Recombinant Biogenesis and Analysis of Cytochrome c Species.

Recombinant expression and biogenesis of cytochrome c species is a simple and efficient method for the production of holocytochrome c species, thus presenting an avenue for the study of cytochrome c or the cytochrome c biogenesis pathways responsible for heme attachment. Here, we describe a method for recombinant E. coli production of holocytochrome c utilizing the System I (CcmABCDEFGH) bacterial cytochrome c biogenesis pathway, followed by analysis of cytochrome c species by cell lysis and heme stain.

NapB Restores cytochrome c biosynthesis in bacterial dsbD-deficient mutants.

Cytochromes c (cyts c), essential for respiration and photosynthesis in eukaryotes, confer bacteria respiratory versatility for survival and growth in natural environments. In bacteria having a cyt c maturation (CCM) system, DsbD is required to mediate electron transport from the cytoplasm to CcmG of the Ccm apparatus. Here with cyt c-rich Shewanella oneidensis as the research model, we identify NapB, a cyt c per se, that suppresses the CCM defect of a dsbD mutant during anaerobiosis, when NapB is produced at elevated levels, a result of activation by cAMP-Crp. Data are then presented to suggest that NapB reduces CcmG, leading to the suppression. We further show that NapB proteins capable of rescuing CCM in the dsbD mutant form a small distinct clade. The study sheds light on multifunctionality of cyts c, and more importantly, unravels a self-salvation strategy through which bacteria have evolved to better adjust to the natural world.

Cryo-EM of CcsBA reveals the basis for cytochrome c biogenesis and heme transport.

Although the individual structures and respiratory functions of cytochromes are well studied, the structural basis for their assembly, including transport of heme for attachment, are unknown. We describe cryo-electron microscopy (cryo-EM) structures of CcsBA, a bifunctional heme transporter and cytochrome c (cyt c) synthase. Models built from the cryo-EM densities show that CcsBA is trapped with heme in two conformations, herein termed the closed and open states. The closed state has heme located solely at a transmembrane (TM) site, with a large periplasmic domain oriented such that access of heme to the cytochrome acceptor is denied. The open conformation contains two heme moieties, one in the TM-heme site and another in an external site (P-heme site). The presence of heme in the periplasmic site at the base of a chamber induces a large conformational shift that exposes the heme for reaction with apocytochrome c (apocyt c). Consistent with these structures, in vivo and in vitro cyt c synthase studies suggest a mechanism for transfer of the periplasmic heme to cytochrome.

Protective Effects of a Lutein Ester Prodrug, Lutein Diglutaric Acid, against H2O2-Induced Oxidative Stress in Human Retinal Pigment Epithelial Cells.

Oxidative stress-induced cell damage and death of the retinal pigmented epithelium (RPE), a polarized monolayer that maintains retinal health and homeostasis, lead to the development of age-related macular degeneration (AMD). Several studies show that the naturally occurring antioxidant Lutein (Lut) can protect RPE cells from oxidative stress. However, the poor solubility and low oral bioavailability limit the potential of Lut as a therapeutic agent. In this study, lutein diglutaric acid (Lut-DG), a prodrug of Lut, was synthesized and its ability to protect human ARPE-19 cells from oxidative stress was tested compared to Lut. Both Lut and Lut-DG significantly decreased H2O2-induced reactive oxygen species (ROS) production and protected RPE cells from oxidative stress-induced death. Moreover, the immunoblotting analysis indicated that both drugs exerted their protective effects by modulating phosphorylated MAPKs (p38, ERK1/2 and SAPK/JNK) and downstream molecules Bax, Bcl-2 and Cytochrome c. In addition, the enzymatic antioxidants glutathione peroxidase (GPx) and catalase (CAT) and non-enzymatic antioxidant glutathione (GSH) were enhanced in cells treated with Lut and Lut-DG. In all cases, Lut-DG was more effective than its parent drug against oxidative stress-induced damage to RPE cells. These findings highlight Lut-DG as a more potent compound than Lut with the protective effects against oxidative stress in RPE cells through the modulation of key MAPKs, apoptotic and antioxidant molecular pathways.

