In vitro antiproliferative activity of 2,3-dihydroxy-9,10-anthraquinone induced apoptosis against COLO320 cells through cytochrome c release caspase mediated pathway with PI3K/AKT and COX-2 inhibition

Sequential activation of caspases is critical for the execution of apoptosis. Recent evidence suggests caspase 2 is a significant upstream caspase capable of initiating mitochondrial events, such as the release of cytochrome c. In particular, in vitro studies using recombinant proteins have shown that cleaved caspase 2 can induce mitochondrial outer membrane permeabilization directly or by cleaving the BH3-only protein BID (BH3 interacting domain death agonist). However, whether interchain cleavage or activation of procaspase 2 occurs prior to Apaf-1-mediated procaspase 9 activation under more natural conditions remains unresolved. In the present study, we show that Apaf-1-deficient Jurkat T-lymphocytes and mouse embryonic fibroblasts were highly resistant to DNA-damage-induced apoptosis and failed to cleave or activate any apoptotic procaspase, including caspase 2. Significantly, drug-induced cytochrome c release and loss of mitochondrial membrane potential were inhibited in cells lacking Apaf-1. By comparison, procaspase proteolysis and apoptosis were only delayed slightly in Apaf-1-deficient Jurkat cells upon treatment with anti-Fas antibody. Our data support a model in which Apaf-1 is necessary for the cleavage or activation of all procaspases and the promotion of mitochondrial apoptotic events induced by genotoxic drugs.

Download Full-text

The warburg effect and tumour cell survival in human GBMs

Clinical & Investigative Medicine ◽

10.25011/cim.v30i4.2878 ◽

2007 ◽

Vol 30 (4) ◽

pp. 97 ◽

Cited By ~ 1

Author(s):

A Wolf ◽

J Mukherjee ◽

A Guha

Keyword(s):

Cytochrome C ◽

Cell Survival ◽

Expression Profile ◽

Tumour Cell ◽

Warburg Effect ◽

Glycolytic Enzymes ◽

Cytochrome C Release ◽

Apoptotic Pathway ◽

The Warburg Effect

Introduction: GBMs are resistant to apoptosis induced by the hypoxic microenvironment and standard therapies including radiation and chemotherapy. We postulate that the Warburg effect, a preferential glycolytic phenotype of tumor cells even under aerobic conditions, plays a role in these aberrant pro-survival signals. In this study we quantitatively examined the expression profile of hypoxia-related glycolytic genes within pathologically- and MRI-defined “centre” and “periphery” of GBMs. We hypothesize that expression of hypoxia-induced glycolytic genes, particularly hexokinase 2 (HK2), favours cell survival and modulates resistance to tumour cell apoptosis by inhibiting the intrinsic mitochondrial apoptotic pathway. Methods: GBM patients underwent conventional T1-weighted contrast-enhanced MRI and MR spectroscopy studies on a 3.0T GE scanner, prior to stereotactic sampling (formalin and frozen) from regions which were T1-Gad enhancing (“centre”) and T2-positive, T1-Gad negative (“periphery”). Real-time qRT-PCR was performed to quantify regional gene expression of glycolytic genes including HK2. In vitro functional studies were performed in U87 and U373 GBM cell lines grown in normoxic (21% pO2) and hypoxic (< 1%pO2) conditions, transfected with HK2 siRNA followed by measurement of cell proliferation (BrdU), apoptosis (activated caspase 3/7, TUNEL, cytochrome c release) and viability (MTS assay). Results: There exists a differential expression profile of glycolytic enzymes between the hypoxic center and relatively normoxic periphery of GBMs. Under hypoxic conditions, there is increased expression of HK2 at the mitochondrial membrane in GBM cells. In vitro HK2 knockdown led to decreased cell survival and increased apoptosis via the intrinsic mitochondrial pathway, as seen by increased mitochondrial release of cytochrome-C. Conclusions: Increased expression of HK2 in the centre of GBMs promotes cell survival and confers resistance to apoptosis, as confirmed by in vitro studies. In vivo intracranial xenograft studies with injection of HK2-shRNA are currently being performed. HK2 and possibly other glycolytic enzymes may provide a target for enhanced therapeutic responsiveness thereby improving prognosis of patients with GBMs.

