Release Of Mitochondrial Cytochrome Research Articles

The effects of restraint stress on ceramide metabolism disorders in the rat liver: the role of CerS6 in hepatocyte injury

BackgroundStress is implicated in various pathological conditions leading to liver injury. Existing evidence suggests that excessive stress can induce mitochondrial damage in hepatocytes, yet the underlying mechanism remains unclear. Ceramide synthase 6 (CerS6)-derived C16:0 ceramide is recognised as a lipotoxic substance capable of causing mitochondrial damage. However, the role of CerS6 in stress has received insufficient attention. This study aimed to explore the involvement of CerS6 in stress-induced hepatic damage and its associated mechanisms.MethodsThe rat restraint stress model and a corticosterone (CORT)-induced hepatocyte stress model were employed for in vivo and in vitro experimental analyses, respectively. Changes in mitochondrial damage and ceramide metabolism in hepatocytes induced by stress were evaluated. The impact of CORT on mitochondrial damage and ceramide metabolism in hepatocytes was assessed following CerS6 knockdown. Mitochondria were isolated using a commercial kit, and ceramides in liver tissue and hepatocytes were detected by LC–MS/MS.ResultsIn comparison to the control group, rats subjected to one week of restraint exhibited elevated serum CORT levels. The liver displayed significant signs of mitochondrial damage, accompanied by increased CerS6 and mitochondrial C16:0 ceramide, along with activation of the AMPK/p38 MAPK pathway. In vitro studies demonstrated that CORT treatment of hepatocytes resulted in mitochondrial damage, concomitant with elevated CerS6 and mitochondrial C16:0 ceramide. Furthermore, CORT induced sequential phosphorylation of AMPK and p38 MAPK proteins, and inhibition of the p38 MAPK pathway using SB203580 mitigated the CORT-induced elevation in CerS6 protein. Knocking down CerS6 in hepatocytes inhibited both the increase in C16:0 ceramide and the release of mitochondrial cytochrome c induced by CORT.ConclusionsCerS6-associated C16:0 ceramide plays a mediating role in stress-induced mitochondrial damage in hepatocytes. The molecular mechanism is linked to CORT-induced activation of the AMPK/p38 MAPK pathway, leading to upregulated CerS6.

A Thia-Analogous Indirubin N-Glycoside Disrupts Mitochondrial Function and Causes the Death of Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells.

Skin cancer is the most common malignant disease worldwide and, therefore, also poses a challenge from a pharmacotherapeutic perspective. Derivatives of indirubin are an interesting option in this context. In the present study, the effects of 3-[3'-oxo-benzo[b]thiophen-2'-(Z)-ylidene]-1-(β-d-glucopyranosyl)-oxindole (KD87), a thia-analogous indirubin N-glycoside, on the viability and mitochondrial properties of melanoma (A375) and squamous cell carcinoma cells (A431) of the skin were investigated. In both cell lines, KD87 caused decreased viability, the activation of caspases-3 and -7, and the inhibition of colony formation. At the mitochondrial level, a concentration-dependent decrease in both the basal and ATP-linked oxygen consumption rate and in the reserve capacity of oxidative respiration were registered in the presence of KD87. These changes were accompanied by morphological alterations in the mitochondria, a release of mitochondrial cytochrome c into the cytosol and significant reductions in succinate dehydrogenase complex subunit B (SDHB, subunit of complex II) in A375 and A431 cells and NADH:ubiquinone oxidoreductase subunit B8 (NDUFB8, subunit of complex I) in A375 cells. The effect of KD87 was accompanied by a significant upregulation of the enzyme heme oxygenase-1, whose inhibition led to a partial but significant reduction in the metabolic-activity-reducing effect of KD87. In summary, our data show a mitochondria-targeting effect of KD87 as part of the cytotoxic effect of this compound on skin cancer cells, which should be considered in future studies with this class of compounds.