Licochalcone C induces cell cycle G1 arrest and apoptosis in human esophageal squamous carcinoma cells by activation of the ROS/MAPK signaling pathway.

Along with changes in dietary habits and lifestyle, the incidence of esophageal cancer is increasing around the world. Since chemotherapy for esophageal cancer has significant side effects, phytochemicals have attracted attention as an alternative medicine. Licochalcone C (LCC) is a flavonoid compound extracted from Licorice, with a variety of clinical uses including anti-cancer, anti-inflammatory and anti-oxidant effects. Treatment with LCC for 48 h significantly decreased cell viability of esophageal squamous cell carcinoma (ESCC) cells in a dose- and time-dependent manner with IC50 values of 28 µM (KYSE 30), 36 µM (KYSE 70), 19 µM (KYSE 410), 28 µM (KYSE 450) and 26 µM (KYSE 510). LCC induced G1 arrest accompanied by decreased cyclin D1 expression and an increase in the levels of p21 and p27. LCC increased the levels of intracellular ROS, cytochrome C release, and multi-caspase activity, and decreased mitochondrial membrane potential. LCC induced the protein expression of ER stress markers (GRP78 and CHOP) and phosphorylation JNK, c-Jun and p38. We investigated the expression of pro-apoptotic and anti-apoptotic proteins to elucidate the mechanism of apoptosis. Our findings contribute to the understanding of apoptosis mechanism underlying LCC in ESCC cells and provide new insights into the potential clinical opportunities of LCC for ESCC treatment.

PTEN-mediated mitophagy and APE1 overexpression protects against cardiac hypoxia/reoxygenation injury.

Autophagy plays a critical role in cardiac hypoxia/reoxygenation (H/R). Studies indicated that the phosphatase and tensin homolog (PTEN) influences level of autophagy. This study aims to explore the role of PTEN mediating a specific autophagy, mitophagy, in cardiac H/R injury. H9c2 cells were cultured and suffered hypoxia and reoxygenation treatment. To inhibit function of PTEN protein, bpv (phen) was added into medium throughout the process of H/R injury. In addition, we overexpressed the apurinic/apyrimidinic endonuclease 1 (APE1) in H/R-injured H9c2 cells. Then the cell viability, apoptosis, and release of Cytochrome C were determined through CCK-8 assay, flow cytometry, and western blotting, respectively. The results indicated that H/R significantly induced autophagy, as identified by an increased level of microtubule-associated protein 1 light chain 3 beta (LC3B) and a decreased level of sequestosome 1 (P62). After stimulation of bpv (phen), PTEN-induced putative kinase protein 1 (PINK1)/Parkin-mediated mitophagy was inhibited, while apoptosis and releases of Cytochrome C were both significantly increased, indicating an exacerbated H/R injury. Furthermore, the overexpression of APE1 attenuated the apoptosis and releases of Cytochrome C induced by H/R injury, and promoted PINK1/Parkin-mediated mitophagy. Our findings provide an insight into the PTEN and APE1 overexpression protects against cardiac hypoxia/reoxygenation injury, which may be through inducing the PINK1/Parkin-mediated mitophagy.

PM2.5 aggravates the lipid accumulation, mitochondrial damage and apoptosis in macrophage foam cells.

