The antibacterial threaded-lasso peptide capistruin inhibits bacterial RNA polymerase

Capistruin, a ribosomally synthesized, post-translationally modified peptide produced by Burkholderia thailandensis E264, efficiently inhibits growth of Burkholderia and closely related Pseudomonas strains. The functional target of capistruin is not known. Capistruin is a threaded-lasso peptide (lariat peptide) consisting of an N-terminal ring of nine amino acids and a C-terminal tail of 10 amino acids threaded through the ring. The structure of capistruin is similar to that of microcin J25 (MccJ25), a threaded-lasso antibacterial peptide that is produced by some strains of Escherichia coli and targets DNA-dependent RNA polymerase (RNAP). Here, we show that capistruin, like MccJ25, inhibits wild type E. coli RNAP but not mutant, MccJ25-resistant, E. coli RNAP. We show further that an E. coli strain resistant to MccJ25, as a result of a mutation in an RNAP subunit gene, exhibits resistance to capistruin. The results indicate that the structural similarity of capistruin and MccJ25 reflects functional similarity and suggest that the functional target of capistruin, and possibly other threaded-lasso peptides, is bacterial RNAP.     

MccJ25 C-terminal is involved in RNA-polymerase inhibition but not in respiration inhibition

Microcin J25 appears to have two intracellular targets: (1) RNA polymerase, which was described in Escherichia coli and Salmonella enterica serovars, and (2) cell respiration in Salmonella enterica serovars. C-terminal glycine amidation of the threaded segment localized in the MccJ25 lariat ring region specifically blocked the RNA-polymerase inhibition, but not the cell respiration inhibition and peptide uptake. These results suggest that different regions of the molecule are responsible for each cellular effect, they are localized far away from the beta-hairpin region and the C-terminal region is an important determinant for RNAP inhibition.     

Redox-active tyrosine residue in the microcin J25 molecule

Microcin J25 (MccJ25) is a 21 amino acid lasso-peptide antibiotic produced by Escherichia coli and composed of an 8-residues ring and a terminal ‘tail’ passing through the ring. We have previously reported two cellular targets for this antibiotic, bacterial RNA polymerase and the membrane respiratory chain, and shown that Tyr9 is essential for the effect on the membrane respiratory chain which leads to superoxide overproduction. In the present paper we investigated the redox behavior of MccJ25 and the mutant MccJ25 (Y9F). Cyclic voltammetry measurements showed irreversible oxidation of both Tyr9 and Tyr20 in MccJ25, but infrared spectroscopy studies demonstrated that only Tyr9 could be deprotonated upon chemical oxidation in solution. Formation of a long-lived tyrosyl radical in the native MccJ25 oxidized by H₂O₂ was demonstrated by Electron Paramagnetic Resonance Spectroscopy; this radical was not detected when the reaction was carried out with the MccJ25 (Y9F) mutant. These results show that the essential Tyr9, but not Tyr20, can be easily oxidized and form a tyrosyl radical.     

The microcin J25 beta-hairpin region is important for antibiotic uptake but not for RNA polymerase and respiration inhibition

The antibiotic microcin J25 (MccJ25) was cleaved by hydrolysis with thermolysin giving a two-chain peptide (MccJ25-Th19) of 10 and 9 amino acid residues. MccJ25-Th19 with deep modifications in beta-hairpin region had no effect on Escherichia coli growth, but still inhibited RNA polymerase in vitro and oxygen consumption in Salmonella strains. MccJ25-Th19 showed antibiotic activity on E. coli transformed with plasmids containing either fhuA or sbmA genes, which code for proteins involved in MccJ25 transport. These results suggest that an intact beta-hairpin region is crucial for MccJ25 import but not for inhibition of E. coli RNA polymerase or oxygen consumption in Salmonella strains.     

Inhibition of Salmonella enterica serovars by microcin J25

Escherichia coli microcin J25 (MccJ25) is a 2107-Da peptide antibiotic whose uptake into E. coli is mediated by the outer-membrane receptor FhuA and the inner membrane proteins TonB, ExbB, ExbD, and SbmA. A survey of the sensitivity of several Salmonella enterica serovars showed that the antibiotic was highly active against some serovars, while S. Typhimurium, S. Derby, and some S. Enteritidis strains were completely resistant. Resistant strains became hypersensitive to MccJ25 when given the fhuA gene of E. coli, indicating that insensitivity is due to the inability of the FhuA protein to mediate penetration of MccJ25. Whereas in E. coli MccJ25 targets RNA polymerase, in S. Typhimurium it inhibits not only RNA synthesis but also cell respiration. Fluorescence viability staining showed that S. Typhimurium cells exposed to MccJ25 remain viable but are unable to form colonies.     

