Human placenta‐derived mesenchymal stem cells inhibit apoptosis of granulosa cells induced by IRE1α pathway in autoimmune POF mice

Previous studies have shown that the ovarian failure in autoimmune‐induced premature ovarian failure (POF) mice could be improved by the transplantation of human placenta‐derived mesenchymal stem cells (hPMSCs); however, the protective mechanism of hPMSCs transplantation on ovarian dysfunction remains unclear. Ovarian dysfunction is closely related to the apoptosis of granulosa cells (GCs). To determine the effects of hPMSCs transplantation on GCs apoptosis, an autoimmune POF mice model was established with zona pellucida glycoprotein 3 (ZP3) peptide. It is reported that the inositol‐requiring enzyme 1α (IRE1α) and its downstream molecules play a central role in the endoplasmic reticulum (ER) stress‐induced apoptosis pathway. So the aim of this study is to investigate whether hPMSCs transplantation attenuated GCs apoptosis via inhibiting ER stress IRE1α signaling pathway. The ovarian dysfunction, follicular dysplasia, and GCs apoptosis were observed in the POF mice. And the IRE1α pathway was activated in ovaries of POF mice, as demonstrated by, increased X‐box binding protein 1 (XBP1), up‐regulated 78 kDa glucose‐regulated protein (GRP78) and caspase‐12. Following transplantation of hPMSCs, the ovarian structure and function were significantly improved in POF mice. In addition, the GCs apoptosis was obviously attenuated and IRE1α pathway was significantly inhibited. Transplantation of hPMSCs suppressed GCs apoptosis‐induced by ER stress IRE1α signaling pathway in POF mice, which might contribute to the hPMSCs transplantation‐mediating ovarian function recovery.     

Resveratrol-induced mitochondrial dysfunction and apoptosis are associated with Ca2+ and mCICR-mediated MPT activation in HepG2 cells

Resveratrol, a natural polyphenolic antioxidant, has been reported to possess the cancer chemopreventive potential in wide range by means of triggering tumor cells apoptosis through various pathways. It induced apoptosis through the activation of the mitochondrial pathway in some kinds of cells. In the present reports, we showed that resveratrol-induced HepG2 cell apoptosis and mitochondrial dysfunction was dependent on the induction of the mitochondrial permeability transition (MPT), because resveratrol caused the collapse of the mitochondrial membrane potential (ΔΨ_m) with the concomitant release of cytochrome c (Cyt.c). In addition, resveratrol induced a rapid and sustained elevation of intracellular [Ca²⁺], which compromised the mitochondrial ΔΨ_m and triggered the process of HepG2 cell apoptosis. In permeabilized HepG2 cells, we further demonstrated that the effect of the resveratrol was indeed synergistic with that of Ca²⁺ and Ca²⁺ is necessary for resveratrol-induced MPT opening. Calcium-induced calcium release from mitochondria (mCICR) played a key role in mitochondrial dysfunction and cell apoptosis: (1) mCICR inhibitor, ruthenium red (RR), prevent MPT opening and Cyt.c release; and (2) RR attenuated resveratrol-induced HepG2 cell apoptotic death. Furthermore, resveratrol promotes MPT opening by lowering Ca²⁺-threshold. These data suggest modifying mCICR and Ca²⁺ threshold to modulate MPT opening may be a potential target to control cell apoptosis induced by resveratrol. Xuemei Tian—Foundation item: Chinese National Natural Science Foundation (No.30300455).     