Epidemiological evidence showed that the particulate matter exposure is associated with atherosclerotic plaque progression, which may be related to foam cell formation, but the mechanism is still unknown. The study was aimed to investigate the toxic effects and possible mechanism of PM2.5 on the formation of macrophage foam cells induced by oxidized low density lipoprotein (ox-LDL). Results showed that PM2.5 induced cytotoxicity by decreasing the cell viability and increasing the LDH level in macrophage foam cells. PM2.5 aggravated the lipid accumulation in ox-LDL-stimulated macrophage RAW264.7 within markedly increasing level of intracellular lipid by Oil red O staining. The level of ROS increased obivously after co-exposure to PM2.5 and ox-LDL than single exposure group. In addition, serious mitochondrial damage such as the mitochondrial swelling, cristae rupturing and disappearance were observed in macrophage foam cells. The loss of the mitochondrial membrane potential (MMP) further exacerbated the mitochondrial damage in PM2.5-induced macrophage foam cells. The apoptotic rate increased more severely via up-regulated protein level of Bax, Cyt C, Caspase-9, Caspase-3, and down-regulated that of Bcl-2, indicating that PM2.5 activated the mitochondrial-mediated apoptosis pathway. In summary, our results demonstrated that PM2.5 aggravated the lipid accumulation, mitochondrial damage and apoptosis in macrophage foam cells, suggesting that PM2.5 was a risk factor of atherosclerosis progression.

XC_0531 encodes a c-type cytochrome biogenesis protein and is required for pathogenesis in Xanthomonas campestris pv. campestris.

BACKGROUND: The phytopathogenic Xanthomonas campestris pv.campestris is a gram-negative bacterium and the causal agent of black-rot disease of cruciferous crops. Many gram-negative bacteria possess a family of proteins, called Dsbs, which are involved in disulfide bond formation in certain periplasmic proteins. In our preliminary screening of the virulence to the plants we identified that gene XC_0531 which annotated gene dsbD of Xanthomonas campestris pv. campestris (Xcc) is related to the virulence to the host plants. RESULTS: Here, we found XC_0531 encoded a DsbD like protein. Its deletion is sensitive to DTT and copper, decreased accumulation of free thiols in periplasm. Its deletion also affected heme synthesis, position of Soret band and the production of peak c550. This suggests that XC_0531 is related to c-type cytochromes biogenesis. XC_0531 mutation decreased the utilization of different carbon sources (such as galactose, xylose, maltose, saccharose and glucose), reduced extracellular polysaccharide (EPS) production, decreased extracellular enzyme activities (protease, cellulose and amylase), slowed down growth rate of Xcc and weakened virulence to the plants. These results suggest that these phenotypes caused by XC_0531 mutation is possibly due to deficient biosynthesis of c-type cytochromes in respiration chain and the formation of disulfide bonds. Our work confirmed the function of XC_0531 and provide theory basis for scientists working on molecular mechanisms of cytochrome c biogenesis, pathogenesis of Xcc, development of EPS commercial values and protecting plant from black rot. CONCLUSION: We confirmed the function of gene XC_0531, which encodes a DsbD like protein, a protein correlated with c-type cytochrome biogenesis. This gene is related to the virulence to plants by affecting funtion of cytochromes c and probably disulfide bonds modification of proteins in type II secretion system (T2SS).

Treatment with Enriched Environment Reduces Neuronal Apoptosis in the Periinfarct Cortex after Cerebral Ischemia/Reperfusion Injury.

BACKGROUND/AIMS: Enriched environment (EE) has been reported to exert neuroprotective effect in animal models of ischemic stroke. However, the underlying mechanism remains unclear. The purpose of this study was to investigate the effect of EE treatment on neuronal apoptosis in the periinfarct cortex after cerebral ischemia/reperfusion (I/R) injury. METHODS: The cerebral I/R injury was established by middle cerebral artery occlusion (MCAO). A set of behavioral tests including the modified neurological severity score (mNSS), limb-placing test and foot-fault test were conducted. The infarct volume and the neuronal survival rate were evaluated by Nissl staining. The morphology and ultrastructure of ischemic neurons was examined by transmission electron microscopy. Neuronal apoptosis was assessed by double labeling of TdT-mediated dUTP-biotin nick end labeling (TUNEL) with NeuN. The expressions of apoptosis-related proteins were tested by western blotting and immunohistochemical labeling. RESULTS: EE treatment improved neurological function, reduced infarct volume, increased neuronal survival rate and alleviated the morphological and ultrastructural damage of neurons (especially mitochondria) after I/R injury. EE treatment reduced the neuronal apoptosis, increased B cell lymphoma/leukemia-2 (Bcl-2) protein levels while decreased Bcl-2-associated X protein (Bax), cytochrome c, caspase-3 expressions and Bax/Bcl-2 ratio in the periinfarct cortex after cerebral I/R injury. CONCLUSION: Our findings suggest that EE treatment inhibits neuronal apoptosis in the periinfarct cortex after focal cerebral I/R injury, which may be one of the possible mechanisms underlying the neuroprotective effects of EE.