Microcin J25 triggers cytochrome c release through irreversible damage of mitochondrial proteins and lipids

We previously showed that the antimicrobial peptide microcin J25 induced the over-production of reactive oxygen species with the concomitant release of cytochrome c from rat heart mitochondria via the opening of the mitochondrial permeability transition pore. Here, we were able to demonstrate that indeed, as a consequence of the oxidative burst, MccJ25 induces carbonylation of mitochondrial proteins, which may explain the irreversible inhibition of complex III and the partial inhibition of superoxide dismutase and catalase. Moreover, the peptide raised the levels of oxidized membrane lipids, which triggers the release of cytochrome c. From in silico analysis, we hypothesize that microcin would elicit these effects through interaction with heme c1 at mitochondrial complex III. On the other hand, under an excess of l-arginine, MccJ25 caused nitric oxide overproduction with no oxidative damage and a marked inhibition in oxygen consumption. Therefore, a beneficial anti-oxidative activity could be favored by the addition of l-arginine. Conversely, MccJ25 pro-oxidative–apoptotic effect can be unleashed in either an arginine-free medium or by suppressing the nitric oxide synthase activity.     

ROS-Ca is associated with mitochondria permeability transition pore involved in surfactin-induced MCF-7 cells apoptosis

The surfactin can inhibit proliferation and induce apoptosis in cancer cells. Moreover, surfactin can induce cell death in human breast cancer MCF-7 cells through mitochondrial pathway. However, the molecular mechanism involved in this pathway remains to be elucidated. Here, the reactive oxygen species (ROS) and Ca²⁺ on mitochondria permeability transition pore (MPTP) activity, and MCF-7 cell apoptosis which induced by surfactin were investigated. It is found that surfactin evoked mitochondrial ROS generation, and the surfactin-induced cell death was prevented by N-acetylcysteine (NAC, an inhibitor of ROS). An increasing cytoplasmic Ca²⁺ concentration was detected in surfactin-induced MCF-7 apoptosis, which was inhibited by 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM, a chelator of calcium). In addition, the relationship between ROS generation and the increase of cytoplasm Ca²⁺ was determined. The results showed that surfactin initially induced the ROS formation, leading to the MPTP opening accompanied with the collapse of mitochondrial membrane potential (ΔΨ_m). Then the cytoplasmic Ca²⁺ concentration increased in virtue of the changes of mitochondrial permeability, which was prevented by BAPTA-AM. Besides, cytochrome c (cyt c) was released from mitochondria to cytoplasm through the MPTP and activated caspase-9, eventually induced apoptosis. In summary, surfactin has notable anti-tumor effect on MCF-7 cells, however, there was no obvious cytotoxicity on normal cells.     

The Ile13 residue of microcin J25 is essential for recognition by the receptor FhuA, but not by the inner membrane transporter SbmA

Entry of the peptide antibiotic microcin J25 (MccJ25) into target cells is mediated by the outer membrane receptor FhuA and the inner membrane protein SbmA. The latter also transports MccB17 into the cell cytoplasm. Comparison of MccJ25 and MccB17 revealed a tetrapeptide sequence (VGIG) common to both antibiotics. We speculated that this structural feature in MccJ25 could be a motif recognized by SbmA. To test this hypothesis, we used a MccJ25 variant in which the isoleucine in VGIG (position 13 in the MccJ25 sequence) was replaced by lysine (I13K). In experiments in which the FhuA receptor was bypassed, the substituted microcin showed an inhibitory activity similar to that of the wild-type peptide. Moreover, MccJ25 interfered with colicin M uptake by FhuA in a competition assay, while the I13K mutant did not. From these results, we propose that the Ile¹³ residue is only required for interaction with FhuA, and that VGIG is not a major recognition element by SbmA.     

Mitochondrial dysfunction leads to reduced chronological lifespan and increased apoptosis in yeast

We previously isolated a Saccharomyces cerevisiae mutant (HsTnII), which displays 40% reduced chronological lifespan as compared to the wild type (WT). In this study, we found HsTnII cultures to be characterized by fragmented and dysfunctional mitochondria, and by increased initiation of apoptosis during chronological aging as compared to WT. Expression of genes encoding subunits of mitochondrial electron transport chain and ATP synthase is significantly downregulated in HsTnII, and as a consequence, HsTnII is not able to respire ethanol. All these data confirm the importance of functional mitochondria and respiration in determining yeast chronological lifespan and apoptosis.     

