptotic pathway has varying specificity across different cell lines. Squamous cell carcinoma (SCC) arises from bronchiolar epithelium cells, therefore, we set out to investigate the pulmonary toxicity and apoptotic effect of BPDE in human bronchiolar epithelium cells (16HBE). Our results show BPDE induces mitochondrial-mediated apoptosis in a dose-dependent manner in 16HBE cells. The cleavage of caspase-3,-9 and release of Cytochrome c (cyt c) was regulated during apoptotic stimulation. However, the opening of mitochondria permeability transition pore (mPTP) has not been detected. Furthermore, our data also indicate that the formation of reactive oxygen species (ROS), decline of mitochondrial membrane potential (ΔΨ_m), increasing p53 and decreasing c-Myc levels play important roles in response to BPDE toxicity. In conclusion, these results suggest that BPDE-mediated apoptosis occurs via caspase-9 dependent mitochondria pathway associated with ROS formation, loss of ΔΨ_m, up-regulation of p53 and down-regulation of c-Myc, but independent of the opening of mPTP in 16HBE cells.     

Total flavonoids from Rosa Laevigata Michx fruit attenuates hydrogen peroxide induced injury in human umbilical vein endothelial cells

The aim of the present study was to evaluate the protective effect and possible mechanism of the total flavonoids (TFs) from Rosa Laevigata Michx fruit (RLMF) against hydrogen peroxide (H₂O₂) induced damage in human umbilical vein endothelial cells (HUVECs). The cell injury caused by H₂O₂ was protected by pretreatment with the TFs for 1 h. Compared with the model group, the TFs decreased S phase cells, suppressed nuclear morphological damage, inhibited the collapse of mitochondrial membrane potentials (ΔΨm), attenuated excessive reactive oxygen species generation, reduced glutathione depletion, impacted the mitochondrial morphology change, decreased caspase-3, -9 activities, and decreased fragmented DNA. Further mechanism investigation showed that the TFs could increase the protein expressions of Procaspase-3, Bcl-2, and decrease the expressions of Bak, Bax, Bid and p53. Generally, the TFs from RLMF is an effective natural product for the treatment of cardiovascular and cerebrovascular diseases.     

Emodin induces apoptosis in human lung adenocarcinoma cells through a reactive oxygen species-dependent mitochondrial signaling pathway

Emodin, a natural anthraquinone derivative isolated from Rheum palmatum L., has been reported to exhibit anti-cancer effect on several human cancers such as liver cancers and lung cancers. However, the molecular mechanisms of emodin-mediated tumor regression have not been fully defined. In this study, we show that treatment with 50 μM emodin resulted in a pronounced release of cytochrome c, activation of caspase-2, -3, and -9, and apoptosis in human lung adenocarcinoma A549 cells. These events were accompanied by the inactivation of ERK and AKT, generation of reactive oxygen species (ROS), disruption of mitochondrial membrane potential (Δψ_m), decrease of mitochondrial Bcl-2, and increase of mitochondrial Bax content. Ectopic expression of Bcl-2, or treatment with aurintricarboxylic acid, furosemide or caspase inhibitors markedly blocked emodin-induced apoptosis. Conversely, pharmacologic ERK and AKT inhibition promoted emodin-induced apoptosis. Furthermore, the free radical scavenger ascorbic acid and N-acetylcysteine attenuated emodin-mediated ROS production, ERK and AKT inactivation, mitochondrial dysfunction, Bcl-2/Bax modulation, and apoptosis. Take together, these findings suggest that in A549 cells, emodin-mediated oxidative injury acts as an early and upstream change in the cell death cascade to antagonize cytoprotective ERK and AKT signaling, triggers mitochondrial dysfunction, Bcl-2 and Bax modulation, mitochondrial cytochrome c release, caspase activation, and consequent leading to apoptosis.     

The apoptotic effect of brucine from the seed of Strychnos nux-vomica on human hepatoma cells is mediated via Bcl-2 and Ca2+ involved mitochondrial pathway.