RNA editing of cytochrome c maturation transcripts is responsive to the energy status of leaf cells in Arabidopsis thaliana.

Overexpression of AtPAP2, a phosphatase located on the outer membranes of chloroplasts and mitochondria, leads to higher energy outputs from these organelles. AtPAP2 interacts with seven MORF proteins of the editosome complex. RNA-sequencing analysis showed that the editing degrees of most sites did not differ significantly between OE and WT, except some sites on the transcripts of several cytochrome c maturation (Ccm) genes. Western blotting of 2D BN-PAGE showed that the patterns of CcmFN1 polypeptides were different between the lines. We proposed that AtPAP2 may influence cytochrome c biogenesis by modulating RNA editing through its interaction with MORF proteins.

Power provides protection: Genetic robustness in yeast depends on the capacity to generate energy.

The functional basis of genetic robustness, the ability of organisms to suppress the effects of mutations, remains incompletely understood. We exposed a set of 15 strains of Saccharomyces cerevisiae form diverse environments to increasing doses of the chemical mutagen EMS. The number of the resulting random mutations was similar for all tested strains. However, there were differences in immediate mortality after the mutagenic treatment and in defective growth of survivors. An analysis of gene expression revealed that immediate mortality was lowest in strains with lowest expression of transmembrane proteins, which are rich in thiol groups and thus vulnerable to EMS. A signal of genuine genetic robustness was detected for the other trait, the ability to grow well despite bearing non-lethal mutations. Increased tolerance of such mutations correlated with high expression of genes responsible for the oxidative energy metabolism, suggesting that the negative effect of mutations can be buffered if enough energy is available. We confirmed this finding in three additional tests of the ability to grow on (i) fermentable or non-fermentable sources of carbon, (ii) under chemical inhibition of the electron transport chain and (iii) during overexpression of its key component, cytochrome c. Our results add the capacity to generate energy as a general mechanism of genetic robustness.

Neuronal Effects of Sugammadex in combination with Rocuronium or Vecuronium.

Rocuronium (ROC) and Vecuronium (VEC) are the most currently used steroidal non-depolarizing neuromuscular blocking (MNB) agents. Sugammadex (SUG) rapidly reverses steroidal NMB agents after anaesthesia. The present study was conducted in order to evaluate neuronal effects of SUG alone and in combination with both ROC and VEC. Using MTT, CASP-3 activity and Western-blot we determined the toxicity of SUG, ROC or VEC in neurons in primary culture. SUG induces apoptosis/necrosis in neurons in primary culture and increases cytochrome C (CytC), apoptosis-inducing factor (AIF), Smac/Diablo and Caspase 3 (CASP-3) protein expression. Our results also demonstrated that both ROC and VEC prevent these SUG effects. The protective role of both ROC and VEC could be explained by the fact that SUG encapsulates NMB drugs. In BBB impaired conditions it would be desirable to control SUG doses to prevent the excess of free SUG in plasma that may induce neuronal damage. A balance between SUG, ROC or VEC would be necessary to prevent the risk of cell damage.

Chemosensitivity of MCF-7 cells to eugenol: release of cytochrome-c and lactate dehydrogenase.