The role of endoplasmic reticulum in cadmium-induced mesangial cell apoptosis

Cd is an industrial and environmental pollutant that affects many organs in humans and other mammals. However, the molecular mechanisms of Cd-induced nephrotoxicity are unclear. In this study, we show that endoplasmic reticula (ER) played a pivotal role in Cd-induced apoptosis in mesangial cells. Using Fluo-3 AM, the intracellular concentration of calcium ([Ca²⁺]_i) was detected as being elevated as time elapsed after Cd treatment. Co-treatment with BAPTA-AM, a calcium chelator, was able to significantly suppress Cd-induced apoptosis. Calcineurin is a cytosolic phosphatase, which was able to dephosphorylate the inositol-1,4,5-triphosphate receptor (IP₃R) calcium channel to prevent the release of calcium from ER. Cyclosporine A, a calcineurin inhibitor, increased both [Ca²⁺]_i and the percentage of Cd-induced apoptosis. However, EGTA and the IP₃R inhibitor, 2-APB, were able to partially modulate Cd cytotoxicity. These results led us to suggest that the extracellular and ER-released calcium plays a crucial role in Cd-induced apoptosis in mesangial cells. Following this line, we further detected the ER stress after Cd treatment since ER is one of the major calcium storage organelles. After Cd exposure, GADD153, a hallmark of ER stress, was upregulated (at 4 h of exposure), followed by activation of ER-specific caspase-12 and its downstream molecule caspase-3 (at 16 h of exposure). The pan caspase inhibitor, Z-VAD, and BAPTA-AM were able to reverse the Cd-induced cell death and ER stress, respectively. Furthermore, the mitochondrial membrane potential (ΔΨ_m) was depolarized significantly and cytochrome c was released after 24 h of exposure to Cd and followed by mild activation of caspase-9 at the 36-h time point, indicating that mitochondria stress is a late event. Therefore, we concluded that ER is the major killer organelle in Cd-induced mesangial cell apoptosis and that calcium oscillation plays a pivotal role.     

Tyrosine 9 is the key amino acid in microcin J25 superoxide overproduction

Escherichia coli microcin J25 (MccJ25) is a lasso-peptide antibiotic comprising 21 l -amino acid residues (G¹-G-A-G-H⁵-V-P-E-Y-F¹⁰-V-G-I-G-T¹⁵-P-I-S-F-Y²⁰-G). MccJ25 has two independent substrates: RNA-polymerase (RNAP) and the membrane respiratory chain. The latter is mediated by oxygen consumption inhibition together with an increase of superoxide production. In the present paper, the antibiotic MccJ25 was engineered by substituting Tyr⁹ or Tyr²⁰ with phenylalanine. Both mutants were well transported into the cells and remained active on RNAP. Only the Y9F mutant lost the ability to overproduce superoxide and inhibit oxygen consumption. The last results confirm that the Tyr⁹, and not Tyr²⁰, is involved in the MccJ25 action on the respiratory chain target.     

A proteomic investigation into the human cervical cancer cell line HeLa treated with dicitratoytterbium (III) complex

Lanthanides have been reported to induce apoptosis in cancer cell lines. Human cervical cancer cell line HeLa was found to be more sensitive to dicitratolanthanum (III) complex ([LaCit₂]³⁻) than other cancer cell lines. However, the effect and mechanism of dicitratoytterbium (III) complex ([YbCit₂]³⁻) on HeLa cells is unknown. Using biochemical and comparative proteomic analyses, [YbCit₂]³⁻ was found to inhibit HeLa cell growth and induce apoptosis. Similar to the effects of [LaCit₂]³⁻, proteomics results from [YbCit₂]³⁻-treated cells revealed profound changes in proteins relating to mitochondria and oxidative stress, suggesting that mitochondrial dysfunction plays a key role in [YbCit₂]³⁻-induced apoptosis. This was confirmed by the decreased mitochondrial transmembrane potential and the increased generation of reactive oxygen species in [YbCit₂]³⁻-treated cells. Western blot analysis showed that [YbCit₂]³⁻-induced apoptosis was accompanied by the activation of caspase-9 and specific proteolytic cleavage of PARP, leading to an increase in the pro-apoptotic protein Bax and a decrease in the anti-apoptotic protein Bcl-2. These results suggest a mitochondrial pathway of cell apoptosis in [YbCit₂]³⁻-treated cells, which will help us understand the molecular mechanisms of lanthanide-induced apoptosis in tumor cells.     