Download Full-text

Prevention of TNF-α-induced apoptosis in polyamine-depleted IEC-6 cells is mediated through the activation of ERK1/2

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00342.2003 ◽

2004 ◽

Vol 286 (3) ◽

pp. G479-G490 ◽

Cited By ~ 36

Author(s):

Sujoy Bhattacharya ◽

Ramesh M. Ray ◽

Leonard R. Johnson

Keyword(s):

Epithelial Cells ◽

Cytochrome C ◽

Critical Role ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Cytochrome C Release ◽

Erk Phosphorylation ◽

Tnf Α ◽

Induced Apoptosis ◽

Jnk Activation

It has been documented that polyamines play a critical role in the regulation of apoptosis in intestinal epithelial cells. We have recently reported that protection from TNF-α/cycloheximide (CHX)-induced apoptosis in epithelial cells depleted of polyamines is mediated through the inactivation of a proapoptotic mediator, JNK. In this study, we addressed the involvement of the MAPK pathway in the regulation of apoptosis after polyamine depletion of IEC-6 cells. Polyamine depletion by α-difluromethylornithine (DFMO) resulted in the sustained activation of ERK in response to TNF-α/CHX treatment. Pretreatment of polyamine-depleted IEC-6 cells with a cell membrane-permeable MEK1/2 inhibitor, U-0126, significantly inhibited TNF-α/CHX-induced ERK phosphorylation and significantly increased DNA fragmentation, JNK activity, and caspase-3 activity in response to TNF-α/CHX. Moreover, the dose dependency of U-0126-mediated inhibition of TNF-α/ CHX-induced ERK phosphorylation correlated with the reversal of the antiapoptotic effect of DFMO. IEC-6 cells expressing constitutively active MEK1 had decreased TNF-α/CHX-induced JNK phosphorylation and were significantly protected from apoptosis. Conversely, a dominant-negative MEK1 resulted in high basal activation of JNK, cytochrome c release, and spontaneous apoptosis. Polyamine depletion of the dominant-negative MEK1 cells did not prevent JNK activation or cytochrome c release and failed to confer protection from both TNF-α/CHX and camptothecin-induced apoptosis. Finally, expression of a dominant-negative mutant of JNK significantly protected IEC-6 cells from TNF-α/CHX-induced apoptosis. These data indicate that polyamine depletion results in the activation of ERK, which inhibits JNK activation and protects cells from apoptosis.

Download Full-text

Bid BH3 peptide, but not its mutant form G94E, induces per-meability transition pore opening and cytochrome c release in vitro

Chinese Science Bulletin ◽

10.1360/02tb9127 ◽

2002 ◽

Vol 47 (7) ◽

pp. 553 ◽

Cited By ~ 3

Author(s):

Tian XIA

Keyword(s):

Cytochrome C ◽

Mutant Form ◽

Cytochrome C Release ◽

Pore Opening ◽

Release In Vitro

Download Full-text

Contribution of Mitochondrial Ion Channels to Chemo-Resistance in Cancer Cells

Cancers ◽

10.3390/cancers11060761 ◽

2019 ◽

Vol 11 (6) ◽

pp. 761 ◽

Cited By ~ 10

Author(s):

Roberta Peruzzo ◽

Ildiko Szabo

Keyword(s):

Ion Channels ◽

Cytochrome C ◽

Cancer Cells ◽

Cytochrome C Release ◽

Drug Induced ◽

Point Of No Return ◽

Different Types ◽

Subsequent Activation ◽

Induced Apoptosis ◽

Potential Efficiency

Mitochondrial ion channels are emerging oncological targets, as modulation of these ion-transporting proteins may impact on mitochondrial membrane potential, efficiency of oxidative phosphorylation and reactive oxygen production. In turn, these factors affect the release of cytochrome c, which is the point of no return during mitochondrial apoptosis. Many of the currently used chemotherapeutics induce programmed cell death causing damage to DNA and subsequent activation of p53-dependent pathways that finally leads to cytochrome c release from the mitochondrial inter-membrane space. The view is emerging, as summarized in the present review, that ion channels located in this organelle may account in several cases for the resistance that cancer cells can develop against classical chemotherapeutics, by preventing drug-induced apoptosis. Thus, pharmacological modulation of these channel activities might be beneficial to fight chemo-resistance of different types of cancer cells.

