Puerarin attenuates cisplatin-induced apoptosis of hair cells through the mitochondrial apoptotic pathway

Puerarin, one of the main components of Pueraria lobata, has been reported to possess a wide range of pharmacological activities, including anti-inflammatory, antioxidative and anti-apoptotic effects. However, the role of puerarin in ototoxic drug-induced hair cell injury has not been well characterized. This study explored whether puerarin protects against cisplatin-induced hair cell damage and its potential mechanisms. The viability of puerarin-treated HEI-OC1 cells was assessed by CCK8 assay. Reactive oxygen species (ROS) was estimated with flow cytometric analysis using Cellrox Green fluorescent probe. Apoptosis-related protein levels were detected by western blot analysis. Immunostaining of the organ of Corti was performed to determine mice cochlear hair cell survival. Our results showed that puerarin improved cell viability and suppressed apoptosis in the cisplatin-damaged HEI-OC1 cells and cochlear hair cells. Mechanistic studies revealed that puerarin attenuated mitochondrial apoptosis pathway by regulating apoptotic related proteins, such as Bax and cleaved caspase-3, and attenuated ROS accumulation after cisplatin damage. Moreover, puerarin was involved in regulating the Akt pathway in HEI-OC1 cells in response to cisplatin. Our results demonstrated that puerarin administration decreased the sensitivity to apoptosis dependent on the mitochondrial apoptotic pathway by reducing ROS generation, which could be used as a new protective agent against cisplatin-induced ototoxicity.     

TIEG1 induces apoptosis through mitochondrial apoptotic pathway and promotes apoptosis induced by homoharringtonine and velcade

Overexpression of TGFbeta inducible early gene (TIEG1) mimics TGFbeta action and induces apoptosis. In this study, we found that TIEG1 was significantly up-regulated during apoptosis induced by homoharringtonine or velcade. Overexpression of TIEG1 could induce apoptosis in K562 cells and promote apoptosis induced by HHT or velcade. TIEG1-induced apoptosis was shown to involve Bax and Bim up-regulation, Bcl-2 and Bcl-XL down-regulation, release of cytochrome c from mitochondria into the cytosol, activation of caspase 3 and disruption of the mitochondrial membrane potential (DeltaPsim). We concluded that TIEG1 is a key regulator which induces and promotes apoptosis through the mitochondrial apoptotic pathway.     

Shikonin selectively induces apoptosis in human prostate cancer cells through the endoplasmic reticulum stress and mitochondrial apoptotic pathway

Background

Despite the recent progress in screening and therapy, a majority of prostate cancer cases eventually attain hormone refractory and chemo-resistant attributes. Conventional chemotherapeutic strategies are effective at very high doses for only palliative management of these prostate cancers. Therefore chemo-sensitization of prostate cancer cells could be a promising strategy for increasing efficacy of the conventional chemotherapeutic agents in prostate cancer patients. Recent studies have indicated that the chemo-preventive natural agents restore the pro-apoptotic protein expression and induce endoplasmic reticulum stress (ER stress) leading to the inhibition of cellular proliferation and activation of the mitochondrial apoptosis in prostate cancer cells. Therefore reprogramming ER stress-mitochondrial dependent apoptosis could be a potential approach for management of hormone refractory chemoresistant prostate cancers. We aimed to study the effects of the natural naphthoquinone Shikonin in human prostate cancer cells.

Results

The results indicated that Shikonin induces apoptosis in prostate cancer cells through the dual induction of the endoplasmic reticulum stress and mitochondrial dysfunction. Shikonin induced ROS generation and activated ER stress and calpain activity. Moreover, addition of antioxidants attenuated these effects. Shikonin also induced the mitochondrial apoptotic pathway mediated through the enhanced expression of the pro-apoptotic Bax and inhibition of Bcl-2, disruption of the mitochondrial membrane potential (MMP) followed by the activation of caspase-9, caspase-3, and PARP cleavage.

Conclusion

The results suggest that shikonin could be useful in the therapeutic management of hormone refractory prostate cancers due to its modulation of the pro-apoptotic ER stress and mitochondrial apoptotic pathways.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0127-1) contains supplementary material, which is available to authorized users.     