The BCL-2 family protein BCL-RAMBO interacts and cooperates with GRP75 to promote its apoptosis signaling pathway

The BCL-2 family protein BCL-RAMBO, also known as BCL2-like 13, anchors at the outer mitochondrial membrane and regulates apoptosis, mitochondrial fragmentation, and mitophagy. However, the mechanisms underlying the proapoptotic role of BCL-RAMBO remain unclear. In the present study, we demonstrated that BCL-RAMBO interacted with glucose-regulated protein 75 (GRP75), also known as heat shock protein family A member 9, and mortalin using co-immunoprecipitation and glutathione S-transferase-based pull-down assays. BCL-RAMBO interacted with GRP75 via its No BCL-2 homology domain. The interaction between BCL-RAMBO and GRP75 was confirmed by genetic interactions in Drosophila because a rough eye phenotype caused by the ectopic expression of BCL-RAMBO was partially suppressed by mutations in Hsc70-5, a mammalian GRP75 ortholog. In human embryonic kidney 293T cells, the co-expression of BCL-RAMBO and GRP75 facilitated an elevation in executioner caspase activity and poly (ADP-ribose) polymerase 1 (PARP-1) cleavage. In contrast, the knockdown of GRP75 suppressed elevated executioner caspase activity and PARP-1 cleavage in BCL-RAMBO-transfected cells. The mitochondrial release of cytochrome c induced by BCL-RAMBO was also attenuated by the knockdown of GRP75. These results indicate that GRP75 interacts with BCL-RAMBO and plays a crucial role in the BCL-RAMBO-dependent apoptosis signaling pathway.

Photobiomodulation Improves Anti-Tumor Efficacy of Photodynamic Therapy against Resistant MCF-7 Cancer Cells.

Cancer resistance is a primary concern in cancer treatment, and developing an effective modality or strategy to improve therapeutic outcomes is imperative. Photodynamic therapy (PDT) is a treatment modality that targets the tumor with a photoactive molecule and light for the specific destruction of cancer cells. Photobiomodulation (PBM) is a light exposure of cells to energize their biomolecules to respond to therapy. In the present study, we used PBM to mediate and improve the anti-tumor efficacy of zinc phthalocyanine tetrasulfonic acid (ZnPcS4)-PDT on resistant MCF-7 breast cancer cells and explore molecular changes associated with cell death. Different laser irradiation models were used for PBM and PDT combination. The combined treatment demonstrated an additive effect on the viability and Annexin-V/PI-staining cell death assessed through MTT assay and mitochondrial release of cytochrome c. Rhodamine (Rh123) showed increased affinity to mitochondrial disruption of the strategic treatment with PBM and PDT. Results from the autophagy assay indicate an interplay between the mitochondrial and autophagic proteins. These findings were indicative that PBM might improve the anti-tumor of PDT by inducing autophagy in resistant MCF-7 breast cancer cells that evade apoptosis.

Temporal relationship of the orphan receptor TR3 translocation and expression with zinc-induced apoptosis in prostate cancer cells.

This study aimed to investigate the precise mechanism of zinc-induced apoptosis in human prostate cell lines PC-3 and LNCaP and explore its relationships with the translocation and expression of the orphan receptor TR3 and cytochrome c. The effects of zinc exposure on apoptosis levels were examined in both PC-3 and LNCaP cells. These cells were treated with exogenous ZnCl2 (100 µM) for 0, 4, 8, 12, and 24 h. Dansylaminoethyl-cyclen (DAEC) fluorescent probe was applied to visualize cellular zinc localization. Zinc-induced apoptosis was identified by Hoechst nuclear staining and flow cytometry analysis. TR3 messenger RNA (mRNA) expression levels were detected by real time-quantitative polymerase chain reaction (PCR). TR3 protein localization and mitochondrial release of cytochrome c were identified by immunofluorescence microscopy. Mitochondrial membrane potential collapse under fluorescence microscopy was examined with a MitoLight probe. Zinc exposure led to gradually increasing apoptosis levels in both LNCaP and PC-3 cells over the 24-h treatment period. The apoptotic effects could be observed as early as after 4 h of zinc treatment in PC-3 cells, with this being seen at 8 h in LNCaP cells. The apoptosis levels of both PC-3 and LNCaP cells began to increase significantly from 8 to 24 h when necrotic cells were also found. TR3 protein translocation from the nucleus to the mitochondria was noted and was accompanied by cytochrome c release into the cytosol from the mitochondria. Interestingly, no significant changes in TR3 mRNA expression levels were observed after zinc treatment. However, the mitochondrial membrane potential of both PC-3 and LNCaP cells gradually disappeared following extended zinc exposure. Zinc-induced apoptosis could be regulated through TR3 protein translocation from the nucleus to the mitochondria in prostate cancer cells without notable changes in TR3 mRNA levels. The direct effect of zinc on the mitochondria was associated with the release of cytochrome c into the cytosol through the mitochondrial targeting of TR3 protein.

