Release Of Mitochondrial Proteins Research Articles

Sinularin stabilizes FOXO3 protein to trigger prostate cancer cell intrinsic apoptosis

Sinularin, a natural product that purified from soft coral, exhibits anti-tumor effects against various human cancers. However, the mechanisms are not well understood. In this study, we demonstrated that Sinularin inhibited the viability of human prostate cancer cells in a dose-dependent manner and displayed significant cytotoxicity only at high concentration against normal prostate epithelial cell RWPE-1. Flow cytometry assay demonstrated that Sinularin induced tumor cell apoptosis. Further investigations revealed that Sinularin exerted anti-tumor activity through intrinsic apoptotic pathway along with up-regulation of pro-apoptotic protein Bax and PUMA, inhibition of anti-apoptotic protein Bcl-2, mitochondrial membrane potential collapses, and release of mitochondrial proteins. Furthermore, we illustrated that Sinularin induced cell apoptosis via up-regulating PUMA through inhibition of FOXO3 degradation by the ubiquitin–proteasome pathway. To explore how Sinularin suppress FOXO3 ubiquitin–proteasome degradation, we tested two important protein kinases AKT and ERK that regulate FOXO3 stabilization. The results revealed that Sinularin stabilized and up-regulated FOXO3 via inhibition of AKT- and ERK1/2-mediated FOXO3 phosphorylation and subsequent ubiquitin–proteasome degradation. Our findings illustrated the potential mechanisms by which Sinularin induced cell apoptosis and Sinularin may be applied as a therapeutic agent for human prostate cancer.

Gasdermin D permeabilization of mitochondrial inner and outer membranes accelerates and enhances pyroptosis

Gasdermin D (GSDMD)-activated inflammatory cell death (pyroptosis) causes mitochondrial damage, but its underlying mechanism and functional consequences are largely unknown. Here, we show that the N-terminal pore-forming GSDMD fragment (GSDMD-NT) rapidly damaged both inner and outer mitochondrial membranes (OMMs) leading to reduced mitochondrial numbers, mitophagy, ROS, loss of transmembrane potential, attenuated oxidative phosphorylation (OXPHOS), and release of mitochondrial proteins and DNA from the matrix and intermembrane space. Mitochondrial damage occurred as soon as GSDMD was cleaved prior to plasma membrane damage. Mitochondrial damage was independent of the B-cell lymphoma 2 family and depended on GSDMD-NT binding to cardiolipin. Canonical and noncanonical inflammasome activation of mitochondrial damage, pyroptosis, and inflammatory cytokine release were suppressed by genetic ablation of cardiolipin synthase (Crls1) or the scramblase (Plscr3) that transfers cardiolipin to the OMM. Phospholipid scramblase-3 (PLSCR3) deficiency in a tumor compromised pyroptosis-triggered anti-tumor immunity. Thus, mitochondrial damage plays a critical role in pyroptosis.

The boundary of life and death: changes in mitochondrial and cytosolic proteomes associated with programmed cell death of Arabidopsis thaliana suspension culture cells.

Despite the critical role of programmed cell death (PCD) in plant development and defense responses, its regulation is not fully understood. It has been proposed that mitochondria may be important in the control of the early stages of plant PCD, but the details of this regulation are currently unknown. We used Arabidopsis thaliana cell suspension culture, a model system that enables induction and precise monitoring of PCD rates, as well as chemical manipulation of this process to generate a quantitative profile of the alterations in mitochondrial and cytosolic proteomes associated with early stages of plant PCD induced by heat stress. The cells were subjected to PCD-inducing heat levels (10 min, 54°C), with/without the calcium channel inhibitor and PCD blocker LaCl3. The stress treatment was followed by separation of cytosolic and mitochondrial fractions and mass spectrometry-based proteome analysis. Heat stress induced rapid and extensive changes in protein abundance in both fractions, with release of mitochondrial proteins into the cytosol upon PCD induction. In our system, LaCl3 appeared to act downstream of cell death initiation signal, as it did not affect the release of mitochondrial proteins, but instead partially inhibited changes occurring in the cytosolic fraction, including upregulation of proteins with hydrolytic activity. We characterized changes in protein abundance and localization associated with the early stages of heat stress-induced PCD. Collectively, the generated data provide new insights into the regulation of cell death and survival decisions in plant cells.