Melatonin alleviates insulin resistance through the PI3K/AKT signaling pathway in ovary granulosa cells of polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is a common endocrine gynecological disorder. Insulin resistance (IR) is a major cause of PCOS. Melatonin, a critical endogenous hormone, has beneficial effects on the female reproductive system. This study aims to investigate the molecular effect of melatonin on IR in human ovarian granulosa cells (GCs). Hormone levels of the subjects were determined through clinical examination. The expression levels of insulin receptor substrate (IRS)-1 and glucose transporter (GLUT4) in GCs from PCOS patients and a human granulosa cell line (SVOG) were examined using qRT-PCR and western blot. The IR cell model was established by inducing SVOG cells with palmitic acid (PA). IR was detected in GCs of PCOS patients and SVOG by measuring glucose content and glucose uptake. Cell viability and apoptosis levels were detected by CCK-8 assay and flow cytometry. PI3K/Akt pathway expression in SVOG was assessed by western blot. PCOS patients had higher levels of luteinizing hormone (LH), testosterone, and LH/follicle-stimulating hormone. PA decreased cell viability, promoted apoptosis, and reduced glucose uptake in SVOG cells. IRS-1 and GLUT4 mRNA and protein expression was downregulated, and glucose uptake capacity was reduced in PCOS GCs and SVOG cells. Melatonin significantly upregulated IRS-1 and GLUT4 expression, downregulated p-IRS-1 (Ser307), and improved glucose uptake in PCOS patients' GCs and SVOG cells. PA decreased PI3K and Akt phosphorylation, whereas melatonin increased p-PI3K and p-Akt levels. Melatonin can reduce IR in GCs and PA-induced SVOG cells via the PI3K/Akt signaling pathway, providing more evidence for treating polycystic ovary syndrome.     

Mir-484 contributes to diminished ovarian reserve by regulating granulosa cell function via YAP1-mediated mitochondrial function and apoptosis

Women with diminished ovarian reserve (DOR) have reduced fertility, but the underlying regulation of ovarian function remains unknown. Although differential microRNA (miRNA) expression has been described in several ovarian disorders, little is known about the role of miRNAs in the pathogenesis of DOR. In this study, we investigated the expression levels of miR-484 in granulosa cells (GCs) derived from human follicular fluid, and explored their correlation with female ovarian reserve function as well as clinical outcomes of assisted reproduction technology (ART). Additionally, we investigated the effects of miR-484 on the biological functions of GC cell lines in vitro. We found that miR-484 was highly expressed in GCs from DOR patients and was correlated with decreasing AMH levels and AFC, as well as increasing FSH levels, but not with LH, progesterone, or estradiol. Additionally, miR-484 was negatively related to the number of retrieved oocytes and the ratio of high-quality embryos. Moreover, we found that miR-484 repressed the proliferation of GCs and induced apoptosis, which can in part be attributed to mitochondrial dysfunction. Conversely, silencing miR-484 had the opposite effect. Multiple approaches, including bioinformatic analysis, RNA-seq, qPCR, immunofluorescence, western blotting and luciferase reporter assays, identified YAP1 as a direct target of miR-484 in GCs. Additionally, reintroduction of YAP1 rescued the effects of miR-484 in GCs. The present study indicates that miR-484 can directly target the mRNA of YAP1, induce mitochondrial dysfunction, and consequently reduce the viability and promote the apoptosis of granulosa cells, which contributes to the pathogenesis of DOR.     

Mitochondrial peroxiredoxin‐5 as potential modulator of mitochondria‐ER crosstalk in MPP+‐induced cell death

Peroxiredoxin‐5 (PRDX5) is an antioxidant enzyme which differs from the other peroxiredoxins with regards to its enzymatic mechanism, its high affinity for organic peroxides and peroxynitrite and its wide subcellular distribution. In particular, the mitochondrial isoform of PRDX5 confers a remarkable cytoprotection toward oxidative stress to mammalian cells. Mitochondrial dysfunction and disruption of Ca²⁺ homeostasis are implicated in neurodegeneration. Growing evidence supports that endoplasmic reticulum (ER) could operate in tandem with mitochondria to regulate intracellular Ca²⁺ fluxes in neurodegenerative processes. Here, we overexpressed mitochondrial PRDX5 in SH‐SY5Y cells to dissect the role of this enzyme in 1‐methyl‐4‐phenylpyridinium (MPP)⁺‐induced cell death. Our data show that mitochondria‐dependent apoptosis triggered by MPP⁺, assessed by the measurement of caspase‐9 activation and mitochondrial DNA damage, is prevented by mitochondrial PRDX5 overexpression. Moreover, PRDX5 overexpression blocks the increase in intracellular Ca²⁺, Ca²⁺‐dependent activation of calpains and Bax cleavage. Finally, using Ca²⁺ channel inhibitors (Nimodipine, Dantrolene and 2‐APB), we show that Ca²⁺ release arises essentially from ER stores through 1,4,5‐inositol‐trisphosphate receptors (IP₃R). Altogether, our results suggest that the MPP⁺ mitochondrial pathway of apoptosis is regulated by mitochondrial PRDX5 in a process that could involve redox modulation of Ca²⁺ transporters via a crosstalk between mitochondria and ER.     

