scholarly journals Cisplatin Induces Apoptosis in LLC-PK1 Cells via Activation of Mitochondrial Pathways

2002 ◽  
Vol 13 (4) ◽  
pp. 858-865 ◽  
Author(s):  
Moon Soo Park ◽  
Maryely De Leon ◽  
Prasad Devarajan
Keyword(s):  
Cytochrome C ◽  
Molecular Mechanisms ◽  
Mitochondrial Permeability Transition ◽  
Apoptotic Cell ◽  
Permeability Transition ◽  
Dependent Manner ◽  
Caspase 9 ◽  
Western Blots ◽  
Mitochondrial Permeability ◽  
Sequence Of Events

ABSTRACT. Cisplatin, a commonly used chemotherapeutic agent, has a major limitation because of its nephrotoxicity. Recent studies have shown that cisplatin causes apoptotic cell death in renal tubule cells, but the underlying molecular mechanisms remain to be elucidated. In this study, cisplatin was found to induce apoptosis in a dose- and duration-dependent manner in cultured proximal tubule (LLC-PK1) cells, as evidenced by DNA laddering and TdT-mediated dUTP nick end-labeling assay. Pretreatment with the specific caspase 9 inhibitor LEHD-CHO completely prevented the apoptosis, whereas the caspase 8 inhibitor IETD-fmk had no effect. Furthermore, the activity of caspase 9 was upregulated about sixfold by cisplatin in a dose-dependent manner. These results implicated the caspase 9–dependent mitochondrial apoptotic pathways. Indeed, cisplatin triggered a duration-dependent translocation of cytochrome c from the mitochondria to the cytosol, by immunofluorescence and Western blots. Cisplatin treatment also resulted in the duration-dependent activation and mitochondrial translocation of the pro-apoptotic molecule Bax, by immunofluorescence. Finally, cisplatin induced a duration-dependent onset of the mitochondrial permeability transition. Our results indicate that cisplatin induces apoptosis in LLC-PK1 cells via activation of mitochondrial signaling pathways. The sequence of events may be summarized as follows: activation of Bax induces mitochondrial permeability transition, leading to release of cytochrome c, activation of caspase 9, and entry into the execution phase of apoptosis. Inhibition of this specific pathway may provide a strategy to minimize cisplatin-induced nephrotoxicity.

scholarly journals Metformin induces apoptosis via uterus mitochondrial permeability transition pore opening and protects against estradiol benzoate-induced uterine defect and associated pathophysiological disorder in female Wistar rats

2021 ◽  
Vol 45 (1) ◽  
Author(s):  
Adeola Oluwakemi Olowofolahan ◽  
Obinna Matthew Paulinus ◽  
Heritage Mojisola Dare ◽  
Olufunso Olabode Olorunsogo
Keyword(s):  
Cell Death ◽  
Cytochrome C ◽  
Mitochondrial Permeability Transition ◽  
Permeability Transition ◽  
Estradiol Benzoate ◽  
Histological Evaluation ◽  
Mitochondrial Atpase ◽  
Rat Uterus ◽  
Mitochondrial Permeability ◽  
Pore Opening

Abstract Background Some antitumor or anticancer agents have been shown to execute cell death by induction of mitochondrial permeability transition (mPT) pore opening in order to elicit their chemotherapeutic effect. Therefore, this study investigated the effect of metformin on cell death via rat uterus mPT pore and estradiol benzoate-induced uterine defect and associated pathophysiological disorder in female rat. Mitochondria were isolated using differential centrifugation. The mPT pore opening, cytochrome c release and mitochondrial ATPase activity were determined spectrophotometrically. Caspases 9 and 3 activities, MDA and estradiol levels and SOD, GSH activities, were determined using ELISA technique. Histological and histochemical assessments of the uterine section were carried out using standard methods. Results Metformin at concentrations 10–90 μg/mL, showed no significant effect on mPT pore opening, mATPase activity and release of cytochrome c. However, oral administration of metformin caused mPT pore opening, enhancement of mATPase activity and activation of caspases 9 and 3 significantly at 300 and 400 mg/kg. Metformin protected against estradiol benzoate (EB)-induced uterine defect and other associated pathophysiological disorder. It also improved the antioxidant defense system. The histological evaluation revealed the protective effect of metformin on the cellular architecture of the uterus while the histochemical examination showed severe hyperplasia in the uterine section of EB-treated rats, remarkably reversed by metformin co-treatment. Conclusion This study suggests that metformin at high doses induces apoptosis via rat uterus mPT pore opening and protects against EB-induced uterine defect (hyperplasia) and associated pathophysiological disorder.