Viral attenuation by Endonuclease G during yeast gametogenesis: insights into ancestral roles of programmed cell death?

Viruses and other genetic parasites are present in virtually all forms of life. This chronic condition has led to diverse host cell adaptations such as CRISPR and RNAi, whose functions attenuate these parasites. It is hypothesized that programmed cell death (PCD) is an additional adaptation whose origins reside in viral defense. A core event of apoptotic PCD is the regulated release of mitochondrial inter-membrane space proteins into the cytosol, following which these apoptogenic proteins bring about the demise of the cell. The most well studied example of this is found in animals, where the release of mitochondrial cytochrome C nucleates the formation of the apoptosome, which then activates caspase mediated cell death. The release of mitochondrial proteins contributes to PCD in diverse organisms lacking the apoptosome, indicating that regulated mitochondrial release predates the evolution of canonical apoptosis. Using the budding yeast Saccharomyces cerevisiae, we recently confirmed an early study showing that Nuc1, a homolog of the mitochondrial apoptotic driver protein Endonuclease G, attenuates cytosolic double stranded RNA (dsRNA) viruses, which are endemic to yeast and many other organisms. Viral attenuation by Nuc1 occurs most prominently during meiosis and in association with its developmentally programmed relocation from the mitochondria to the cytosol. Intriguingly, meiotic viral attenuation by Nuc1 occurs within the context of meiotic PCD of the superfluous mother cell that we have also discovered. These findings are discussed here.

Sinularin exerts anti-tumor effects against human renal cancer cells relies on the generation of ROS.

Sinularin, a soft corals-derived natural product, exerts anti-tumorigenic activity in various types of human cancer cells. However, the action of Sinularin and its mechanism in renal carcinoma is not well understood. In the current study, we demonstrated that Sinularin inhibited the viability of human renal cancer cells 786-O and ACHN in a dose- and time-dependent manner, but did not show significant toxicity against non-malignant HRCEpic cells. Cell cycle analysis revealed that Sinularin induced G2/M arrest significantly. In addition, Sinularin could induce apoptosis in cells along with caspase-3/-9 activation, release of mitochondrial proteins, up-regulation of pro-apoptotic Bcl-2 family proteins and inhibition of anti-apoptotic Bcl-2 family proteins. Sinularin could also repress the activation of PI3K/Akt/mTOR signaling pathway. Moreover, Sinularin triggered the activation of MAPKs and p38 activation was essential for the anti-tumor effect of Sinularin. The generation of ROS (reactive oxygen species) was critical for Sinularin-induced apoptosis since ROS scavenger NAC (N-acetyl cysteine) could block the Sinularin-triggered apoptosis. In conclusion, all the results indicated that Sinularin may be applied as a therapeutic natural agent for human renal cancer.

6′-Benzyloxy-4-bromo-2′-hydroxychalcone is cytotoxic against human leukaemia cells and induces caspase-8- and reactive oxygen species-dependent apoptosis

In this study, we investigated the effects of synthetic 6′-benzyloxy-4-bromo-2′-hydroxychalcone on viabilities of seven human leukaemia cells. It was cytotoxic against U-937, HL-60, K-562, NALM-6, MOLT-3 cells, and also against Bcl-2-overexpressing U-937/Bcl-2 cells and P-glycoprotein-overexpressing K-562/ADR, but had no significant cytotoxic effects against quiescent or proliferating human peripheral blood mononuclear cells. This chalcone is a potent apoptotic inducer in human leukaemia U-937 cells. Cell death was (i) mediated by the activation and the cleavage of initiator and executioner caspases and poly(ADP-ribose) polymerase; (ii) prevented by the pan-caspase inhibitor z-VAD-fmk, and by the selective caspase-3/7, −6 and −8 inhibitors, and by a cathepsins B/L inhibitor; (iii) associated with the release of mitochondrial proteins, including cytochrome c and Smac/DIABLO; (iv) accompanied by dissipation of the mitochondrial membrane potential, (v) partially blocked by the inhibition of p38MAPK and (vi) mostly abrogated by catalase. In conclusion, the synthetic chalcone is cytotoxic against several types of human leukaemia cell with apoptosis being induced by activation of the extrinsic pathway and the generation of reactive oxygen species.