In an attempt to dissect the mechanism of Strychnos nux-vomica, a commonly used Chinese folk medicine in the therapy of liver cancer, the cytotoxic effects of four alkaloids in Strychnos nux-vomica, brucine, brucine N-oxide, strychnine, and isostrychnine, on human hepatoma cells (HepG2) were screened by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrasolium bromide (MTT) assay. Brucine, among the four alkaloids, exhibited the strongest toxic effect, the mechanism of which was found to cause HepG2 cell apoptosis, since brucine caused HepG2 cell shrinkage, the formation of apoptotic bodies, DNA fragmentation, cell cycle arrest, as well as phosphatidylserine externalization, all of which are typical characteristics of apoptotic programmed cell death. Brucine-induced HepG2 cell apoptosis was caspase dependent, with caspase-3 activated by caspase-9. Brucine also caused the proteolytic processing of caspase-9. In addition, brucine caused depolarization of the mitochondrial membrane of HepG2 cells, the inhibition of which by cyclosporine A completely abrogated the activation of casapses and release of cytochrome c in brucine-treated HepG2 cells. These findings suggested a pivotal role of mitochondrial membrane depolarization in HepG2 cell apoptosis elicited by brucine. Furthermore, brucine induced a rapid and sustained elevation of intracellular [Ca2+], which compromised the mitochondrial membrane potential and triggered the process of HepG2 cell apoptosis. Finally, Bcl-2 was found to predominately control the whole event of cell apoptosis induced by brucine. The elevation of [Ca2+]i caused by brucine was also suppressed by overexpression of Bcl-2 protein in HepG2 cells. From the facts given above, Ca2+ and Bcl-2 mediated mitochondrial pathway were found to be involved in brucine-induced HepG2 cell apoptosis.     

In vitro anti-cancer activity of chamaejasmenin B and neochamaejasmin C isolated from the root of Stellera chamaejasme L

Aim:

To examine the anti-cancer effects of chamaejasmenin B and neochamaejasmin C, two biflavonones isolated from the root of Stellera chamaejasme L (known as the traditional Chinese herb Rui Xiang Lang Du) in vitro.

Methods:

Human liver carcinoma cell lines (HepG2 and SMMC-7721), a human non-small cell lung cancer cell line (A549), human osteosarcoma cell lines (MG63, U2OS, and KHOS), a human colon cancer cell line (HCT-116) and a human cervical cancer cell line (HeLa) were used. The anti-proliferative effects of the compounds were measured using SRB cytotoxicity assay. DNA damage was detected by immunofluorescence and Western blotting. Apoptosis and cell cycle distribution were assessed using flow cytometry analysis. The expression of the related proteins was examined with Western blotting analysis.

Results:

Both chamaejasmenin B and neochamaejasmin C exerted potent anti-proliferative effects in the 8 human solid tumor cell lines. Chamaejasmenin B (the IC₅₀ values ranged from 1.08 to 10.8 μmol/L) was slightly more potent than neochamaejasmin C (the IC₅₀ values ranged from 3.07 to 15.97 μmol/L). In the most sensitive A549 and KHOS cells, the mechanisms underlying the anti-proliferative effects were characterized. The two compounds induced prominent expression of the DNA damage marker γ-H2AX as well as apoptosis. Furthermore, treatment of the cells with the two compounds caused prominent G₀/G₁ phase arrest.

Conclusion:

Chamaejasmenin B and neochamaejasmin C are potential anti-proliferative agents in 8 human solid tumor cell lines in vitro via inducing cell cycle arrest, apoptosis and DNA damage.     

Ginsenoside Rbl protects cardiomyocytes against CoCl2-induced apoptosis in neonatal rats by inhibiting mitochondria permeability transition pore opening

Aim:

To investigate whether mitochondria permeability transition pore (mPTP) opening was involved in ginsenoside Rb1 (Gs-Rb1) induced anti-hypoxia effects in neonatal rat cardiomyocytes ex vivo.

Methods:

Cardiomyocytes were randomly divided into 7 groups: control group, hypoxia group (500 μmol/L CoCl₂), Gs-Rb1 200 μmol/L group (CoCl₂ intervention+Gs-Rb1), wortmannin (PI3K inhibitor) 0.5 μmol/L group, wortmannin+Gs-Rb1 group, adenine 9-β-D-arabinofuranoside (Ara A, AMPK inhibitor) 500 μmol/L group, and Ara A and Gs-Rb1 group. Apoptosis rate was determined by using flow cytometry. The opening of the transient mPTP was assessed by using co-loading with calcein AM and CoCl₂ in high conductance mode. Expression of GSK-3β, cytochrome c, caspase-3 and poly (ADP-ribose) polymerase (PARP) was measured by using Western blotting. ΔGSK-3β was defined as the ratio of p-Ser9-GSK-3β to total GSK-3β.