Antioxidative activity of the olive oil constituent hydroxy-1-aryl-isochromans in cells and cell-free systems

Background

Hydroxy-1-aryl-isochromans (HAIC) are newly emerging natural polyphenolic antioxidants, enriched in extravirgin olive oil, whose antioxidative potency was only scarcely characterized using cell-free systems and cells.

Methods

We characterized the activity of HAIC to inactivate reactive oxygen species (ROS) generated by the xanthine/xanthine oxidase system, mitochondria (rat brain) and neural cells. ROS levels were estimated using ROS-sensitive probes, such as Amplex Red, MitoSOX^RED.

Results

HAIC (with 2, 3 or 4 hydroxyl substituents) effectively scavenge ROS released from mitochondria. EC₅₀ values estimated with mitochondria and submitochondrial particles were around 20 μM. Moreover, in PC12 and cultured neural primary cells, HAIC buffered cytosolic ROS. Although HAIC permeate biological membranes, HAIC fail to buffer matrix ROS in isolated mitochondria. We show that hydrogen peroxide was effectively abolished by HAIC, whereas the production of superoxide was not affected.

Conclusion

HAIC exert high antioxidative activity to reduce hydrogen peroxide. The antioxidative activity of HAIC is comparable with that of the stilbene-like, polyphenolic resveratrol, but much higher than that of trolox, N-acetylcysteine or melatonin.

General significance

Unlike resveratrol, HAIC do not impair mitochondrial ATP synthesis or Ca²⁺ retention by mitochondria. Thus, HAIC have the decisive advantage to be potent antioxidants with no detrimental side effects on mitochondrial functions.     

Mitochondrial localization of cyclooxygenase-2 and calcium-independent phospholipase A2 in human cancer cells: implication in apoptosis resistance

Cyclooxygenase-2 (COX-2) is inducible by myriad stimuli. The inducible COX-2 in primary cultured human cells has been reported to localize to nuclear envelope, endoplasmic reticulum, nucleus and caveolae. As COX-2 plays an important role in tumor growth, we were interested in its subcellular location in cancer cells. We examined COX-2 localization in several cancer cell lines by confocal microscopy. A majority of COX-2 was colocalized with heat shock protein 60, a mitochondrial protein, in colon cancer (HT-29, HCT-15 and DLD-1), breast cancer (MCF7), hepatocellular cancer (HepG2) and lung cancer cells (A549) with a similar distribution pattern. By contrast, COX-2 was not localized to mitochondria in human foreskin fibroblasts or endothelial cells. Immunoblot analysis of COX-2 in mitochondrial and cytosolic fractions confirmed localization of COX-2 to mitochondria in HT-29 and DLD-1 cells but not in fibroblasts. Calcium-independent phospholipase A2 was colocalized with heat shock protein 60 to mitochondria not only in cancer cells (HT-29 and DLD-1) but also in fibroblasts. HT-29 which expressed more abundant mitochondrial COX-2 than DLD-1 was highly resistant to arachidonic acid and H2O2-induced apoptosis whereas DLD-1 was less resistant and human fibroblasts were highly susceptible. Treatment of HT-29 cells with sulindac or SC-236, a selective COX-2 inhibitor, resulted in loss of resistance to apoptosis. These results suggest that mitochondrial COX-2 in cancer cells confer resistance to apoptosis by reducing the proapoptotic arachidonic acid.     

The Human SLC25A33 and SLC25A36 Genes of Solute Carrier Family 25 Encode Two Mitochondrial Pyrimidine Nucleotide Transporters

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport inorganic anions, amino acids, carboxylates, nucleotides, and coenzymes across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. Here two members of this family, SLC25A33 and SLC25A36, have been thoroughly characterized biochemically. These proteins were overexpressed in bacteria and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that SLC25A33 transports uracil, thymine, and cytosine (deoxy)nucleoside di- and triphosphates by an antiport mechanism and SLC25A36 cytosine and uracil (deoxy)nucleoside mono-, di-, and triphosphates by uniport and antiport. Both carriers also transported guanine but not adenine (deoxy)nucleotides. Transport catalyzed by both carriers was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. In confirmation of their identity (i) SLC25A33 and SLC25A36 were found to be targeted to mitochondria and (ii) the phenotypes of Saccharomyces cerevisiae cells lacking RIM2, the gene encoding the well characterized yeast mitochondrial pyrimidine nucleotide carrier, were overcome by expressing SLC25A33 or SLC25A36 in these cells. The main physiological role of SLC25A33 and SLC25A36 is to import/export pyrimidine nucleotides into and from mitochondria, i.e. to accomplish transport steps essential for mitochondrial DNA and RNA synthesis and breakdown.     