Download Full-text

Volatile Anesthetics Induce Caspase-dependent, Mitochondria-mediated Apoptosis in Human T Lymphocytes In Vitro

Anesthesiology ◽

10.1097/00000542-200506000-00014 ◽

2005 ◽

Vol 102 (6) ◽

pp. 1147-1157 ◽

Cited By ~ 102

Author(s):

Torsten Loop ◽

David Dovi-Akue ◽

Michael Frick ◽

Martin Roesslein ◽

Lotti Egger ◽

...

Keyword(s):

Membrane Potential ◽

T Lymphocytes ◽

Cytochrome C ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Caspase 3 ◽

Volatile Anesthetics ◽

Human T Lymphocytes ◽

Induced Apoptosis

Background Volatile anesthetics modulate lymphocyte function during surgery, and this compromises postoperative immune competence. The current work was undertaken to examine whether volatile anesthetics induce apoptosis in human T lymphocytes and what apoptotic signaling pathway might be used. Methods Effects of sevoflurane, isoflurane, and desflurane were studied in primary human CD3 T lymphocytes and Jurkat T cells in vitro. Apoptosis and mitochondrial membrane potential were assessed using flow cytometry after green fluorescent protein-annexin V and DiOC6-fluorochrome staining. Activity and proteolytic processing of caspase 3 was measured by cleaving of the fluorogenic effector caspase substrate Ac-DEVD-AMC and by anti-caspase-3 Western blotting. Release of mitochondrial cytochrome c was studied after cell fractionation using anti-cytochrome c Western blotting and enzyme-linked immunosorbent assays. Results Sevoflurane and isoflurane induced apoptosis in human T lymphocytes in a dose-dependent manner. By contrast, desflurane did not exert any proapoptotic effects. The apoptotic signaling pathway used by sevoflurane involved disruption of the mitochondrial membrane potential and release of cytochrome c from mitochondria to the cytosol. In addition, the authors observed a proteolytic cleavage of the inactive p32 procaspase 3 to the active p17 fragment, increased caspase-3-like activity, and cleavage of the caspase-3 substrate poly-ADP-ribose-polymerase. Sevoflurane-induced apoptosis was blocked by the general caspase inhibitor Z-VAD.fmk. Death signaling was not mediated via the Fas/CD95 receptor pathway because neither anti-Fas/CD95 receptor antagonism nor FADD deficiency or caspase-8 deficiency were able to attenuate sevoflurane-mediated apoptosis. Conclusion Sevoflurane and isoflurane induce apoptosis in T lymphocytes via increased mitochondrial membrane permeability and caspase-3 activation, but independently of death receptor signaling.

Download Full-text

Sirt5 Attenuates Cisplatin-Induced Acute Kidney Injury through Regulation of Nrf2/HO-1 and Bcl-2

BioMed Research International ◽

10.1155/2019/4745132 ◽

2019 ◽

Vol 2019 ◽

pp. 1-11 ◽

Cited By ~ 6

Author(s):

Wei Li ◽

Yuanyuan Yang ◽

You Li ◽

Yueyue Zhao ◽

Hong Jiang

Keyword(s):