Does Oxidation of Mitochondrial Cardiolipin Trigger a Chain of Antiapoptotic Reactions?

Oxidative stress causes selective oxidation of cardiolipin (CL), a four-tail lipid specific for the inner mitochondrial membrane. Interaction with oxidized CL transforms cytochrome c into peroxidase capable of oxidizing even more CL molecules. Ultimately, this chain of events leads to the pore formation in the outer mitochondrial membrane and release of mitochondrial proteins, including cytochrome c, into the cytoplasm. In the cytoplasm, cytochrome c promotes apoptosome assembly that triggers apoptosis (programmed cell death). Because of this amplification cascade, even an occasional oxidation of a single CL molecule by endogenously formed reactive oxygen species (ROS) might cause cell death, unless the same CL oxidation triggers a separate chain of antiapoptotic reactions that would prevent the CL-mediated apoptotic cascade. Here, we argue that the key function of CL in mitochondria and other coupling membranes is to prevent proton leak along the interface of interacting membrane proteins. Therefore, CL oxidation should increase proton permeability through the CL-rich clusters of membrane proteins (CL islands) and cause a drop in the mitochondrial membrane potential (MMP). On one hand, the MMP drop should hinder ROS generation and further CL oxidation in the entire mitochondrion. On the other hand, it is known to cause rapid fission of the mitochondrial network and formation of many small mitochondria, only some of which would contain oxidized CL islands. The fission of mitochondrial network would hinder apoptosome formation by preventing cytochrome c release from healthy mitochondria, so that slowly working protein quality control mechanisms would have enough time to eliminate mitochondria with the oxidized CL. Because of these two oppositely directed regulatory pathways, both triggered by CL oxidation, the fate of the cell appears to be determined by the balance between the CL-mediated proapoptotic and antiapoptotic reactions. Since this balance depends on the extent of CL oxidation, mitochondria-targeted antioxidants might be able to ensure cell survival in many pathologies by preventing CL oxidation.

MTOR inhibition attenuates glucose deprivation-induced death in photoreceptors via suppressing a mitochondria-dependent apoptotic pathway

Acute energy depletion contributes to ischemia-induced retinal neuronal injury, causing photoreceptor death and subsequent vision loss. The mTOR pathway is a crucial cellular signaling hub modulating RNA transcription, protein synthesis, and metabolic balance. Thus, we mimicked acute energy depletion in photoreceptor cells (661W cells) with glucose deprivation and investigated neuroprotective mechanisms of mTOR inhibition. We found that treatment with rapamycin, an mTOR-specific inhibitor, reduced intracellular ROS, maintained the mitochondrial membrane potential and restored mitochondrial dysfunction. In addition, inhibiting the mTOR signal suppressed DRP1 translocation to the mitochondria, pro-apoptotic mitochondrial protein release, and caspase 3 activation when glucose was deprived. Inhibition of mTOR offers significant neuroprotection against glucose deprivation-induced injury in 661W cells, chiefly via suppressing mitochondrial-dependent pathways. These observations may shed light on treating ischemia-related retinal diseases.

Optogenetic apoptosis: light-triggered cell death.

An optogenetic Bax has been designed that facilitates light-induced apoptosis. We demonstrate that mitochondrial recruitment of a genetically encoded light-responsive Bax results in the release of mitochondrial proteins, downstream caspase-3 cleavage, changes in cellular morphology, and ultimately cell death. Mutagenesis of a key phosphorylatable residue or modification of the C-terminus mitigates background (dark) levels of apoptosis that result from Bax overexpression. The mechanism of optogenetic Bax-mediated apoptosis was explored using a series of small molecules known to interfere with various steps in programmed cell death. Optogenetic Bax appears to form a mitochondrial apoptosis-induced channel analogous to that of endogenous Bax.