Exosomes from mesenchymal stromal cells enhance imatinib-induced apoptosis in human leukemia cells via activation of caspase signaling pathway

Background aims

Imatinib (IM), a tyrosine kinase inhibitor targeting the BCR-ABL oncoprotein, remains a major therapeutic strategy for patients with chronic myelogenous leukemia (CML). However, IM resistance is still a challenge in the treatment of CML. Recently, it was reported that exosomes (Exo) were involved in drug resistance. Therefore, the present study investigated whether Exo secreted by human umbilical cord mesenchymal stromal cells (hUC-MSC-Exo) affected the sensitivity of K562 cells to IM.

Complestatin prevents apoptotic cell death: inhibition of a mitochondrial caspase pathway through AKT/PKB activation

Complestatin, a bicyclo hexapeptide from Streptomyces, was isolated as a possible regulator of neuronal cell death. In this study, we report an anti-apoptotic activity of complestatin and its underlying molecular mechanism. Complestatin blocked TRAIL (TNF-related apoptosis-inducing ligand)-induced apoptosis and activation of caspase-3 and -8 at micromolar concentration levels without inhibiting the catalytic activities of these caspases. Complestatin potently induced a rapid and sustained AKT/PKB activation and Bad phosphorylation, resulting in inhibition of mitochondrial cytochrome c release. These anti-apoptotic activities of complestatin were significantly abrogated in cells expressing dominant negative AKT/PKB. Taken together, our results suggest that complestatin prevents apoptotic cell death via AKT/PKB-dependent inhibition of the mitochondrial apoptosis signal pathway. The novel property of complestatin may be valuable for developing new pharmaceutical means that will control unwanted cell death.     

Canstatin induces apoptosis in gastric cancer xenograft growth in mice through the mitochondrial apoptotic pathway

Canstatin, the non-collagenous domain of collagen type IV α-chains, belongs to a series of collagen-derived angiogenic inhibitors. In this study, the inhibitory effect of recombinant canstatin on tumour growth was investigated using a gastric cancer xenograft model. The volume and weight of tumours in mice treated with canstatin were lower than that in mice treated with PBS. Accordingly, the survival rate of these mice was significantly higher than that of mice bearing tumours treated with PBS. Moreover, valuable insight into the mechanisms mediated by canstatin was obtained.     

Vitamin A treatment induces apoptosis through an oxidant-dependent activation of the mitochondrial pathway

Even though retinoids are widely used as adjuvant in chemotherapeutic interventions to improve cancer cell death, their mechanism(s) of action involves multiple overlapping pathways that remain unclear. We have previously shown that vitamin A, the natural precursor of the retinoids, induces oxidative-dependent cytochrome c release from isolated mitochondria, suggesting a putative mechanism for apoptosis activation. Using Sertoli cells in culture, we show that retinol causes mitochondrial-dependent apoptosis, involving oxidative stress. Apoptosis was evaluated by nuclear morphology, DNA fragmentation, and caspase-3/7 activity. Retinol induced oxidant- and time-dependent imbalance of several mitochondrial parameters, cytochrome c release and caspase-3/7 activation, leading cells to commit apoptosis. All parameters tested were attenuated or blocked by trolox co-administration, suggesting that retinol induces apoptosis through oxidative damage, which mitochondria plays a pivotal role.     

Hydrogen peroxide induces apoptosis in HeLa cells through mitochondrial pathway

Cervical cancer is the most common cancer amongst females in India and is associated with high risk HPVs, reactive oxygen species (ROS), and excessive inflammation in most cases. ROS in turn affects the expression of pro- and anti-apoptotic proteins. The objective of the present study was to elucidate the effect of hydrogen peroxide (H(2)O(2)) on apoptotic signaling molecules in vitro. HeLa cell line expresses the Human papilloma virus - 18, E6 oncoprotein which causes the ubiquitin mediated degradation of p53 protein and is thus p53 deficient. p53 is known to act as a cellular stress sensor and triggers apoptosis. p73, a member of the p53 family also induces apoptosis in response to DNA damaging agents but unlike p53, it is infrequently mutated in human tumors. We demonstrate here, that in HeLa cells, apoptosis is triggered by H(2)O(2) via the mitochondrial pathway involving upregulation of p73, and its downstream target Bax. This was accompanied by upregulation of ERK, JNK, c-Myc, Hsp-70 and down regulation of anti-apoptotic Bcl-XL, release of cytochrome c from mitochondria and activation of caspases-9 and -3.     