Acute Kidney Injury ◽

Cytochrome C ◽

Kidney Injury ◽

Human Kidney ◽

Dependent Manner ◽

Cytochrome C Release ◽

Bax Protein ◽

Intracellular Ros ◽

Induced Apoptosis ◽

Mitotracker Red

Cisplatin- (CDDP) induced acute kidney injury (AKI) limits the clinical use of cisplatin. Several sirtuin (SIRT) family proteins are involved in AKI, while the roles of Sirt5 in cisplatin-induced AKI remain unknown. In the present study, we characterized the role and mechanism of Sirt5 in cisplatin-induced apoptosis using the human kidney 2 (HK-2) cell line. CDDP treatment decreased Sirt5 expression of HK-2 cells in a dose-dependent manner. In addition, Sirt5 overexpression enhanced the metabolic activity in CDDP-treated HK-2 cells while Sirt5 siRNA attenuated it. Forced expression of Sirt5 inhibited CDDP-induced apoptosis while Sirt5 siRNA showed the opposite effects. Accordingly, Sirt5 overexpression inhibited the level of caspase 3 cleavage and cytochrome c levels. Furthermore, we found that Sirt5 increased mitochondrial membrane potentials and ameliorated intracellular ROS production. Mitotracker Red staining indicated that Sirt5 overexpression was able to maintain the mitochondrial density during CDDP treatment. We also investigated possible downstream targets of Sirt5 and found that Sirt5 increased Nrf2, HO-1, and Bcl-2 while it decreased Bax protein expression. Sirt5 siRNA showed the opposite effect on these proteins. The levels of Nrf2, HO-1, and Bcl-2 proteins in HK-2 cells were also decreased after CDDP treatment. Moreover, Nrf2 and Bcl-2 siRNA partly abolished the protecting effect of Sirt5 on CDDP-induced apoptosis and cytochrome c release. Catalase inhibitor 3-AT also abolished the cytoprotective effect of Sirt5. Together, the results demonstrated that Sirt5 attenuated cisplatin-induced apoptosis and mitochondrial injury in human kidney HK-2 cells, possibly through the regulation of Nrf2/HO-1 and Bcl-2.

Download Full-text

Nitric Oxide–Induced Apoptosis in Human Leukemic Lines Requires Mitochondrial Lipid Degradation and Cytochrome C Release

Blood ◽

10.1182/blood.v93.7.2342 ◽

1999 ◽

Vol 93 (7) ◽

pp. 2342-2352 ◽

Cited By ~ 101

Author(s):

Alexey Ushmorov ◽

Frank Ratter ◽

Volker Lehmann ◽

Wulf Dröge ◽

Volker Schirrmacher ◽

...

Keyword(s):

Nitric Oxide ◽

Cytochrome C ◽

Mitochondrial Protein ◽

Jurkat Cells ◽

Leukemic Cells ◽

Human Leukemia ◽

Cytochrome C Release ◽

Mitochondrial Functions ◽

Mitochondrial Lipid ◽

Induced Apoptosis

Abstract We have previously shown that nitric oxide (NO) stimulates apoptosis in different human neoplastic lymphoid cell lines through activation of caspases not only via CD95/CD95L interaction, but also independently of such death receptors. Here we investigated mitochondria-dependent mechanisms of NO-induced apoptosis in Jurkat leukemic cells. NO donor glycerol trinitrate (at the concentration, which induces apoptotic cell death) caused (1) a significant decrease in the concentration of cardiolipin, a major mitochondrial lipid; (2) a downregulation in respiratory chain complex activities; (3) a release of the mitochondrial protein cytochrome c into the cytosol; and (4) an activation of caspase-9 and caspase-3. These changes were accompanied by an increase in the number of cells with low mitochondrial transmembrane potential and with a high level of reactive oxygen species production. Higher resistance of the CD95-resistant Jurkat subclone (APO-R) cells to NO-mediated apoptosis correlated with the absence of cytochrome c release and with less alterations in other mitochondrial parameters. An inhibitor of lipid peroxidation, trolox, significantly suppressed NO-mediated apoptosis in APO-S Jurkat cells, whereas bongkrekic acid (BA), which blocks mitochondrial permeability transition, provided only a moderate antiapoptotic effect. Transfection of Jurkat cells with bcl-2 led to a complete block of apoptosis due to the prevention of changes in mitochondrial functions. We suggest that the mitochondrial damage (in particular, cardiolipin degradation and cytochrome c release) induced by NO in human leukemia cells plays a crucial role in the subsequent activation of caspase and apoptosis.

Download Full-text