The mycotoxin viriditoxin induces leukemia- and lymphoma-specific apoptosis by targeting mitochondrial metabolism

Inhibition of the mitochondrial metabolism offers a promising therapeutic approach for the treatment of cancer. Here, we identify the mycotoxin viriditoxin (VDT), derived from the endophytic fungus Cladosporium cladosporioides, as an interesting candidate for leukemia and lymphoma treatment. VDT displayed a high cytotoxic potential and rapid kinetics of caspase activation in Jurkat leukemia and Ramos lymphoma cells in contrast to solid tumor cells that were affected to a much lesser extent. Most remarkably, human hematopoietic stem and progenitor cells and peripheral blood mononuclear cells derived from healthy donors were profoundly resilient to VDT-induced cytotoxicity. Likewise, the colony-forming capacity was affected only at very high concentrations, which provides a therapeutic window for cancer treatment. Intriguingly, VDT could directly activate the mitochondrial apoptosis pathway in leukemia cells in the presence of antiapoptotic Bcl-2 proteins. The mitochondrial toxicity of VDT was further confirmed by inhibition of mitochondrial respiration, breakdown of the mitochondrial membrane potential (ΔΨm), the release of mitochondrial cytochrome c, generation of reactive oxygen species (ROS), processing of the dynamin-like GTPase OPA1 and subsequent fission of mitochondria. Thus, VDT-mediated targeting of mitochondrial oxidative phosphorylation (OXPHOS) might represent a promising therapeutic approach for the treatment of leukemia and lymphoma without affecting hematopoietic stem and progenitor cells.

Cyclooxygenase-2 activates the free radical-mediated apoptosis of polymorphonuclear leukocytes in the maneb- and paraquat-intoxicated rats

Overproduction of free radicals and inflammation could lead to maneb (MB)- and paraquat (PQ)-induced toxicity in the polymorphonuclear leukocytes (PMNs). Cyclooxygenase-2 (COX-2), an inducible COX, is imperative in the pesticides-induced pathological alterations. However, its role in MB- and PQ-induced toxicity in the PMNs is not yet clearly deciphered. The current study explored the contribution of COX-2 in MB- and PQ-induced toxicity in the PMNs and the mechanism involved therein. Combined MB and PQ augmented the production of free radicals, lipid peroxides and activity of superoxide dismutase (SOD) in the rat PMNs. While combined MB and PQ elevated the expression of COX-2 protein, activation of nuclear factor-kappa B (NF-κB) and phosphorylation of c-Jun N-terminal kinase (JNK), release of mitochondrial cytochrome c and levels of procaspase-3/9 were attenuated in the PMNs. Celecoxib (CXB), a COX-2 inhibitor, ameliorated the combined MB and PQ-induced modulations in the PMNs. MB and PQ augmented the free radical generation, COX-2 protein expression, NF-κB activation and JNK phosphorylation and reduced the cell viability of cultured rat PMNs and human leukemic HL60. MB and PQ elevated mitochondrial cytochrome c release and poly (ADP-ribose) polymerase cleavage whilst procaspase-3/9 levels were attenuated in the cultured PMNs. MB and PQ also increased the levels of phosphorylated c-jun and caspase-3 activity in the HL60 cells. CXB; SP600125, a JNK-inhibitor and pyrrolidine dithiocarbamate (PDTC), a NF-κB inhibitor, rescued from MB and PQ-induced changes in the PMNs and HL60 cells. However, CXB offered the maximum protection among the three. The results show that COX-2 activates apoptosis in the PMNs following MB and PQ intoxication, which could be linked to NF-κB and JNK signaling.