Capsaicin induces mitochondrial dysfunction and apoptosis in anaplastic thyroid carcinoma cells via TRPV1-mediated mitochondrial calcium overload

Anaplastic thyroid cancer (ATC) is a rare malignancy and has a poor prognosis due to its aggressive behavior and resistance to treatments. Calcium (Ca²⁺) serves as a ubiquitous cellular second messenger and influences several tumor behaviors. Therefore, Ca²⁺ modulation is expected to be a novel therapeutic target in cancers. However, whether Ca²⁺ modulation is effective in ATC therapy remains unknown. In this study, we reported that capsaicin (CAP), a transient receptor potential vanilloid type1 (TRPV1) agonist, inhibited the viability of anaplastic thyroid cancer cells. Capsaicin treatment triggered Ca²⁺ influx by TRPV1 activation, resulting in disequilibrium of intracellular calcium homeostasis. The rapidly increased cytosolic Ca²⁺ concentration was mirrored in the mitochondria and caused a severe condition of mitochondrial calcium overload in ATC cells. In addition, the disruption of mitochondrial calcium homeostasis caused by capsaicin led to mitochondrial dysfunction in ATC cells, as evidenced by the production of mitochondrial reactive oxygen species (ROS), depolarization of mitochondrial membrane potential (ΔΨm), and opening of mitochondrial permeability transition pore (mPTP). Next, the resulting release of cyt c into the cytosol triggered apoptosome assembly and subsequent caspase activation and apoptosis. It was worth noting that both TRPV1 antagonist (capsazepine) and calcium chelator (BAPTA) could attenuate aberrant Ca²⁺ homeostasis, mitochondrial dysfunction and apoptosis induced by capsaicin treatment. Thus, our study demonstrated that capsaicin induced mitochondrial calcium overload and apoptosis in ATC cells through a TRPV1-mediated pathway. The better understanding of the anti-cancer mechanisms of calcium modulation provides a potential target for the ATC therapy.     

Enhancement of hyperthermia-induced apoptosis by a free radical initiator, 2,2′-azobis (2-amidinopropane) dihydrochloride,in human histiocytic lymphoma U937 cells

To elucidate the mechanism how a free radical initiator, 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH), induces cell death at hyperthermic temperatures, apoptosis in a human histiocytic lymphoma cell line, U937, was investigated. Free radical formation deriving from the thermal decomposition of AAPH was examined by spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). An assay for DNA fragmentation, observation of nuclear morphological changes, and flow cytometry for phosphatidylserine (PS) externalization were used to detect apoptosis and revealed enhancement of 44.0°C hyperthermia-induced apoptosis by free radicals due to AAPH. However, free radicals alone derived from AAPH did not induce apoptosis. Hyperthermia induced the production of lipid peroxidation (LPO), an increase in intracellular Ca²⁺ concentration ([Ca²⁺]i) and enhanced expression of the type 1 inositol 1,4,5-trisphosphate receptor (IP₃R1). The effects of hyperthermia on LPO and [Ca²⁺]i were enhanced markedly by the combination with AAPH. A significant decrease in Bcl-2 expression, increase in Bax expression, a loss of mitochondrial membrane potential (ΔΨm) and a marked increase in cytochrome c expression were found only in cells treated with hyperthermia and AAPH. Although an intracellular Ca²⁺ ion chelator, BAPTA-AM, completely inhibited DNA fragmentation, water-soluble vitamine E, Trolox, only partially suppressed DNA fragmentation and the increase in [Ca²⁺]i. In contrast, LPO was inhibited completely by Trolox, but no inhibition by BAPTA-AM was found. These results suggest that apoptosis induced by hyperthermia alone is due to the increase in [Ca²⁺]i arising from increased expression of IP₃R1 and LPO. Additional increase in [Ca²⁺]i due to increased LPO and the activation of mitochondria-caspase dependent pathway play a major role in the enhancement of apoptosis by the combination with hyperthermia and AAPH.     