Results:

CoCl₂ significantly stimulated mPTP opening and up-regulated the level of ΔGSK-3β. There was a statistically significant positive correlation between apoptosis rate and mPTP opening, between apoptosis rate and ΔGSK-3β, and between mPTP opening and ΔGSK-3β. Gs-Rb1 significantly inhibited mPTP opening induced by hypoxia (41.3%±2.0%, P<0.001) . Gs-Rb1 caused a 77.3%±3.2% reduction in the expression of GSK-3β protein (P<0.001) and a significant increase of 1.182±0.007–fold (P=0.0001) in p-Ser9-GSK-3β compared with control group. Wortmannin and Ara A significantly inhibited the effect of Gs-Rb1 on mPTP opening and ΔGSK-3β. Gs-Rb1 significantly decreased expression of cytochrome c (66.1%±1.7%, P=0.001), caspase-3 (56.5%±2.7%, P=0.001) and cleaved poly ADP-ribose polymerase (PARP) (57.9%±1.4%, P=0.001).

Conclusion:

Gs-Rb1 exerted anti-hypoxia effect on neonatal rat cardiomyocytes by inhibiting GSK-3β-mediated mPTP opening.     

Silver nanoparticles induce oxidative cell damage in human liver cells through inhibition of reduced glutathione and induction of mitochondria-involved apoptosis

Silver nanoparticles (AgNPs), which have well-known antimicrobial properties, are extensively used in various medical and general applications. Despite the widespread use of AgNPs, relatively few studies have been undertaken to determine the cytotoxic effects of AgNPs exposure. This study investigates possible molecular mechanisms underlying the cytotoxic effects of AgNPs. Here, we show that AgNPs-induced cytotoxicity was higher compared than that observed when AgNO₃ was used as a silver ion source. AgNPs induced reactive oxygen species (ROS) generation and suppression of reduced glutathione (GSH) in human Chang liver cells. ROS generated by AgNPs resulted in damage to various cellular components, DNA breaks, lipid membrane peroxidation, and protein carbonylation. Upon AgNPs exposure, cell viability decreased due to apoptosis, as demonstrated by the formation of apoptotic bodies, sub-G₁ hypodiploid cells, and DNA fragmentation. AgNPs induced a mitochondria-dependent apoptotic pathway via modulation of Bax and Bcl-2 expressions, resulting in the disruption of mitochondrial membrane potential (Δψ_m). Loss of Δψ_m was followed by cytochrome c release from the mitochondria, resulting in the activation of caspases 9 and 3. The apoptotic effect of AgNPs was exerted via the activation of c-Jun NH₂-terminal kinase (JNK) and was abrogated by the JNK-specific inhibitor, SP600125 and siRNA targeting JNK. In summary, the results suggest that AgNPs cause cytotoxicity by oxidative stress-induced apoptosis and damage to cellular components.     

Bcl-2/Bad/mPTP通路介导14-3-3γ对抗脂多糖所致心肌损伤

目的探讨Bcl-2/Bad/mPTP通路在14-3-3γ对抗脂多糖所致心肌损伤中的作用。方法构建pFLAG-14-3-3γ重组质粒,转染至原代乳鼠心肌细胞中,然后行LPS损伤处理。处理结束后,取培养液检测LDH活性,MTT比色法检测细胞存活率,流式细胞术检测细胞凋亡,线粒体肿胀实验检测mPTP开放,Western blot检测14-3-3γ、Bad、phospho-Bad以及线粒体Bcl-2蛋白表达。结果 LPS损伤使心肌细胞LDH活性升高、细胞存活率下降、凋亡细胞增加、mPTP开放加剧,转染pFLAG-14-3-3γ重组质粒后再行LPS损伤,则LDH活性下降、细胞存活率升高、mPTP开放与细胞凋亡减少,同时phospho-Bad蛋白表达增加,线粒体Bcl-2蛋白表达增加。结论 pFLAG-14-3-3γ能够对抗脂多糖所致的心肌细胞损伤,其机制可能与磷酸化Bad,释放Bcl-2至线粒体,抑制mPTP的开放有关。     