scholarly journals Metformin inhibits mitochondrial permeability transition and cell death: a pharmacological in vitro study

2004 ◽  
Vol 382 (3) ◽  
pp. 877-884 ◽  
Author(s):  
Bruno GUIGAS ◽  
Dominique DETAILLE ◽  
Christiane CHAUVIN ◽  
Cécile BATANDIER ◽  
Frédéric De OLIVEIRA ◽  
...  
Keyword(s):  
Cell Death ◽  
Cytochrome C ◽  
Mitochondrial Permeability Transition ◽  
Permeability Transition ◽  
Cytochrome C Release ◽  
Intact Cells ◽  
Human Carcinoma ◽  
Butyl Hydroperoxide ◽  
Mitochondrial Permeability ◽  
New Findings

Metformin, a drug widely used in the treatment of Type II diabetes, has recently received attention owing to new findings regarding its mitochondrial and cellular effects. In the present study, the effects of metformin on respiration, complex 1 activity, mitochondrial permeability transition, cytochrome c release and cell death were investigated in cultured cells from a human carcinoma-derived cell line (KB cells). Metformin significantly decreased respiration both in intact cells and after permeabilization. This was due to a mild and specific inhibition of the respiratory chain complex 1. In addition, metformin prevented to a significant extent mitochondrial permeability transition both in permeabilized cells, as induced by calcium, and in intact cells, as induced by the glutathione-oxidizing agent t-butyl hydroperoxide. This effect was equivalent to that of cyclosporin A, the reference inhibitor. Finally, metformin impaired the t-butyl hydroperoxide-induced cell death, as judged by Trypan Blue exclusion, propidium iodide staining and cytochrome c release. We propose that metformin prevents the permeability transition-related commitment to cell death in relation to its mild inhibitory effect on complex 1, which is responsible for a decreased probability of mitochondrial permeability transition.

scholarly journals Bax-induced Cytochrome C Release from Mitochondria Is Independent of the Permeability Transition Pore but Highly Dependent on Mg2+ Ions

1998 ◽  
Vol 143 (1) ◽  
pp. 217-224 ◽  
Author(s):  
Robert Eskes ◽  
Bruno Antonsson ◽  
Astrid Osen-Sand ◽  
Sylvie Montessuit ◽  
Christoph Richter ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Cyclosporin A ◽  
Mitochondrial Permeability Transition Pore ◽  
Mitochondrial Permeability Transition ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Cytochrome C Release ◽  
Mitochondrial Permeability ◽  
Isolated Mitochondria ◽  
Mammalian Homologue

Bcl-2 family members either promote or repress programmed cell death. Bax, a death-promoting member, is a pore-forming, mitochondria-associated protein whose mechanism of action is still unknown. During apoptosis, cytochrome C is released from the mitochondria into the cytosol where it binds to APAF-1, a mammalian homologue of Ced-4, and participates in the activation of caspases. The release of cytochrome C has been postulated to be a consequence of the opening of the mitochondrial permeability transition pore (PTP). We now report that Bax is sufficient to trigger the release of cytochrome C from isolated mitochondria. This pathway is distinct from the previously described calcium-inducible, cyclosporin A–sensitive PTP. Rather, the cytochrome C release induced by Bax is facilitated by Mg2+ and cannot be blocked by PTP inhibitors. These results strongly suggest the existence of two distinct mechanisms leading to cytochrome C release: one stimulated by calcium and inhibited by cyclosporin A, the other Bax dependent, Mg2+ sensitive but cyclosporin insensitive.