CORM-3, a water soluble CO-releasing molecule,uncouples mitochondrial respiration via interaction with the phosphate carrier

Carbon monoxide is continuously produced in small quantities in tissues and is an important signaling mediator in mammalian cells. We previously demonstrated that CO delivered to isolated rat heart mitochondria using a water-soluble CO-releasing molecule (CORM-3) is able to uncouple mitochondrial respiration. The aim of this study was to explore more in depth the mechanism(s) of this uncoupling effect. We found that acceleration of mitochondrial O₂ consumption and decrease in membrane potential induced by CORM-3 were associated with an increase in mitochondrial swelling. This effect was independent of the opening of the mitochondrial transition pore as cyclosporine A was unable to prevent it. Interestingly, removal of phosphate from the incubation medium suppressed the effects mediated by CORM-3. Blockade of the dicarboxylate carrier, which exchanges dicarboxylate for phosphate, decreased the effects induced by CORM-3 while direct inhibition of the phosphate carrier with N-ethylmaleimide completely abolished the effects of CORM-3. In addition, CORM-3 was able to enhance the transport of phosphate into mitochondria as evidenced by changes in mitochondrial phosphate concentration and mitochondrial swelling that evaluates the activity of the phosphate carrier in de-energized conditions. These results indicate that CORM-3 activates the phosphate carrier leading to an increase in phosphate and proton transport inside mitochondria, both of which could contribute to the non-classical uncoupling effect mediated by CORM-3. The dicarboxylate carrier amplifies this effect by increasing intra-mitochondrial phosphate concentration.     

Salinomycin-induced apoptosis of human prostate cancer cells due to accumulated reactive oxygen species and mitochondrial membrane depolarization

The anticancer activity of salinomycin has evoked excitement due to its recent identification as a selective inhibitor of breast cancer stem cells (CSCs) and its ability to reduce tumor growth and metastasis in vivo. In prostate cancer, similar to other cancer types, CSCs and/or progenitor cancer cells are believed to drive tumor recurrence and tumor growth. Thus salinomycin can potentially interfere with the end-stage progression of hormone-indifferent and chemotherapy-resistant prostate cancer. Androgen-responsive (LNCaP) and androgen-refractive (PC-3, DU-145) human prostate cancer cells showed dose- and time-dependent reduced viability upon salinomycin treatment; non-malignant RWPE-1 prostate cells were relatively less sensitive to drug-induced lethality. Salinomycin triggered apoptosis of PC-3 cells by elevating the intracellular ROS level, which was accompanied by decreased mitochondrial membrane potential, translocation of Bax protein to mitochondria, cytochrome c release to the cytoplasm, activation of the caspase-3 and cleavage of PARP-1, a caspase-3 substrate. Expression of the survival protein Bcl-2 declined. Pretreatment of PC-3 cells with the antioxidant N-acetylcysteine prevented escalation of oxidative stress, dissipation of the membrane polarity of mitochondria and changes in downstream molecular events. These results are the first to link elevated oxidative stress and mitochondrial membrane depolarization to salinomycin-mediated apoptosis of prostate cancer cells.     

Potential Applicability of Chymotrypsin-Susceptible Microcin J25 Derivatives to Food Preservation