Calenduloside E alleviates cerebral ischemia/reperfusion injury by preserving mitochondrial function

Calenduloside E (CE) isolated from Aralia elata (Miq.) Seem. is a natural triterpenoid saponin that can reportedly ameliorate myocardial ischemia/reperfusion injury. However, its potential roles and mechanism in cerebral ischemia/reperfusion injury are barely understood. In this study, we established an oxygen-glucose deprivation/reoxygenation (OGD/R) model in HT22 cells. We found that CE significantly attenuated the OGD/R-induced inhibition of cell viability and apoptotic cell death in HT22 cells. Moreover, CE treatment significantly ameliorated OGD/R-induced mitochondrial fission by inhibiting mitochondrial dynamin-related protein 1 (Drp1) recruitment and increasing Drp1 phosphorylation at Ser637. CE treatment significantly ameliorated OGD/R-induced mitochondrial dysfunction by increasing the mitochondrial membrane potential and reducing the mitochondrial ROS and cellular calcium accumulation. Moreover, CE treatment significantly inhibited the OGD/R-induced release of mitochondrial Cytochrome C and increase in Bax, Cleaved-caspase3 and Cleaved-caspase9 protein levels, whereas CE treatment significantly reversed the OGD/R-induced decrease in Bcl-2 and full length of caspase3 and caspase9 protein levels. In vivo, we found that CE treatment significantly ameliorated ischemic/hypoxic-induced brain infarct volume, neurological deficits, and neuronal apoptosis in mice after middle cerebral artery occlusion and reperfusion. CE treatment also significantly ameliorated the mitochondrial transmembrane potential, decreased Cytochrome C release, and reversed the increase in Bax, Cleaved-caspase3 and Cleaved-caspase9 protein levels and the decrease in Bcl-2 and full length of caspase3 and caspase9 protein levels induced by cerebral ischemia/reperfusion (I/R). All these results indicated that CE treatment exerted a neuroprotective effect by ameliorating mitochondrial dysfunction during cerebral I/R injury.

Duck Tembusu virus infection induces mitochondrial-mediated and death receptor-mediated apoptosis in duck embryo fibroblasts

Duck Tembusu virus (DTMUV) is a pathogenic flavivirus that has caused enormous economic losses in Southeast Asia. Our previous study showed that DTMUV could induce duck embryo fibroblast (DEF) apoptosis, but the specific mechanism was not clear. In this study, we confirmed that DTMUV could induce the apoptosis of DEFs by DAPI staining and TUNEL staining. Furthermore, we found that the expression levels of cleaved-caspase-3/7/8/9 were significantly upregulated after DTMUV infection. After treatment of cells with an inhibitor of caspase-8 or caspase-9, DTMUV-induced apoptosis rates were significantly decreased, indicating that the caspase-8-mediated death receptor apoptotic pathway and caspase-9-mediated mitochondrial apoptotic pathway were involved in DTMUV-induced apoptosis. Moreover, we found that DTMUV infection not only caused the release of mitochondrial cytochrome C (Cyt C) and the downregulation of the apoptosis-inhibiting protein Bcl-2 but also reduced the mitochondrial membrane potential (MMP) and the accumulation of intracellular reactive oxygen species (ROS). Key genes in the mitochondrial apoptotic pathway and death receptor apoptotic pathway were upregulated to varying degrees, indicating the activation of the mitochondrial apoptosis pathway and death receptor apoptosis pathway. In conclusion, this study clarifies the molecular mechanism of DTMUV-induced apoptosis and provides a theoretical basis for revealing the pathogenic mechanism of DTMUV infection.

The Combination of Δ9-Tetrahydrocannabinol and Cannabidiol Suppresses Mitochondrial Respiration of Human Glioblastoma Cells via Downregulation of Specific Respiratory Chain Proteins.

Simple SummaryCannabidiol (CBD) is a phytocannabinoid from Cannabis sativa L. that exhibits no psychoactivity and, like the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC), shows anticancer effects in preclinical cell and animal models. Previous studies have indicated a stronger cancer-targeting effect when THC and CBD are combined. Here, we investigated how the combination of THC and CBD in a 1:1 ratio affects glioblastoma cell survival. The compounds were found to synergistically enhance cell death, which was attributed to mitochondrial damage and disruption of energy metabolism. A detailed look at the mitochondrial electron transfer chain showed that THC/CBD selectively decreased certain subunits of complexes I and IV. These data highlight the fundamental changes in cellular energy metabolism when cancer cells are exposed to a mixture of cannabinoids and underscore the potential of combining cannabinoids in cancer treatment.Phytocannabinoids represent a promising approach in glioblastoma therapy. Previous work has shown that a combined treatment of glioblastoma cells with submaximal effective concentrations of psychoactive Δ9-tetrahydrocannabinol (THC) and non-psychoactive cannabidiol (CBD) greatly increases cell death. In the present work, the glioblastoma cell lines U251MG and U138MG were used to investigate whether the combination of THC and CBD in a 1:1 ratio is associated with a disruption of cellular energy metabolism, and whether this is caused by affecting mitochondrial respiration. Here, the combined administration of THC and CBD (2.5 µM each) led to an inhibition of oxygen consumption rate and energy metabolism. These effects were accompanied by morphological changes to the mitochondria, a release of mitochondrial cytochrome c into the cytosol and a marked reduction in subunits of electron transport chain complexes I (NDUFA9, NDUFB8) and IV (COX2, COX4). Experiments with receptor antagonists and inhibitors showed that the degradation of NDUFA9 occurred independently of the activation of the cannabinoid receptors CB1, CB2 and TRPV1 and of usual degradation processes mediated via autophagy or the proteasomal system. In summary, the results describe a previously unknown mitochondria-targeting mechanism behind the toxic effect of THC and CBD on glioblastoma cells that should be considered in future cancer therapy, especially in combination strategies with other chemotherapeutics.