Optogenetic Apoptosis: Light‐Triggered Cell Death

AbstractAn optogenetic Bax has been designed that facilitates light‐induced apoptosis. We demonstrate that mitochondrial recruitment of a genetically encoded light‐responsive Bax results in the release of mitochondrial proteins, downstream caspase‐3 cleavage, changes in cellular morphology, and ultimately cell death. Mutagenesis of a key phosphorylatable residue or modification of the C‐terminus mitigates background (dark) levels of apoptosis that result from Bax overexpression. The mechanism of optogenetic Bax‐mediated apoptosis was explored using a series of small molecules known to interfere with various steps in programmed cell death. Optogenetic Bax appears to form a mitochondrial apoptosis‐induced channel analogous to that of endogenous Bax.

The abietane diterpenoid parvifloron D from Plectranthus ecklonii is a potent apoptotic inducer in human leukemia cells

BackgroundAbietane diterpenes have attracted much attention because they display a wide range of biological activities, including antitumor activities. These compounds are the most diverse of the diterpenoids isolated from species of Plectranthus. Naturally occurring diterpene parvifloron D is the main phytochemical constituent of Plectranthus ecklonii. To examine the therapeutic potential of the plant, we evaluated whether parvifloron D displays cytotoxicity against human tumor cells. MethodsThe cytotoxicity was analyzed by colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apoptosis was evaluated by fluorescent microscopy, transmission electron microscopy, flow cytometric analysis of annexin V-FITC and propidium iodide-stained cells and DNA fragmentation. Protein expression and processing and release of mitochondrial proteins were analyzed by Western blot. Caspase activity was determined using colorimetric substrates. The membrane potential and intracellular reactive oxygen species were detected by flow cytometry. ResultsParvifloron D displays strong cytotoxic properties against leukemia cells (HL-60, U-937, MOLT-3 and K-562) and in particular P-glycoprotein-overexpressing K-562/ADR cells, but has only weak cytotoxic effects on peripheral blood mononuclear cells (PBMCs). Overexpression of the protective mitochondrial proteins Bcl-2 and Bcl-xL did not confer resistance to parvifloron D-induced cytotoxicity. Growth inhibition of HL-60 cells that was triggered by parvifloron D was found to be caused by a rapid induction of apoptotic cell death. This apoptosis was prevented by the non-specific caspase inhibitor z-VAD-fmk, and by the selective caspase-9 inhibitor z-LEHD-fmk. Cell death induced by parvifloron D was found to be (i) associated with the dissipation of the mitochondrial membrane potential and the release of cytochrome c, (ii) amplified by inhibition of extracellular signal-regulated kinases (ERKs) 1/2 signaling and (iii) caused by a mechanism dependent on intracellular reactive oxygen species generation. ConclusionParvifloron D is a potent cytotoxic compound against several human tumor cells and also a fast and potent apoptotic inducer in leukemia cells.

Targeting Mitochondria with Avocatin B Induces Selective Leukemia Cell Death.

Treatment regimens for acute myeloid leukemia (AML) continue to offer weak clinical outcomes. Through a high-throughput cell-based screen, we identified avocatin B, a lipid derived from avocado fruit, as a novel compound with cytotoxic activity in AML. Avocatin B reduced human primary AML cell viability without effect on normal peripheral blood stem cells. Functional stem cell assays demonstrated selectivity toward AML progenitor and stem cells without effects on normal hematopoietic stem cells. Mechanistic investigations indicated that cytotoxicity relied on mitochondrial localization, as cells lacking functional mitochondria or CPT1, the enzyme that facilitates mitochondria lipid transport, were insensitive to avocatin B. Furthermore, avocatin B inhibited fatty acid oxidation and decreased NADPH levels, resulting in ROS-dependent leukemia cell death characterized by the release of mitochondrial proteins, apoptosis-inducing factor, and cytochrome c. This study reveals a novel strategy for selective leukemia cell eradication based on a specific difference in mitochondrial function.