Bilobalide prevents apoptosis through activation of the PI3K/Akt pathway in SH-SY5Y cells

Bilobalide, a sesquiterpene trilactone constituent of Ginkgo biloba leaf extracts, has been proposed to exert protective and trophic effects on neurons. However, mechanisms underlying the protective effects of bilobalide remain unclear. Using human SH-SY5Y neuroblastoma cells and primary hippocampal neurons, this study investigated the neuroprotective effects of bilobalide. We mimicked aging-associated neuronal impairments by applying external factors (beta amyloid protein (Aβ) 1-42, H₂O₂ and serum deprivation) consequently inducing cell apoptosis. As markers for apoptosis, cell viability, DNA fragmentation, mitochondrial membrane potential and levels of cleaved caspase 3 were measured. We found that, bilobalide prevented Aβ 1-42-, H₂O₂- and serum deprivation-induced apoptosis. To better understand the neuroprotective effects of bilobalide, we also tested the ability of bilobalide to modulate pro-survival signaling pathways such as protein kinase C (PKC), extracellular-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. It was found that, bilobalide dose-dependently increased PI3K activity and levels of phosphorylated Akt (p-Akt Ser473 and Thr308), which could be maintained up to at least 2 h after bilobalide withdrawal in cells treated with or without Aβ 1-42, H₂O₂ or serum-free medium. In addition, application of PI3K/Akt inhibitor LY294002 could abrogate both the protective effects of bilobalide against Aβ 1-42-, H₂O₂- and serum deprivation-induced apoptotic cell damage and bilobalide-induced increase in PI3K activity and levels of p-Akt (Ser473 and Thr308). In contrast, application of PKC inhibitor staurosporine (STS) did not affect the protective effects of bilobalide. Moreover, no change in levels of phosphorylated ERK1/2 (p-ERK1/2) was observed in bilobalide-treated cells. These results further suggested that the PI3K/Akt pathway might be involved in the protective effects of bilobalide. Since modern technology allows production of purified bilobalide with high bioavailability, bilobalide may be useful in developing therapy for diseases involving age-associated neurodegeneration.     

Baculoviral p35 inhibits oxidant-induced activation of mitochondrial apoptotic pathway

In this study we report that the baculovirus p35 anti-apoptotic protein prevents cell death by quenching free radicals at a very upstream step in the apoptotic pathway. Mitochondria of activated rat peritoneal macrophages as well as Spodoptera frugiperda (Sf9) insect cells, following treatment with oxidants, H(2)O(2)/UVB irradiation, release cytochrome c followed by activation of caspase-3. Transfection of macrophages/Sf9 cells with a construct carrying the p35 gene under the CMV/HSP promoters resulted in p35 expression and consequent arrest of oxidative stress-induced apoptosis. p35 expression also inhibited cytochrome c release from the mitochondria of oxidant-exposed cells and blocked caspase-3 activation.     

Induction of apoptosis by Legionella pneumophila in mammalian cells requires the mitochondrial pathway for caspase activation

Legionella pneumophila, the agent of human Legionnaire's disease is a Gram-negative, rod-shaped bacterium. During infection, the bacteria invade human cells and replicate intracellularly. L. pneumophila can induce apoptosis in human myeloid and epitheloid cells and this may contribute to the development of pathology and disease. However, the molecular mechanism of apoptosis induction is still uncertain. Here we investigate this process. Legionella efficiently induced apoptosis in myeloid cells, T cells and fibroblasts. Induction of apoptosis involved activation of the initiator caspase-9 and effector caspases. Caspase activity was required for cell death. Analysis of mutant cells showed that the death receptor pathway was not involved in Legionella-induced apoptosis. Surprisingly, caspase activity was found almost exclusively in cells that did not harbor bacteria. Infection with Legionella caused the activation of the pro-apoptotic protein Bax and the release of cytochrome c. Mouse embryonic fibroblasts deficient for Bax and/or Bak were protected from Legionella-induced caspase activation. These results show a clear contribution of the mitochondrial pathway to Legionella-induced apoptosis.     