Transglutaminase 2 Contributes to Apoptosis Induction in Jurkat T Cells by Modulating Ca2+ Homeostasis via Cross-Linking RAP1GDS1

Background

Transglutaminase 2 (TG2) is a protein cross-linking enzyme known to be associated with the in vivo apoptosis program of T cells. However, its role in the T cell apoptosis program was not investigated yet.

Results

Here we report that timed overexpression of both the wild type (wt) and the cross-linking mutant of TG2 induced apoptosis in Jurkat T cells, the wt being more effective. Part of TG2 colocalised with mitochondria. WtTG2-induced apoptosis was characterized by enhanced mitochondrial Ca²⁺ uptake. Ca²⁺-activated wtTG2 cross-linked RAP1, GTP-GDP dissociation stimulator 1, an unusual guanine exchange factor acting on various small GTPases, to induce a yet uncharacterized signaling pathway that was able to promote the Ca²⁺ release from the endoplasmic reticulum via both Ins₃P and ryanodine sensitive receptors leading to a consequently enhanced mitochondrial Ca²⁺uptake.

Conclusions

Our data indicate that TG2 might act as a Ca²⁺ sensor to amplify endoplasmic reticulum-derived Ca²⁺ signals to enhance mitochondria Ca²⁺ uptake. Since enhanced mitochondrial Ca²⁺ levels were previously shown to sensitize mitochondria for various apoptotic signals, our data demonstrate a novel mechanism through which TG2 can contribute to the induction of apoptosis in certain cell types. Since, as compared to knock out cells, physiological levels of TG2 affected Ca²⁺ signals in mouse embryonic fibroblasts similar to Jurkat cells, our data might indicate a more general role of TG2 in the regulation of mitochondrial Ca²⁺ homeostasis.     

Sodium Butyrate Induces Apoptosis in MSN Neuroblastoma Cells in a Calcium Independent Pathway

Sodium butyrate (NaBt), a histone deacetylase inhibitor, can cause apoptosis in a number of cancer cells. However, the mechanism of this action is poorly understood. Increased intracellular [Ca²⁺] level has been suggested as a likely mechanism, but there is little corroborating data. In this report we provide evidence that NaBt-treated MSN neuroblastoma cells undergo massive apoptosis in the presence of serum and regardless of external or internal [Ca²⁺] levels. Presented data suggest that apoptotic effect of NaBt is both time- and dose-dependent (LD₅₀ 1 mM); and that, presence of serum or cAMP, a second messenger molecule that modulates the apoptotic program in a wide variety of cells could not circumvent the apoptotic effect of NaBt. Our findings suggest that NaBt-induced apoptosis in MSN neuroblastoma cells occurs via a pathway that is independent of Ca²⁺flux, intracellular [Ca²⁺] or cAMP levels. Further, we also present data that exclude a role for PKC or histones acetylation.Special issue dedicated to Lawrence F. Eng     

Echinacoside reverses myocardial remodeling and improves heart function via regulating SIRT1/FOXO3a/MnSOD axis in HF rats induced by isoproterenol

Myocardial remodelling is important pathological basis of HF, mitochondrial oxidative stress is a promoter to myocardial hypertrophy, fibrosis and apoptosis. ECH is the major active component of a traditional Chinese medicine Cistanches Herba, plenty of studies indicate it possesses a strong antioxidant capacity in nerve cells and tumour, it inhibits mitochondrial oxidative stress, protects mitochondrial function, but the specific mechanism is unclear. SIRT1/FOXO3a/MnSOD is an important antioxidant axis, study finds that ECH binds covalently to SIRT1 as a ligand and up-regulates the expression of SIRT1 in brain cells. We hypothesizes that ECH may reverse myocardial remodelling and improve heart function of HF via regulating SIRT1/FOXO3a/MnSOD signalling axis and inhibit mitochondrial oxidative stress in cardiomyocytes. Here, we firstly induce cellular model of oxidative stress by ISO with AC-16 cells and pre-treat with ECH, the level of mitochondrial ROS, mtDNA oxidative injury, MMP, carbonylated protein, lipid peroxidation, intracellular ROS and apoptosis are detected, confirm the effect of ECH in mitochondrial oxidative stress and function in vitro. Then, we establish a HF rat model induced by ISO and pre-treat with ECH. Indexes of heart function, myocardial remodelling, mitochondrial oxidative stress and function, expression of SIRT1/FOXO3a/MnSOD signalling axis are measured, the data indicate that ECH improves heart function, inhibits myocardial hypertrophy, fibrosis and apoptosis, increases the expression of SIRT1/FOXO3a/MnSOD signalling axis, reduces the mitochondrial oxidative damages, protects mitochondrial function. We conclude that ECH reverses myocardial remodelling and improves cardiac function via up-regulating SIRT1/FOXO3a/MnSOD axis and inhibiting mitochondrial oxidative stress in HF rats.     