Synthesis of hydroxycinnamic acid derivatives as mitochondria-targeted antioxidants and cytotoxic agents

In order to develop agents with superior chemopreventive and chemotherapeutic properties against hepatocellular carcinomas, mitochondria-targeted hydroxycinnamic acids (MitoHCAs) were synthesized by conjugation with a triphenylphosphonium cation. These synthetic compounds were evaluated for their antioxidant activities in hepatic mitochondria, including against OH^∙− and ROO^∙− induced lipid peroxidation. H₂O₂ production was decreased significantly by increasing glutathione peroxidase and catalase activities. In addition, cell proliferation data from three cell lines (HepG2, L02 and WI38) indicated that the MitoHCAs were selective for cancer cells. Interestingly, the MitoHCAs both with or without Ca²⁺ triggered mitochondrial dysfunction by inducing mitochondrial swelling, collapsing the mitochondrial membrane potential and causing cytochrome c release. In particular, an inhibitor of the mitochondrial permeability transition pore (mPTP), cyclosporin A, attenuated mitochondrial damage and cell apoptosis, indicating that mPTP may be involved in the antiproliferative activity of MitoHCAs. Further studies focused on structural optimization of these compounds are onging.     

The activation of p38 and JNK by ROS,contribute to OLO-2-mediated intrinsic apoptosis in human hepatocellular carcinoma cells

In this study, we describe that a novel synthesized compound, olean-28,13β-olide 2 (OLO-2), exhibits selective cytotoxic activity via inducing apoptosis in human hepatocellular carcinoma (HCC) cell lines but not normal human hepatic cells in vitro. Exposure of human HCC HepG2 cells to OLO-2 results in significant loss of mitochondrial transmembrane potential (ΔΨ_m), the release of cytochrome c, the recruitment of B-cell lymphoma 2 (Bcl-2) assaciated X protein (Bax) and the downregulation of Bcl-2. The apoptosis induced by OLO-2 is associated with the activation of caspase-3/9 and the nuclear translocation of apoptosis inducing factor (AIF). Moreover, the increase of phosphorylated p38 and c-Jun N-terminal kinase (JNK) is observed. OLO-2-induced the externalization of phosphatidyl-serine (PS) and the loss of ΔΨ_m are blocked by p38 inhibitor SB203580 or JNK inhibitor SP600125. In addition, OLO-2 provokes the generation of reactive oxygen species (ROS) in HepG2 cells, while the antioxidant N-acetyl cysteine (NAC) almost completely blocks OLO-2-induced apoptosis and the activation of p38 and JNK. Taken together, the present study demonstrates that OLO-2 exhibits its cytotoxic activity through intrinsic apoptosis via ROS generation and the activation of p38 and JNK. Its potential to be a candidate of anti-cancer agent is worth being further investigated.     

Modulation of Bax/Bcl-2 and caspases by probiotics during acetaminophen induced apoptosis in primary hepatocytes

Oxidative stress is an important factor in drug induced hepatotoxicity and antioxidants from natural sources have potential to ameliorate it. The present study was aimed to investigate cyto-protective potential of probiotic Enterococcus lactis IITRHR1 (El_SN) and Lactobacillus acidophilus MTCC447 (La_SN) lysate against acetaminophen (APAP) induced hepatotoxicity. Cultured rat hepatocytes pretreated with El_SN/La_SN showed higher cell viability under APAP stress. Pre-treatment with El_SN, restored glutathione level and reduced ROS generation significantly which are major biomarkers of oxidative stress. It also reduced NO level, MDA formation and enhanced SOD activity. Pre-treatment with probiotic lysates significantly inhibited the translocation of pro-apoptotic protein (Bax), enhanced anti-apoptotic (Bcl-2) protein levels and prevented release of cyt c to cytosol; suggesting involvement of mitochondrial proteins in protection against APAP induced oxidative cellular damage. Loss in mitochondrial membrane potential due to APAP treatment was prevented in the presence of probiotic lysates. Protective action of El_SN/La_SN pretreatment was further supported by prevention of procaspase-3 activation, DNA fragmentation and chromatin condensation, in turn inhibiting APAP induced apoptotic cell death. The results indicate that probiotic preparations modulate crucial end points of oxidative stress induced apoptosis and may be used for management of drug induced liver injury.     