N-methyl-D-aspartate blocks activation of JNK and mitochondrial apoptotic pathway induced by potassium deprivation in cerebellar granule cells

During the postnatal development of cerebellum, lack of excitatory innervation from the mossy fibers results in cerebellar granule cell (CGC) apoptosis during the migration of the cells toward the internal granule cell layer. Accordingly, CGCs die by apoptosis when cultured in physiological KCl concentrations (5 mm; K5), and they survive in the presence of depolarizing conditions such as high KCl concentration (25 mm; K25) or N-methyl-D-aspartate (NMDA). We have recently shown that NMDA is able to exert a long lasting neuroprotective effect when added to immature (2 days in vitro) CGC cultures by inhibition of caspase-3 activity. Here we show that NMDA- and K25-mediated neuroprotection is associated with an increase in the levels of Bcl-2, an inhibition of K5-mediated increase in Bax, and the inhibition of the release of apoptogenic factors from mitochondria such as Smac/DIABLO and cytochrome c. Moreover, we have shown that similar effects are observed when c-Jun N-terminal kinases (JNKs) are inhibited and that treatment of CGC cultures with NMDA blocks K5-mediated JNK activation. These results allow us to postulate that the inhibition of JNK-mediated release of apoptogenic factors from mitochondria is involved in the NMDA protection from K5-mediated apoptosis of CGCs.     

MicroRNA-15b enhances hypoxia/reoxygenation-induced apoptosis of cardiomyocytes via a mitochondrial apoptotic pathway

Myocardial ischemia reperfusion (I/R) can induce altered expression of microRNAs (miRNAs). The miRNAs—miR-15a, miR-15b and miR-16 have been shown to play a role in apoptosis, although not in cardiac-related models. We investigated the roles of miR-15b in hypoxia/reoxygenation (H/R)-induced apoptosis of cardiomyocytes. Quantitative real time polymerase chain reaction results showed that the expression of miR-15a and miR-15b were up-regulated in Sprague–Dawley rat hearts subjected to I/R. Expression levels of miR-15b increased more than four fold above basal levels. Similar results were obtained for cardiomyocytes exposed to H/R. Recombinant adenoviral vectors were generated to explore the functional role of miR-15b in cultured cardiomyocytes exposed to H/R. Overexpression of miR-15b enhanced cell apoptosis and the loss of mitochondrial membrane potential, as determined by flow cytometric analysis. Conversely, down-regulated expression was cytoprotective. The effects of miR-15b can by mimicked by Bcl-2 short-interfering RNAs. The inhibition of miR-15b increased expression levels of the Bcl-2 protein without affecting Bcl-2 mRNA levels, suppressed the release of mitochondrial cytochrome c to the cytosol and decreased the activities of caspase-3 and 9. It is possible that miR-15b is the upstream regulator of a mitochondrial signaling pathway for H/R induced apoptosis.     

Endotoxin induces luteal cell apoptosis through the mitochondrial pathway

The effect of endotoxin (lipopolysacharide, LPS) exposure on luteal cells was studied using an in vitro cell culture system. Buffalo luteal cells were isolated from corpora lutea of the late luteal phase (days 14-16 post estrus) and exposed to various LPS doses (5, 10 and 100 microg/ml) for different time periods (6, 12, 18 or 24 h). The cultured cells were subsequently evaluated for oxidative stress (super oxide, nitric oxide, inducible nitric oxide synthase activity, reduced glutathione depletion and lipid peroxidation) and apoptotic markers (mitochondrial membrane potential, DNA fragmentation, apoptotic cells and cell viability). LPS exposure significantly increased the production of super oxide (P<0.05) and nitric oxide (P<0.01) and increased inducible nitric oxide synthase activity (P<0.01). LPS exposure further depleted reduced glutathione (P<0.05) levels and induced lipid peroxidation (P<0.05). LPS exposure also induced the loss of mitochondrial membrane potential (P<0.05), increased DNA fragmentation (P<0.01) and apoptosis (P<0.01) and decreased cell viability (P<0.01). LPS mediated apoptotic pathway in luteal cells was further characterized using a selected LPS dose (10 microg/ml). It was observed that LPS exposure induced mitochondrial translocation of proapoptotic protein Bax, increased the total Bad expression and down regulated the expression of antiapoptotic proteins Bcl2 and BclXL. LPS exposure further induced cytochrome c release and increased Caspase-9 (P<0.01) and Caspase-3 (P<0.01) activities. LPS exposure also inhibited luteal progesterone secretion (P<0.01). It was evident that the LPS mediated apoptotic effects could be prevented by the coincubation of luteal cells with mitochondrial permeability transition pore blocker Cyclosporine A, inducible nitric oxide synthase inhibitor N-[3-(aminomethyl)benzyl]acetamidine and oxidative stress scavenger N-acetyl cysteine. Our study clearly indicates that LPS induces oxidative stress mediated apoptosis in luteal cells through the mitochondrial pathway.     