Phytochemicals have been extensively researched for their potential anticancer effects. In previous study, direct exposure of rat liver mitochondria to eugenol main ingredient of clove, uncoupled mitochondria and increased F0F1ATPase activity. In the present study, we further investigated the effects of eugenol on MCF-7 cells in culture. Eugenol demonstrated: a dose-dependent decrease in viability (MTT assay), and proliferation (real time cell analysis) of MCF-7 cells, (EC50: 0.9 mM); an increase in reactive oxygen species; a decrease in ATP level and mitochondrial membrane potential (MitoPT JC-1 assay); and a release of cytochrome-c and lactate dehydrogenase (Cytotoxicity Detection Kit PLUS) into culture media at eugenol concentration >EC50. Pretreatment with the antioxidants Trolox and N-acetyl cysteine partially restored cell viability and decreased ROS, with Trolox being more potent. Expression levels of both anti- and pro-apoptotic markers (Bcl-2 and Bax, respectively) decreased with increasing eugenol concentration, with no variation in their relative ratios. Eugenol-treated MCF-7 cells overexpressing Bcl-2 exhibited results similar to those of MCF-7. Our findings indicate that eugenol toxicity is non-apoptotic Bcl-2 independent, affecting mitochondrial function and plasma membrane integrity with no effect on migration or invasion. We report here the chemo-sensitivity of MCF-7 cells to eugenol, a phytochemical with anticancer potential.

Engineered holocytochrome c synthases that biosynthesize new cytochromes c.

Cytochrome c (cyt c), required for electron transport in mitochondria, possesses a covalently attached heme cofactor. Attachment is catalyzed by holocytochrome c synthase (HCCS), leading to two thioether bonds between heme and a conserved CXXCH motif of cyt c In cyt c, histidine (His19) of CXXCH acts as an axial ligand to heme iron and upon release of holocytochrome c from HCCS, folding leads to formation of a second axial interaction with methionine (Met81). We previously discovered mutations in human HCCS that facilitate increased biosynthesis of cyt c in recombinant Escherichia coli Focusing on HCCS E159A, novel cyt c variants in quantities that are sufficient for biophysical analysis are biosynthesized. Cyt c H19M, the first bis-Met liganded cyt c, is compared with other axial ligand variants (M81A, M81H) and single thioether cyt c variants. For variants with axial ligand substitutions, electronic absorption, near-UV circular dichroism, and electron paramagnetic resonance spectroscopy provide evidence that axial ligands are changed and the heme environment is altered. Circular dichroism spectra in far UV and thermal denaturation analyses demonstrate that axial ligand changes do not affect secondary structures and stability. Redox potentials span a 400-mV range (+349 mV vs. standard hydrogen electrode, H19M; +252 mV, WT; -19 mV, M81A; -69 mV, M81H). We discuss the results in the context of a four-step mechanism for HCCS, whereby HCCS mutants such as E159A are enhanced in release (step 4) of cyt c from the HCCS active site; thus, we term these "release mutants."

SLC25A26 overexpression impairs cell function via mtDNA hypermethylation and rewiring of methyl metabolism.

Cancer cells down-regulate different genes to give them a selective advantage in invasiveness and/or metastasis. The SLC25A26 gene encodes the mitochondrial carrier that catalyzes the import of S-adenosylmethionine (SAM) into the mitochondrial matrix, required for mitochondrial methylation processes, and is down-regulated in cervical cancer cells. In this study we show that SLC25A26 is down-regulated due to gene promoter hypermethylation, as a mechanism to promote cell survival and proliferation. Furthermore, overexpression of SLC25A26 in CaSki cells increases mitochondrial SAM availability and promotes hypermethylation of mitochondrial DNA, leading to decreased expression of key respiratory complex subunits, reduction of mitochondrial ATP and release of cytochrome c. In addition, increased SAM transport into mitochondria leads to impairment of the methionine cycle with accumulation of homocysteine at the expense of glutathione, which is strongly reduced. All these events concur to arrest the cell cycle in the S phase, induce apoptosis and enhance chemosensitivity of SAM carrier-overexpressing CaSki cells to cisplatin.

Mitochondrial hyperpolarization and cytochrome-c release in microwave-exposed MCF-7 cells.