Structure-activity relationships reveal a 2-furoyloxychalcone as a potent cytotoxic and apoptosis inducer for human U-937 and HL-60 leukaemia cells

Synthetic flavonoids with new substitution patterns have attracted attention as potential anticancer drugs. Here, twelve chalcones were synthesized and their antiproliferative activities against five human tumour cells were evaluated. This series of chalcone derivatives was characterized by the presence of an additional aromatic or heterocyclic ring linked by an ether, in the case of a benzyl radical, or an ester or amide functional group in the case of a furoyl radical. In addition, the influence on cytotoxicity by the presence of one or three methoxy groups or a 2,4-dimethoxy-3-methyl system on the B ring of the chalcone scaffold was also explored. The results revealed that the most cytotoxic chalcones contain a furoyl substituent linked by an ester or an amide through the 2′-hydroxy or the 2′-amino group of the A ring of the chalcone skeleton, with IC50 values between 0.2 ± 0.1 μM and 1.3 ± 0.1 μM against human leukaemia cells. The synthetic chalcone 2′-furoyloxy-4-methoxychalcone (FMC) was, at least, ten-fold more potent than the antineoplastic agent etoposide against U-937 cells and displayed less cytotoxicity against human peripheral blood mononuclear cells. Treatment of U-937 and HL-60 cells with FMC induced cell cycle arrest at the G2-M phase, an increase in the percentage of sub-G1 and annexin-V positive cells, the release of mitochondrial cytochrome c, activation of caspase and poly(ADP-ribose) polymerase cleavage. In addition, it inhibited tubulin polymerization in vitro in a concentration dependent manner. Cell death triggered by this chalcone was decreased by the pan-caspase inhibitor z-VAD-fmk and was dependent of the generation of reactive oxygen species. We conclude that this furoyloxychalcone may be useful in the development of a potential anti-leukaemia strategy.

Echinacoside Induces Mitochondria-Mediated Pyroptosis through Raf/MEK/ERK Signaling in Non-Small Cell Lung Cancer Cells.

Background Various natural compounds are effective in cancer prevention and treatment with fewer side effects than conventional radiotherapy and chemotherapy. Considering the uncertainty of the antitumor mechanism of Echinacoside (Ech) and the fact that no study on Ech against non-small cell lung cancer (NSCLC) has been explored previously, this study inquired into the anti-NSCLC effect of Ech and explored its potential mechanisms. Methods The IC50 to Ech of the NSCLC cells was calculated based on a series of cell viability assays. Different concentrations of Ech were used to treat the cells; the proliferation activity of the cells was evaluated using EdU staining. Mitochondrial membrane potential was detected by JC-1 staining. Levels of cytokines IL-1β and IL-18 were measured by ELISA. GSH and MDA levels were measured by microplate reader. Expression of cytochrome c, NLRP3, caspase-1, IL-1β, c-Myc, c-Fos, and Raf/MEK/ERK pathway proteins was evaluated by western blot. Meanwhile, we used xenograft, immunohistochemical staining, and H&E staining to evaluate the pharmacological effects of Ech in mice in vivo. Results ECH inhibited the proliferation of NSCLC cells. Ech increased the expression of pyroptosis-related proteins. Besides, Ech perturbed the mitochondrial membrane potential with the release of mitochondrial cytochrome c, accompanied by increased oxidative stress. Ech inhibited the phosphorylation levels of Raf/MEK/ERK signaling pathway and subsequently reduced c-myc and c-fos protein expression. In addition, Ech effectively restrained the growth of tumors in vivo. Conclusions Ech inhibited the Raf/MEK/ERK signaling. Impaired mitochondria activated inflammasome, which in turn led to the pyroptosis of NSCLC cells. These findings can provide some ideas on how to use pyroptosis to treat NSCLC.