Microcin J25 (MccJ25) is a 21-residue ribosomally synthesized lariat peptide antibiotic. MccJ25 is active against such food-borne disease-causing pathogens as Salmonella spp., Shigella spp., and Escherichia coli, including E. coli O157:H7 and non-O157 strains. MccJ25 is highly resistant to digestion by proteolytic enzymes present in the stomach and intestinal contents. MccJ25 would therefore remain active in the gastrointestinal tract, affecting normal intestinal microbiota, and this limits the potential use of MccJ25 as a food preservative. In the present paper, we describe a chymotrypsin-susceptible MccJ25 derivative with a mutation of Gly¹² to Tyr that retained almost full antibiotic activity and efficiently inhibited the growth of pathogenic Salmonella enterica serovar Newport and Escherichia coli O157:H7 in skim milk and egg yolk. However, unlike the wild-type MccJ25, the MccJ25(G12Y) variant was inactivated by digestive enzymes both in vitro and in vivo. To our knowledge, our results represent the first example of a rational modification of a microcin aimed at increasing its potential use in food preservation.Escherichia coli microcin J25 (MccJ25) is a plasmid-encoded antibiotic peptide consisting of 21 amino acid residues (G¹-G-A-G-H⁵-V-P-E-Y-F¹⁰-V-G-I-G-T¹⁵-P-I-S-F-Y²⁰-G) (4, 12). Four genes (mcjA, mcjB, mcjC, and mcjD) are required for MccJ25 synthesis, export, and immunity (14, 15). The mcjA gene encodes a 58-amino-acid MccJ25 precursor, which is processed by the products of mcjC and mcjB (7). Once synthesized, the mature MccJ25 is excreted to the medium by McjD, an ABC-type transporter (6, 14). The tertiary structure of MccJ25 was elucidated as a lariat peptide (1, 10, 17). It contains an eight-residue ring (G¹ to E⁸) and a tail (Y⁹ to G²¹) whose C-terminal end is sterically trapped within the ring. MccJ25 amino acids F¹⁰ to P¹⁶ form a β-hairpin structure, comprising two β-strands (F¹⁰-V¹¹ and T¹⁵-P¹⁶) and a β-turn (V¹¹ to G¹⁴).MccJ25 is active on gram-negative bacteria related to the producer strain, and among them are several human pathogens (11, 12, 16). It was previously shown that the E. coli RNA polymerase (5, 18) and the bacterial respiratory chain (2, 9) are the targets for MccJ25 action. MccJ25 is active on pathogenic strains of Salmonella spp., Shigella spp. (12), and E. coli, including O157:H7 (11) as well as non-O157 strains (data not shown), that frequently cause outbreaks of food-borne diseases. In addition, Sable et al. (11) showed that MccJ25 was the most active microcin against 12 out of 15 diarrheagenic E. coli strains tested. These authors also demonstrated that MccJ25 inhibits E. coli O157:H7 in biological products such as milk, egg yolk, and meat extract. These findings suggest that MccJ25 could be an efficient complement to nisin for food preservation. However, the potential usefulness of MccJ25 is compromised by the fact that it is highly resistant to digestion by proteolytic enzymes of the stomach (pepsin) and intestinal (trypsin, chymotrypsin, and carboxypeptidases) contents. Thus, the antibiotic would most likely remain active in the intestine, and this could lead to disturbance of the normal microbiota. Therefore, for potential use of MccJ25 as a food additive, it would be desirable to render MccJ25 susceptible to at least one of these proteases. In the present work, we describe a chymotrypsin-susceptible MccJ25 derivative that remains fully active on S. Newport and E. coli O157:H7 in biological products, namely milk and egg yolk. In addition, we demonstrate that the peptide is inactivated by rat intestinal contents.     

Importance of cytochrome c redox state for ceramide-induced apoptosis of human mammary adenocarcinoma cells

Background

Ceramides are intracellular lipid mediator implicated in various cellular responses, including oxidative stress and programmed cell death. Studies demonstrated strong links between ceramide and the mitochondria in the regulation of apoptosis. However, the mechanism of apoptosis induced by ceramides is not fully understood. The present study delineates importance of the redox state of cytochrome c for release of cytochrome c and apoptosis of human mammary adenocarcinoma MCF-7 and MDA-MB-231 cells induced by ceramides.

Methods

The study uses MCF-7 and MDA-MB-231 cells, isolated mitochondria, submitochondrial particles, and oxidized and reduced cytochrome c. Methods used include flow cytometry, immunoblotting, spectroscopy, and respirometry.

Results

We show that ceramides induce mitochondrial oxidative stress and release of cytochrome c from the mitochondria of these cells. Our findings show that ceramides react with oxidized cytochrome c whereas reduced cytochrome c does not react with ceramides. We also show that oxidized cytochrome c reacted with ceramides exerts lower reducibility and function to support mitochondrial respiration. Furthermore, our data show that glutathione protects cytochrome c of reacting with ceramides by increasing the reduced state of cytochrome c.

Conclusions

Ceramides induce oxidative stress and apoptosis in human mammary adenocarcinoma cells by interacting with oxidized cytochrome c leading to the release of cytochrome c from the mitochondria. Our findings suggest a novel mechanism for protective role of glutathione.

General significance

Our study suggests that the redox state of cytochrome c is important in oxidative stress and apoptosis induced by ceramides.