This study examines the effects of a 2.1-GHz WCDMA-modulated microwave (MW) radiation on apoptotic activity and mitochondrial membrane potential (ΔΨm) in MCF-7 cells. The cells were exposed to the MW at a specific absorption rate (SAR) of 0.528 W/kg for 4 or 24 h. The antiproliferative effect of MW exposure was determined by the MTT test. Cytochrome-c and p53 levels were determined by an ELISA method. The relative ΔΨm was analysed by JC-1 staining using flow cytometer. Apoptotic rate of the cells was measured by Annexin-V-FITC staining. All assays were performed after certain time of incubations (15 min-4 h) following MW exposure. MW-exposed cells showed a significant decrease in viability when compared to unexposed cells. A significantly larger decrease was observed after longer exposure. The percentage of apoptotic cells, amount of cytochrome-c, and relative ΔΨm were significantly higher in MW-exposed cells. The percent of apoptotic cells and relative ΔΨm in 24 h MW-exposed group was significantly higher than those in 4 h MW-exposed group. However, no significant change was observed in p53 levels. These results demonstrated that exposure to 2.1-GHz WCDMA-modulated MW radiation caused hyperpolarization of mitochondria that in turn induced apoptosis in MCF-7 cells.

New PCR primers targeting hydrazine synthase and cytochrome c biogenesis proteins in anammox bacteria.

PCR primers targeting genes encoding the two proteins of anammox bacteria, hydrazine synthase and cytochrome c biogenesis protein, were designed and tested in this study. Three different ecotypes of samples, namely ocean sediments, coastal wetland sediments, and wastewater treatment plant (WWTP) samples, were used to assess the primer efficiency and the community structures of anammox bacteria retrieved by 16S ribosomal RNA (rRNA) and the functional genes. Abundances of hzsB gene of anammox bacteria in South China Sea (SCS) samples were significantly correlated with 16S rRNA gene by qPCR method. And hzsB and hzsC gene primer pair hzsB364f-hzsB640r and hzsC745f-hzsC862r in combination with anammox bacterial 16S rRNA gene primers were recommended for quantifying anammox bacteria. Congruent with 16S rRNA gene-based community study, functional gene hzsB could also delineate the coastal-ocean distributing pattern, and seawater depth was positively associated with the diversity and abundance of anammox bacteria from shallow- to deep-sea. Both hzsC and ccsA genes could differentiate marine samples between deep and shallow groups of the Scalindua sp. clades. As for WWTP samples, non-Scalindua anammox bacteria reflected by hzsB, hzsC, ccsA, and ccsB gene-based libraries showed a similar distribution pattern with that by 16S rRNA gene. NH4+ and NH4+/Σ(NO3- + NO2-) positively correlated with anammox bacteria gene diversity, but organic matter contents correlated negatively with anammox bacteria gene diversity in SCS. Salinity was positively associated with diversity indices of hzsC and ccsB gene-harboring anammox bacteria communities and could potentially differentiate the distribution patterns between shallow- and deep-sea sediment samples. SCS surface sediments harbored considerably diverse community of Scalindua. A new Mai Po clade representing coastal estuary wetland anammox bacteria group based on 16S rRNA gene phylogeny is proposed. Existence of anammox bacteria within wider coverage of genera in Mai Po wetland indicates this unique niche is very complex, and species of anammox bacteria are niche-specific with different physiological properties towards substrates competing and chemical tolerance capability.

Regulation of the Ku70 and apoptosis-related proteins in experimental diabetic nephropathy.