Augmenter of liver regeneration plays a protective role against hydrogen peroxide-induced oxidative stress in renal proximal tubule cells.

Oxidative stress plays an important role in cellular destruction. Augmenter of liver regeneration (ALR) is an anti-apoptotic factor that is expressed in all mammalian cells and functions as an anti-oxidant by stimulating the expression of a secretory isoform of clusterin and inhibiting reactive oxygen species (ROS) generation. Previous work from our group showed that ALR expression is upregulated in acute kidney injury (AKI) rats, and recombinant human ALR reduces tubular injury. In the present study, we used small interfering RNA (siRNA) silencing of ALR to examine its role in H2O2 induced mitochondrial injury and apoptosis. Knockdown of ALR increased ROS levels, reduced mitochondrial membrane potential, and increased the release of mitochondrial proteins and the rate of apoptosis in response to H2O2. In addition, the ratio of Bax/Bcl-2 was increased in siRNA/ALR groups treated with H2O2. These data confirm the protective role of ALR against oxidative stress-induced mitochondrial injury and suggest a potential mechanism underlying the protective role of ALR in AKI.

Inhibition of Fatty Acid Oxidation with Avocatin B Selectively Targets AML Cells and Leukemia Stem Cells

Acute myeloid leukemia (AML) is an aggressive malignant disease characterized by poor patient outcome and suboptimal front-line chemotherapy. To identify novel anti-AML compounds, we performed a high-throughput screen of a natural products library (n=800). This screen was performed against the AML cell line (TEX), which has several properties of leukemia stem cells, the cells responsible for disease pathophysiology and patient relapse. Here, avocatin B was identified as a potent and novel anti-leukemia agent. Avocatin B, at concentrations as high as 20µM, had no effect on normal peripheral blood stem cell viability. In contrast, it induced death of primary AML cells with an EC50 of 1.5-5.0 µM. Selective toxicity towards a functionally defined subset of primitive leukemia cells was also demonstrated. Avocatin B (3µM) reduced clonogenic growth of AML progenitor cells with no effect on clonogenic growth of normal hematopoietic stem cells. Further, treatment of primary AML cells with avocatin B (3µM) diminished their ability to engraft into the bone marrow of pre-conditioned, NOD/SCID mice (t18=6.5; p<0.001). Together, these results confirm that avocatin B is a novel anti-AML agent with selective toxicity toward leukemia and leukemia stem cells.Mechanistically, avocatin B-induced reactive oxygen species (ROS)-dependent leukemia cell apoptosis that was characterized by the release of mitochondrial proteins, cytochrome c and apoptosis inducing factor (AIF). Cytochrome c and AIF were detected in the cytosol of avocatin B treated TEX cells by flow cytometry. Avocatin B-induced apoptosis, as measured by the Annexin V/Propidium iodide assay, DNA fragmentation and PARP cleavage, was abolished in the presence of anti-oxidants confirming the functional importance of ROS. Next, we further evaluated the role of mitochondria in avocatin B-induced apoptosis. First, we generated leukemia cells lacking mitochondria by successive culturing in media containing ethidium bromide. The drastic (>80%) reduction in mitochondria were confirmed by nonyl acridine orange staining and flow cytometry and a near absence of the mitochondria specific proteins ANT and ND1, as measured by Western blotting. Avocatin B’s activity was abolished in leukemia cells lacking mitochondria. Next, using lentiviral knockdown, we generated leukemia cells lacking CPT1, the enzyme that facilitates transport of 16-20 carbon lipids into mitochondria. Avocatin B’s activity was abolished in cells with reduced CPT1 expression (>70% as measured by qPCR analysis). To further confirm the importance of CPT1 in avocatin B-induced death, we chemically inhibited CPT1 with etomoxir. Avocatin B’s activity was blocked in the presence of etomoxir, further demonstrating that avocatin B accumulates in mitochondria.Since avocatin B is a lipid that targets mitochondria and that mitochondria can oxidize fatty acids for energy, we next assessed the impact of avocatin B on fatty acid oxidation, using the Seahorse Bioanalyzer. Avocatin B inhibited leukemia cell fatty acid oxidation (>40% reduction in oxygen consumption at 10µM) and this occurred at a 10-fold less concentration than etomoxir, the standard experimental molecule used to probe this pathway. Further, avocatin B resulted in a 50% reduction in levels of NADPH, an important co-factor generated during fatty acid oxidation that participates in catabolic processes during cell proliferation.These results show that avocatin B accumulates in mitochondria to inhibit fatty acid oxidation and reduce NADPH to result in ROS-mediated leukemia cell apoptosis. This highlights a novel AML-therapeutic strategy by which mitochondria are targeted to impair cellular metabolism leading directly to AML cell death. DisclosuresNo relevant conflicts of interest to declare.