Quenching or Misalignment? Confocal Microscopy Onset of the Mitochondrial Permeability Transition in Cultured Hepatocytes

1999 ◽  
Vol 5 (S2) ◽  
pp. 468-469
Author(s):  
Ting Qian ◽  
Lawrence C. Trost ◽  
John J. Lemasters
Keyword(s):  
Confocal Microscopy ◽  
Mitochondrial Permeability Transition ◽  
Apoptotic Cell ◽  
Negative Contrast ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Dark Spot ◽  
Cultured Hepatocytes ◽  
Mitochondrial Permeability ◽  
Calcein Am

INTRODUCTION: The mitochondrial permeability transition (MPT) has been implicated in mediating both necrotic and apoptotic cell death. Opening of the permeability transition pore in the mitochondrial inner membrane causes the MPT. Previously, our laboratory developed a method to detect the MPT in cultured hepatocytes by visualizing redistribution of calcein fluorescence from the cytosol into the mitochondria after permeability transition pore opening, using confocal microscopy. (1). However, a recent paper suggests that unstained mitochondria are unlikely to be detected against bright cytosolic signal, because fluorescence spreading magnifies the size of emitting objects while reducing that of dark spot (2). Rather, it was suggested that dark mitochondrial voids in green calcein images were the result of fluorescent quenching by concurrent use of a red-fluorescing mitochondrial dye, tetramethylrhodamine (TMRM). The AIMS of the present study were to determine 1) whether small voids can be visualized in the absence of any potential quenching agent; 2) whether quenching can produce dark mitochondrial voids and 3) the importance of alignment in visualizing mitochondria as negative contrast by confocal microscopy. METHODS: Overnight cultured rat hepatocytes were used. To load calcein exclusively into cytosolic space, hepatocytes were incubated with 1 μM calcein AM for 15 min at 37°C. To load calcein into both cytosol and mitochondria, cells were incubated with 1 μM calcein AM for 1 hour at 4°C.

scholarly journals Bisindolylpyrrole triggers transient mitochondrial permeability transitions to cause apoptosis in a VDAC1/2 and cyclophilin D-dependent manner via the ANT-associated pore

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Masami Koushi ◽  
Yasunori Aoyama ◽  
Yoshiko Kamei ◽  
Rei Asakai
Keyword(s):  
Mitochondrial Permeability Transition ◽  
Permeability Transition ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Dependent Manner ◽  
Cyclophilin D ◽  
Cytoprotective Effect ◽  
Mitochondrial Permeability ◽  
Sirna Knockdown ◽  
Pkc Activation

Abstract Bisindolylpyrrole at 0.1 μM is cytoprotective in 2% FBS that is counteracted by cyclosporin-A (CsA), an inhibitor of cyclophilin-D (CypD). We hypothesized that the cytoprotective effect might be due to transient mitochondrial permeability transition (tPT). This study tested the hypothesis that bisindolylpyrrole can trigger tPT extensively, thereby leading to cell death under certain conditions. Indeed, CsA-sensitive tPT-mediated apoptosis could be induced by bisindolylpyrrole at > 5 μM in HeLa cells cultured in 0.1% FBS, depending on CypD and VDAC1/2, as shown by siRNA knockdown experiments. Rat liver mitochondria also underwent swelling in response to bisindolylpyrrole, which proceeded at a slower rate than Ca2+-induced swelling, and which was blocked by the VDAC inhibitor tubulin and the ANT inhibitor bongkrekate, indicating the involvement of the ANT-associated, smaller pore. We examined why 0.1% FBS is a prerequisite for apoptosis and found that apoptosis is blocked by PKC activation, which is counteracted by the overexpressed defective PKCε. In mitochondrial suspensions, bisindolylpyrrole triggered CsA-sensitive swelling, which was suppressed selectively by pretreatment with PKCε, but not in the co-presence of tubulin. These data suggest that upon PKC inactivation the cytoprotective compound bisindolylpyrrole can induce prolonged tPT causing apoptosis in a CypD-dependent manner through the VDAC1/2-regulated ANT-associated pore.

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