Inhibition of proteasomal function by curcumin induces apoptosis through mitochondrial pathway

Curcumin is a natural polyphenolic compound having an antiproliferative property, which recent evidence suggests is due to its ability to induce apoptosis. However, the molecular mechanisms through which curcumin induces apoptosis are not fully understood. Here, we report that the curcumin-induced apoptosis is mediated through the impairment of the ubiquitin-proteasome system. Exposure of curcumin to the mouse neuro 2a cells causes a dose-dependent decrease in proteasome activity and an increase in ubiquitinated proteins. Curcumin exposure also decreases the turnover of the destabilized enhanced green fluorescence protein, a model substrate for proteasome and cellular p53 protein. Like other proteasome inhibitors, curcumin targets proliferative cells more efficiently than differentiated cells and induces apoptosis via mitochondrial pathways. Addition of curcumin to neuro 2a cells induces a rapid decrease in mitochondrial membrane potential and the release of cytochrome c into cytosol, followed by activation of caspase-9 and caspase-3.     

Cell death via mitochondrial apoptotic pathway due to activation of Bax by lysosomal photodamage

Lysosomal photosensitizers have been used in photodynamic therapy. The combination of such photosensitizers and light causes lysosomal photodamage, inducing cell death. Lysosomal disruption can lead to apoptosis but its signaling pathways remain to be elucidated. In this study, N-aspartyl chlorin e6 (NPe6), an effective photosensitizer that preferentially accumulates in lysosomes, was used to study the mechanism of apoptosis caused by lysosomal photodamage. Apoptosis in living human lung adenocarcinoma cells (ASTC-a-1) after NPe6-photodynamic treatment (NPe6-PDT) was studied using real-time single-cell analysis. Our results demonstrated that NPe6-PDT induced rapid generation of reactive oxygen species (ROS). The photodynamically produced ROS caused a rapid destruction of lysosomes, leading to release of cathepsins, and the ROS scavengers vitamin C and NAC prevent the effects. Then the following spatiotemporal sequence of cellular events was observed during cell apoptosis: Bcl-2-associated X protein (Bax) activation, cytochrome c release, and caspase-9/-3 activation. Importantly, the activation of Bax proved to be a crucial event in this apoptotic machinery, because suppressing the endogenous Bax using siRNA could significantly inhibit cytochrome c release and caspase-9/-3 activation and protect the cell from death. In conclusion, this study demonstrates that PDT with lysosomal photosensitizer induces Bax activation and subsequently initiates the mitochondrial apoptotic pathway.     

Methylseleninic acid induces apoptosis of human bladder cancer cells through the ROS-mediated mitochondrial pathway

As the most common selenium derivative, methylseleninic acid (MSA) has attracted wide attention. Its apoptotic induction ability and the possible molecular mechanism in human bladder cancer (BC) J82 and T24 cells were investigated in the present study. We found that the survival of J82 and T24 cells were inhibited in a dose-dependent manner after MSA treatment. Propidium iodide (PI) staining and Annexin V-fluorescein isothiocyanate/PI double staining clarified that MSA stocked cells at G₂/M phase and caused apoptosis in J82 and T24 cells. Further, typical morphological features of apoptotic cells were also observed. Accumulation of reactive oxygen species (ROS) and loss of mitochondrial membrane potential were also detected by dichlorodihydrofluorescein diacetate and Rhodamin123 staining. Meanwhile, pretreatment with N-acetylcysteine, an ROS scavenging agent, found that the apoptosis of BC cells induced by MSA was related to the production of ROS. Western blot analysis results showed that MSA interrupted Bax/Bcl-2 balance, stimulated cytochrome c release into the cytoplasm, activated caspase-9 and caspase-3, and finally induced the apoptosis of the BC cells. These findings demonstrated that MSA was able to induce apoptosis in J82 and T24 cells through ROS-mediated mitochondrial apoptosis.