Mitochondrial Regulation of Cell Death

Although required for life, paradoxically, mitochondria are often essential for initiating apoptotic cell death. Mitochondria regulate caspase activation and cell death through an event termed mitochondrial outer membrane permeabilization (MOMP); this leads to the release of various mitochondrial intermembrane space proteins that activate caspases, resulting in apoptosis. MOMP is often considered a point of no return because it typically leads to cell death, even in the absence of caspase activity. Because of this pivotal role in deciding cell fate, deregulation of MOMP impacts on many diseases and represents a fruitful site for therapeutic intervention. Here we discuss the mechanisms underlying mitochondrial permeabilization and how this key event leads to cell death through caspase-dependent and -independent means. We then proceed to explore how the release of mitochondrial proteins may be regulated following MOMP. Finally, we discuss mechanisms that enable cells sometimes to survive MOMP, allowing them, in essence, to return from the point of no return.

Antiproliferative Activity of Abietane Diterpenoids against Human Tumor Cells

In the present study, the cytotoxicity of 30 diterpenoids with an abietane or a halimane skeleton was determined against five human tumor cell lines (HL-60, U937, Molt-3, SK-MEL-1, and MCF-7). Diterpenoids containing an abietane skeleton including taxodone (1) and taxodione (2), as well as the semisynthetic derivatives 12, 14, 15, 17, and 22, were the most cytotoxic compounds for human leukemia cells. Overexpression of the protective mitochondrial proteins Bcl-2 and Bcl-x(L) did not confer resistance to abietane diterpene-induced cytotoxicity. Studies performed on HL-60 cells indicated that growth inhibition triggered by compounds 1, 12, 14, and 15 was caused by induction of apoptosis. This was prevented by the nonspecific caspase inhibitor Z-VAD-FMK and, in the case of compounds 14 and 15, reduced by the selective caspase-8 inhibitor Z-IETD-FMK. Cell death induced by these abietane diterpenes was found to be associated with the release of mitochondrial proteins, including cytochrome c, Smac/DIABLO, and AIF (apoptosis-inducing factor), accompanied by dissipation of the mitochondrial membrane potential (ΔΨ), and modulated by inhibition of extracellular signal-regulated kinases signaling and the p38 mitogen-activated protein kinase pathway.

High temporal resolution fluorescence measurements of a mitochondrial dye for detection of early stage apoptosis

In the present study, early stage apoptosis is explored with high temporal resolution. In addition to monitoring early apoptosis induction in single cells by ultrasensitive confocal fluorescence microscopy (UCFM), the mitochondrial protein release kinetics was explored. The current study shows development and optimization of a novel, rapid apoptosis assay to explore the earliest changes in cells by the intrinsic apoptosis pathway. We show that early apoptotic changes in the mitochondria begin nearly simultaneously with the addition of an apoptosis-inducing drug, such as staurosporine. With a temporal resolution of five minutes, this non-invasive analytical technique can elucidate the earliest apoptotic events in living cells. Moreover, our results show that the mitochondrial inter-membrane proteins are not involved in the extrinsic pathway of Ramos cells mediated by an anti-CD95 antibody. Additional techniques such as light microscopy and flow cytometry were employed to confirm the results obtained by ultrasensitive confocal fluorescence microscopy. The results of this study help to understand the earliest mechanisms of apoptosis induction in cells, enabling new methods of drug testing and dose-response analyses.