Mitochondrial Dysfunction and Reactive Oxygen Species in Excitotoxicity and Apoptosis: Implications for the Pathogenesis of Neurodegenerative Diseases

In recent years we have witnessed a major interest in the study of the role of mitochondria, not only as ATP producers through oxidative phosphorylation but also as regulators of intracellular Ca²⁺ homeostasis and endogenous producers of reactive oxygen species (ROS). Interestingly, the mitochondria have been also implicated as central executioners of cell death. Increased mitochondrial Ca²⁺ overload as a result of excitotoxicity has been associated with the generation of superoxide and may induce the release of proapoptotic mitochondrial proteins, proceeding through DNA fragmentation/condensation and culminating in cell demise by apoptosis and/or necrosis. In addition, these processes have been implicated in the pathogenesis of many neurodegenerative diseases, which share several features of cell death: selective brain areas undergo neurodegeneration, involving mitochondrial dysfunction (mitochondrial complexes are affected), loss of intracellular Ca²⁺ homeostasis, excitotoxicity, and the extracellular or intracellular accumulation of insoluble protein aggregates in the brain.     

The Nitric Oxide Prodrug JS‐K Induces Ca2+‐Mediated Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells

Hepatocellular carcinoma is one of the most common and deadly forms of human malignancies. JS‐K, O²‐(2, 4‐dinitrophenyl) 1‐ [(4‐ethoxycarbonyl) piperazin‐1‐yl] diazen‐1‐ium‐1, 2‐diolate, has the ability to induce apoptosis of tumor cell lines. In the present study, JS‐K inhibited the proliferation of HepG2 cells in a time‐ and concentration‐dependent manner and significantly induced apoptosis. JS‐K enhanced the ratio of Bax‐to‐Bcl‐2, released of cytochrome c (Cyt c) from mitochondria and the activated caspase‐9/3. JS‐K caused an increasing cytosolic Ca²⁺ and the loss of mitochondrial membrane potential. Carboxy‐PTIO (a NO scavenger) and BAPTA‐AM (an intracellular Ca²⁺ chelator) significantly blocked an increasing cytosolic Ca²⁺ in JS‐K‐induced HepG2 cells apoptosis, especially Carboxy‐PTIO. Meanwhile, Carboxy‐PTIO and BAPTA‐AM treatment both attenuate JS‐K‐induced apoptosis through upregulation of Bcl‐2, downregulation of Bax, reduction of Cyt c release from mitochondria to cytoplasm and inactivation of caspase‐9/3. In summary, JS‐K induced HepG2 cells apoptosis via Ca²⁺/caspase‐3‐mediated mitochondrial pathway.     

Musca Domestica Larva Lectin Induces Apoptosis in BEL-7402 Cells Through a Ca2+/JNK-mediated Mitochondrial Pathway

Although Musca domestica larvae lectin (MLL) is able to inhibit cancer cell proliferation and to induce cancer cell apoptosis, the molecular mechanism(s) responsible for these processes remain elusive. In the current study, the signaling network underlying the MLL-induced apoptosis of human hepatoma BEL-7402 cell was investigated. Our data found out that MLL causes a sustained increase of the intracellular Ca²⁺ and this process was prevented by the intracellular calcium chelator, BAPTA-AM, suggesting the involvement of intracellular Ca²⁺ in MLL-induced cell apoptosis. MLL also causes the production of reactive oxygen species and elevates the phosphorylation status of JNK, processes associated with the increased cytoplasmic Ca²⁺. The mitochondrial permeability transition pore (MPTP) opening study showed that MLL treatment of BEL-7402 cells results in the opening of MPTP and a reduction of mitochondrial transmembrane potential. In such condition, cytochrome-c was detected to be released from mitochondria to cytoplasm through the MPTP. This eventually activates caspase-3 and thus results in apoptosis of the tested BEL-7402 cells. According to a comprehensive review of all the evidence, it is concluded that MLL induces apoptosis of BEL-7402 cells through a Ca²⁺/JNK-mediated MPTP pathway.     