Alpha-Mangostin Activates MOAP-1 Tumor Suppressor and Mitochondrial Signaling in MCF-7 Human Breast Cancer Cells.

α-Mangostin, one of the major constituents of Garcinia mangostana, has been reported to possess several biological activities, including antioxidant, anti-inflammatory, antibacterial, and cytotoxic activities associated with the inhibition of cell proliferation and activation of apoptosis. However, the cellular signaling pathway mediated by α-mangostin has not been firmly established. To investigate the cellular activities of α-mangostin, human cancer cells, MCF-7 and MCF-7-CR cells, were treated with α-mangostin to measure the cellular responses, including cytotoxicity, protein-protein interaction, and protein expression. Cancer cells stably expressed Myc-BCL-XL and HA-MOAP-1 were also included in the studies to delineate the cell signaling events mediated by α-mangostin. Our results showed that the apoptosis signaling mediated by α-mangostin involves the upregulation of endogenous MOAP-1, which interacts with α-mangostin activated BAX (act-BAX) while downregulating the expression of BCL-XL. Moreover, α-mangostin was found to induce BAX oligomerization, the release of mitochondrial cytochrome C, and activation of caspase in MCF-7 cells. In overexpression studies, MCF-7 cells and spheroids stably expressed HA-MOAP-1 and Myc-BCL-XL exhibited differential chemosensitivity toward α-mangostin in which the stable clones expressing HA-MOAP-1 and MYC-BCL-XL were chemosensitive and chemoresistant to the apoptosis signaling events mediated by α-mangostin, respectively, when compared to untreated cells. Together, the data suggest that the cytotoxicity of α-mangostin involves the activation of MOAP-1 tumor suppressor and its interaction with act-BAX, leading to mitochondria dysfunction and cell death.

Wogonin reduces cardiomyocyte apoptosis from mitochondrial release of cytochrome c to improve doxorubicin-induced cardiotoxicity.

Doxorubicin (DOX) has powerful anticancer properties, but its clinical application is affected by its serious cardiotoxicity. Wogonin (WG) has been shown to have marked cardiovascular protection potential. However, it is not known whether this potential can protect the heart from DOX damage. The aim of the present study was to investigate whether WG could ameliorate the cardiotoxicity of DOX. DOX and WG were used to establish a model of cardiac damage. Echocardiography, brain natriuretic peptide, creatine kinase MB and cardiac troponin T were used to detect the degree of cardiac damage. The levels of superoxide dismutase, malondialdehyde, glutathione and catalase in serum were measured to observed oxidative stress state. The mRNA levels of cyclophilin D, voltage-dependent anion-selective channel 1 and adenine nucleotide transporter 1 were detected by reverse transcription-quantitative PCR. Western blotting was used to detect the expression of cytochrome c in mitochondria and cytoplasm and cleaved-caspase-9 and pro/cleaved-caspase-3 in cytoplasm in cardiac tissue and primary cardiomyocytes to verify the related signaling pathways. DOX rats showed a series of cardiac damage. However, these damages were alleviated following WG treatment. Further studies showed that WG antagonized DOX cardiotoxicity through inhibiting the release of cytochrome c. WG protected rat heart from DOX damage. The mechanism may be closely related to inhibiting the release of cytochrome c from mitochondria and reducing cardiomyocyte apoptosis caused by caspase activation.