Anticancer Activity of Silver–N‐Heterocyclic Carbene Complexes: Caspase‐Independent Induction of Apoptosis via Mitochondrial Apoptosis‐Inducing Factor (AIF)

Fourteen silver(I) complexes bearing N-heterocyclic carbene (NHC) ligands were prepared and evaluated for anticancer activity. Some of these were found to exhibit potent antiproliferative activity toward several types of human cancer cell lines, including drug-resistant cell lines, with IC(50) values in the nanomolar range. An initial investigation into the mechanism of cell death induced by this family of silver(I) complexes was carried out. Cell death was shown to result from the activation of apoptosis without involvement of primary necrosis. In HL60 cells, silver-NHCs induce depolarization of the mitochondrial membrane potential (ΔΨ(m)) and likely allow the release of mitochondrial proteins to elicit early apoptosis. This effect is not related to the overproduction of reactive oxygen species (ROS). In addition, apoptosis is not associated with the activation of caspase-3, but is triggered by the translocation of apoptosis-inducing factor (AIF) and caspase-12 from mitochondria and the endoplasmic reticulum, respectively, into the nucleus to promote DNA fragmentation and ultimately cell death. No modification in cell-cycle distribution was observed, indicating that silver-NHCs are not genotoxic. Finally, the use of a fluorescent complex showed that silver-NHCs target mitochondria. Altogether, these results demonstrate that silver-NHCs induce cancer cell death independent of the caspase cascade via the mitochondrial AIF pathway.

Hsp70 promotes chemoresistance by blocking Bax mitochondrial translocation in ovarian cancer cells

Cisplatin can induce apoptosis in ovarian cancer cells through the mitochondrial death pathway, and dysregulation of this pathway contributes to cisplatin resistance in ovarian cancer cells. Here we show that cisplatin induces mitochondrial proteins such as Smac/DIABLO, Cytochrome c, and HrtA2/Omi release to the cytosol and apoptosis in cisplatin-sensitive, but not -resistant ovarian cancer cells. Bax translocation to mitochondria is required for mitochondrial protein release and cisplatin-induced apoptosis in human ovarian cancer cells. Hsp70 is highly expressed in cisplatin-resistant cells. Hsp70 promotes chemoresistance, in part, by blocking Bax translocation to the mitochondria and mitochondrial protein release to cytosol in human ovarian cancer cells.

Effect of XIAP overexpression on sodium butyrate-induced apoptosis in recombinant Chinese hamster ovary cells producing erythropoietin

Previously, overexpression of X-linked inhibitor of apoptosis (XIAP), which is known to inhibit activities of caspase-3, -7, and -9 in CHO-K1 cells offered protection against Sindbis virus-induced apoptosis. In this study, the potential role of XIAP overexpression in recombinant CHO (rCHO) cells treated with sodium butyrate (NaBu), which can increase the specific productivity, was investigated by establishing erythropoietin (EPO)-producing rCHO cells with regulated XIAP overexpression (EPO-off-XIAP). The XIAP overexpression in EPO-off-XIAP was tightly regulated by doxycycline. The XIAP overexpression could simultaneously reduce the activation of caspase-3, -7, and -9 induced by NaBu addition. However, XIAP overexpression could not inhibit NaBu-induced apoptosis, as evidenced by DNA fragmentation. In addition, it also did not help the maintenance of the mitochondrial membrane potential in the presence of NaBu, suggesting that the release of mitochondrial proteins might induce caspase-independent apoptosis. As a result, XIAP overexpression did not affect cell growth and EPO production significantly. Taken together, XIAP overexpression, which was reported to inhibit Sindbis virus-induced apoptosis, could not inhibit the NaBu-induced apoptosis in rCHO cells.