BACKGROUND: Apoptosis contributes nephropathy pathogenesis in diabetes. However, its mechanisms still remain unclear. We examined the extent to which the angiotensin-II type 1 receptor blocker (AT1RB) irbesartan and the angiotensin converting enzyme inhibitor (ACEI) perindopril affected the apoptosis-related proteins Bcl-2, Bax, caspase-3, cytochrome-c and Ku70 in streptozotocin (STZ)-diabetic rats. MATERIALS AND METHODS: Animals were divided into five groups of eight each, four of which received STZ (60mg/kg in a single dose, i.p.) to induce diabetes. The groups were performed as untreated diabetic; non-diabetic control; daily irbesartan (15mg/kg/day) or perindopril (6mg/kg/day) and also combined irbesartan and perindopril (respectively, 5mg/kg/day, 3mg/kg/day) were applied by gavage for 30days to STZ-diabetic rats. The kidney tissue analysis was performed by using immunohistochemical staining with Bcl-2, Bax, caspase-3, cytochrome-c and Ku70 antibodies and by using Western blot analysis with caspase-3 and cytochrome-c antibodies. RESULTS: Immunoreactivity of Bax, caspase-3, cytochrome-c and Ku70 was increased in the tubuli and glomeruli of the untreated diabetic group, but decreased in all treated diabetic groups. In the irbesartan and perindopril treated diabetic groups Bcl-2 immunoreactivity was higher than that of the untreated diabetic group. Caspase-3 and cytoplasmic cytochrome-c protein levels increased in the untreated diabetic group. CONCLUSIONS: We conclude that the increased expression of Bax and caspase-3, and the increased level of cytoplasmic cytochrome-c relate to renal tissue injury. This case is also seen in the early stages of diabetes as a result of the damage caused by local increased expression of renin angiotensin system (RAS) in the renal tissue, which is induced by hyperglycemia. The increase of the cytosolic cytochrome-c, caspase-3 and Ku70 expression in the tubuli is suggestive of apoptosis. Overall, our results show that treatments of irbesartan and perindopril are effective and efficient in preventing renal tissue injury and apoptosis by blocking the RAS in experimental diabetic nephropathy and reducing the expression of proteins associated with apoptosis.

A mitochondria-targeted antioxidant can inhibit peroxidase activity of cytochrome c by detachment of the protein from liposomes.

Interaction of cytochrome c with cardiolipin converts this respiratory chain electron-transfer protein into a peroxidase, supposedly involved in mitochondria-mediated apoptosis initiation. Liposome membrane permeabilization provoked by peroxidase activity of the cytochrome c/cardiolipin complex has been previously shown to be suppressed by conventional antioxidants. Here, the mitochondria-targeted antioxidant SkQ1 (plastoquinonyl-decyl-triphenylphosphonium) was found to strongly inhibit both cytochrome c/cardiolipin peroxidase activity and the permeabilization of liposomes composed of phosphatidylcholine and cardiolipin. A number of binding assays revealed a significant inhibiting effect of SkQ1 on cytochrome c binding to liposomes, thus suggesting that SkQ1-mediated protection of liposomes from the cytochrome c/H2 O2 -induced permeabilization involved distortion of the cytochrome c-membrane binding. It is suggested that antioxidant and antiapoptotic effects of alkyltriphenylphosphonium cations can be related to the prevention of cytochrome c/cardiolipin interaction.

An Ingenol Derived from Euphorbia kansui Induces Hepatocyte Cytotoxicity by Triggering G0/G1 Cell Cycle Arrest and Regulating the Mitochondrial Apoptosis Pathway in Vitro.

Natural product lingenol, a purified diterpenoid compound derived from the root of Euphorbia kansui, exerts serious hepatotoxicity; however, the molecular mechanisms remain to be defined. In the present study, cell counting Kit-8 (CCK-8), inverted phase contrast microscope and flow cytometry were used to demonstrate that lingenol significantly inhibited L-O2 cells proliferation, and induced cell cycle arrest and apoptosis. Moreover, the results investigated that lingenol markedly disrupted mitochondrial functions by high content screening (HCS). In addition, the up-regulation of cytochrome c, AIF and Apaf-1 and activation of caspases were found in L-O2 cells detected by Western blotting and ELISA assay, which was required for lingenol activation of cytochrome c-mediated caspase cascades and AIF-mediated DNA damage. Mechanistic investigations revealed that lingenol significantly down-regulated the Bcl-2/Bax ratio and enhanced the reactive oxygen species (ROS) in L-O2 cells. These data collectively indicated that lingenol modulation of ROS and Bcl-2/Bax ratio led to cell cycle arrest and mitochondrial-mediated apoptosis in L-O2 cells in vitro. All of these results will be helpful to reveal the hepatotoxicity mechanism of Euphorbia kansui and to effectively guide safer and better clinical application of this herb.