Reactive oxygen species‐dependent JNK downregulated olaquindox‐induced autophagy in HepG2 cells

Autophagy plays an important role in response to intracellular and extracellular stress to sustain cell survival. However, dysregulated or excessive autophagy may lead to cell death, known as “type II programmed cell death,” and it is closely associated with apoptosis. In our previous study, we proposed that olaquindox induced apoptosis of HepG2 cells through a caspase‐9 dependent mitochondrial pathway. In this study, we investigated autophagy induced by olaquindox and explored the crosstalk between apoptosis and autophagy in olaquindox‐treated HepG2 cells. Olaquindox‐induced autophagy was demonstrated by the accumulation of monodansylcadervarine, as well as elevated expression of autophagy‐related MAP‐LC3 and Beclin 1 proteins. The autophagy inhibitor 3‐methyladenine significantly increased the apoptotic rate induced by olaquindox, which was correlated with increased ratio of Bax/Bcl‐2. The further studies showed that olaquindox increased the levels of reactive oxygen species (ROS), and antioxidant N‐acetyl‐L ‐cysteine (NAC) effectively blocked the accumulation of ROS but failed to block autophagy. Moreover, olaquindox induced the activation of c‐Jun N‐terminal protein kinase (JNK), and JNK inhibitor SP600125 failed to block autophagy. Instead, olaquindox‐induced autophagy was enhanced by NAC or SP600125. Meanwhile, JNK activation was remarkably blocked by NAC, indicating that ROS may be the upstream signaling molecules of JNK activation and involved in the negative regulation of olaquindox‐induced autophagy. These results suggest that olaquindox induces autophagy in HepG2 cells and that olaquindox‐induced apoptosis can be enhanced by 3‐methyladenine. Olaquindox‐induced autophagy in HepG2 cells is upregulated by Beclin 1 but downregulated by ROS‐dependent JNK. Copyright © 2014 John Wiley & Sons, Ltd.     

Cytotoxic effects of procyanidins from Castanea mollissima Bl. shell on human hepatoma G2 cells in vitro

Significant cytotoxic effects of procynadins from chestnut (Castanea mollissima Bl.) shell (CSPC) on human hepatoma G2 (HepG2) cells were found in vitro. CSPC could inbibit HepG2 proliferation in a dose-dependent manner (100–400 μg/mL), arrest cell cycle in the G₀/G₁ phase, induce apoptosis and trigger necrosis of HepG2. Proapoptotic effect of CSPC was evidenced by nuclear condensation, internucleosomal DNA fragmentation. Treatment of HepG2 cells with CSPC caused a loss of mitochondrial membrane potential and stimulated reactive oxidative species (ROS) generation. These results suggested CSPC could trigger apoptosis and necrotic cell death in HepG2 cell, which might be associated with ROS generation through the mitochondria-dependent signaling way.     

Pyrogallol-induced calf pulmonary arterial endothelial cell death via caspase-dependent apoptosis and GSH depletion

Pyrogallol (PG) as a polyphenol induces apoptosis in cells. Here, we evaluated the effects of PG on the growth and death of endothelial cells (ECs). PG dose-dependently inhibited the growth of calf pulmonary artery endothelial cells (CPAEC) and human umbilical vein endothelial cells (HUVEC). PG also induced apoptosis in both cells accompanied by the loss of mitochondrial membrane potential (ΔΨ_m). CPAEC were more sensitive to PG than HUVEC concerning cell growth and death. Caspase inhibitors (pan-caspase, caspase-3, -8 or -9 inhibitor) did not affect the growth inhibition of CPAEC by PG. However, pan-caspase inhibitor (Z-VAD) significantly reduced apoptosis and the loss of ΔΨ_m in PG-treated CPAEC. PG reduced ROS level and increased GSH depleted cell numbers in CPAEC. While Z-VAD increased ROS levels in PG-treated CPAEC, it decreased GSH depleted cell numbers. In conclusion, PG inhibited the growth of ECs, especially CPAEC via caspase-dependent apoptosis and GSH depletion.