Role of cardiolipin peroxidation and Ca in mitochondrial dysfunction and disease

Cardiolipin is a unique phospholipid which is almost exclusively located at the level of the inner mitochondrial membrane where it is biosynthesized. This phospholipid is known to be intimately involved in several mitochondrial bioenergetic processes. In addition, cardiolipin also has active roles in several of the mitochondrial-dependent steps of apoptosis and in mitochondrial membrane dynamics. Alterations in cardiolipin structure, content and acyl chains composition have been associated with mitochondrial dysfunction in multiple tissues in several physiopathological conditions, including ischemia/reperfusion, different thyroid states, diabetes, aging and heart failure. Cardiolipin is particularly susceptible to ROS attack due to its high content of unsaturated fatty acids. Oxidative damage to cardiolipin would negatively impact the biochemical function of the mitochondrial membranes altering membrane fluidity, ion permeability, structure and function of components of the mitochondrial electron transport chain, resulting in reduced mitochondrial oxidative phosphorylation efficiency and apoptosis. Diseases in which mitochondrial dysfunction has been linked to cardiolipin peroxidation are described. Ca²⁺, particularly at high concentrations, appears to have several negative effects on mitochondrial function, some of these effects being linked to CL peroxidation. Cardiolipin peroxidation has been shown to participate, together with Ca²⁺, in mitochondrial permeability transition. In this review, we provide an overview of the role of CL peroxidation and Ca²⁺ in mitochondrial dysfunction and disease.     

Ion transporters and ischemic mitochondrial dysfunction

Ischemia-induced ionic imbalance leads to the activation of numerous events including mitochondrial dysfunction and eventual cell death. Dysregulation of mitochondrial Ca²⁺ (Ca²⁺_m) plays a critical role in cell damage under pathological conditions including traumatic brain injury and stroke. High Ca²⁺_m levels can induce the persistent opening of the mitochondrial permeability transition pore and trigger mitochondrial membrane depolarization, Ca²⁺ release, cessation of oxidative phosphorylation, matrix swelling and eventually outer membrane rupture with release of cytochrome c and other apoptogenic proteins. Thus, the dysregulation of mitochondrial Ca²⁺ homeostasis is now recognized to play a crucial role in triggering mitochondrial dysfunction and subsequent apoptosis. Recent studies show that some secondary active transport proteins, such as Na⁺-dependent chloride transporter and Na⁺/Ca²⁺ exchanger, contribute to ischemia-induced dissipation of ion homeostasis including Ca²⁺_m.Key words: ischemia, intracellular Ca²⁺ dysregulation, changes of mitochondrial Ca²⁺, cytochrome c, apoptosis     

Spatiotemporal activation of caspase‐dependent and ‐independent pathways in staurosporine‐induced apoptosis of p53wt and p53mt human cervical carcinoma cells

Background information. Caspase‐dependent and ‐independent death mechanisms are involved in apoptosis in a variety of human carcinoma cells treated with antineoplastic compounds. Our laboratory has reported that p53 is a key contributor of mitochondrial apoptosis in cervical carcinoma cells after staurosporine exposure. However, higher mitochondrial membrane potential dissipation and greater DNA fragmentation were observed in p53^wt (wild‐type p53) HeLa cells compared with p53^mt (mutated p53) C‐33A cells. Here, we have studied events linked to the mitochondrial apoptotic pathway. Results. Staurosporine can induce death of HeLa cells via a cytochrome c/caspase‐9/caspase‐3 mitochondrial‐dependent apoptotic pathway and via a delayed caspase‐independent pathway. In contrast with p53^wt cells, p53^mt C‐33A cells exhibit firstly caspase‐8 activation leading to caspase‐3 activation and Bid cleavage followed by cytochrome c release. Attenuation of PARP‐1 [poly(ADP‐ribose) polymerase‐1] cleavage as well as oligonucleosomal DNA fragmentation in the presence of z‐VAD‐fmk points toward a major involvement of a caspase‐dependent pathway in staurosporine‐induced apoptosis in p53^wt HeLa cells, which is not the case in p53^mt C‐33A cells. Meanwhile, the use of 3‐aminobenzamide, a PARP‐1 inhibitor known to prevent AIF (apoptosis‐inducing factor) release, significantly decreases staurosporine‐induced death in these p53^mt carcinoma cells, suggesting a preferential implication of caspase‐independent apoptosis. On the other hand, we show that p53, whose activity is modulated by pifithrin‐α, isolated as a suppressor of p53‐mediated transactivation, or by PRIMA‐1 (p53 reactivation and induction of massive apoptosis), that reactivates mutant p53, causes cytochrome c release as well as mitochondrio—nuclear AIF translocation in staurosporine‐induced apoptosis of cervical carcinoma cells. Conclusions. The present paper highlights that staurosporine engages the intrinsic mitochondrial apoptotic pathway via caspase‐8 or caspase‐9 signalling cascades and via caspase‐independent cell death, as well as through p53 activity.     

Permeability transition pore of the inner mitochondrial membrane can operate in two open states with different selectivities

Prooxidants induce release of Ca²⁺ from mitochondria through the giant solute pore in the mitochondrial inner membrane. However, under appropriate conditions prooxidants can induce Ca²⁺ release without inducing a nonspecific permeability change. Prooxidant-induced release of Ca²⁺ isselective. Presumably, this is the result of the operation of a permeability pathway for H⁺ coupled to the reversal of the Ca²⁺ uniporter, the latter generating the selectivity. The solute pore and prooxidant-induced Ca²⁺-specific pathways exhibit common sensitivities to a set of inhibitors and activators. It is proposed that the pore can operate in two open states: (1) permeable to H⁺ only and (2) permeable to solutes of M_r<1500. Under some conditions, prooxidants induce the H⁺-selective state which, in turn, collapses the inner membrane potential and permits selective loss of Ca²⁺ via the Ca²⁺ uniporter.     

Development of antithrombotic miniribozymes that target peripheral tryptophan hydroxylase

Transient receptor potential (TRP) proteins have been identified as cation channels that are activated by agonist–receptor coupling and mediate various cellular functions. TRPC7, a homologue of TRP channels, has been shown to act as a Ca²⁺ channel activated by G protein-coupled stimulation and to be abundantly expressed in the heart with an as-yet-unknown function. We studied the role of TRPC7 in G protein-activated signaling in HEK293 cells and cultured cardiomyocytes in vitro transfected with FLAG-tagged TRPC7 cDNA and in Dahl salt-sensitive rats with heart failure in vivo. TRPC7-transfected HEK293 cells showed an augmentation of carbachol-induced intracellular Ca²⁺ transient, which was attenuated under a Ca²⁺-free condition or in the presence of SK&F96365 (a Ca²⁺-permeable channel blocker). Upon stimulation with angiotensin II (Ang II), cultured neonatal rat cardiomyocytes transfected with TRPC7 exhibited a significant increase in apoptosis detected by TUNEL staining, accompanied with a decrease in the expression of atrial natriuretic factor and destruction of actin fibers, as compared with non-transfected cardiomyocytes. Ang II-induced apoptosis was inhibited by CV-11974 (Candesartan; Ang II type 1 [AT1] receptor blocker), SK&F96365, and FK506 (calcineurin inhibitor). In Dahl salt-sensitive rats, apoptosis and TRPC7 expression were increased in the failing myocardium, and a long-term treatment with temocapril, an angiotensin-converting enzyme inhibitor, suppressed both. Our findings suggest that TRPC7 could act as a Ca²⁺ channel activated by AT1 receptors, leading to myocardial apoptosis possibly via a calcineurin-dependent pathway. TRPC7 might be a key initiator linking AT1-activation to myocardial apoptosis, and thereby contributing to the process of heart failure.