The inhibitory effects of selenium nanoparticles modified by fructose-enriched polysaccharide from Codonopsis pilosula on HepG2 cells

Ethanol volume fraction of 80% was widely applied for polysaccharides precipitation from Codonopsis pilosula water-leach liquor, while few researches focused on the dissolved components in 80% alcohol solution. Selenium nanoparticles presented strong biological activities with low toxicity, but the poor stability limited the practical applications. This study isolated a C. pilosula polysaccharide from the supernate after 80% ethanol precipitation, which was used as surface decorator for nano-composites preparation, and then the structural performance, storage stability and inhibitory mechanism on HepG2 cells were investigated. Results showed that the C. pilosula polysaccharide was mainly composed of fructose and glucose (molar ratio of 2.68:1.00) with average molecular weight of 3.21 × 103 Da. Additionally, the nano-composites were successfully prepared under Na2SeO3 to polysaccharide (2.0 mg/mL) ratio (w:w) of 1:20, and exhibited homogeneous distribution with an average particle size of ~ 75 nm and superior stability at 4 °C for at least 35 days. Besides, the nano-composites effectively inhibited HepG2 cells proliferation dose-dependently, and induced the mitochondrial dysfunction via improving Bax expressions and descending Bcl-2 expressions, resulting in the release of mitochondrial cytochrome c to cytoplasm and the activation of caspase-9 / − 3, thereby promoting cells apoptosis. These results would effectively improve utilization rate of C. pilosula and provide theoretical foundation for clinical applications of the composite in hepatoma-bearing patients.

Mitochondrial Permeability Transition Causes Mitochondrial Reactive Oxygen Species- and Caspase 3-Dependent Atrophy of Single Adult Mouse Skeletal Muscle Fibers.

Elevated mitochondrial reactive oxygen species (mROS) and an increase in caspase-3 activity are established mechanisms that lead to skeletal muscle atrophy via the upregulation of protein degradation pathways. However, the mechanisms upstream of an increase in mROS and caspase-3 activity in conditions of muscle atrophy have not been identified. Based upon knowledge that an event known as mitochondrial permeability transition (MPT) causes an increase in mROS emission and the activation of caspase-3 via mitochondrial release of cytochrome c, as well as the circumstantial evidence for MPT in some muscle atrophy conditions, we tested MPT as a mechanism of atrophy. Briefly, treating cultured single mouse flexor digitorum brevis (FDB) fibers from adult mice with a chemical inducer of MPT (Bz423) for 24 h caused an increase in mROS and caspase-3 activity that was accompanied by a reduction in muscle fiber diameter that was able to be prevented by inhibitors of MPT, mROS, or caspase-3 (p < 0.05). Similarly, a four-day single fiber culture as a model of disuse caused atrophy that could be prevented by inhibitors of MPT, mROS, or activated caspase-3. As such, our results identify MPT as a novel mechanism of skeletal muscle atrophy that operates through mROS emission and caspase-3 activation.

Deoxynivalenol Induces Caspase-8-Mediated Apoptosis through the Mitochondrial Pathway in Hippocampal Nerve Cells of Piglet.

Deoxynivalenol (DON) is a common trichothecene mycotoxin found worldwide. DON has broad toxicity towards animals and humans. However, the mechanism of DON-induced neurotoxicity in vitro has not been fully understood. This study investigated the hypothesis that DON toxicity in neurons occurs via the mitochondrial apoptotic pathway. Using piglet hippocampal nerve cells (PHNCs), we evaluated the effects of different concentrations of DON on typical indicators of apoptosis. The obtained results demonstrated that DON treatment inhibited PHNC proliferation and led to morphological, biochemical, and transcriptional changes consistent with apoptosis, including decreased mitochondrial membrane potential, mitochondrial release of cytochrome C (CYCS) and apoptosis inducing factor (AIF), and increased abundance of active cleaved-caspase-9 and cleaved-caspase-3. Increasing concentrations of DON led to decreased B-cell lymphoma-2 (Bcl-2) expression and increased expression of BCL2-associated X (Bax) and B-cell lymphoma-2 homology 3 interacting domain death agonist (Bid), which in turn increased transcriptional activity of the transcription factors AIF and P53 (a tumor suppressor gene, promotes apoptosis). The addition of a caspase-8 inhibitor abrogated these effects. These results reveal that DON induces apoptosis in PHNCs via the mitochondrial apoptosis pathway, and caspase-8 is shown to play an important role during apoptosis regulation.

Structural insights of macromolecules involved in bacteria-induced apoptosis in the pathogenesis of human diseases.

Numbers of pathogenic bacteria can induce apoptosis in human host cells and modulate the cellular pathways responsible for inducing or inhibiting apoptosis. These pathogens are significantly recognized by host proteins and provoke the multitude of several signaling pathways and alter the cellular apoptotic stimuli. This process leads the bacterial entry into the mammalian cells and evokes a variety of responses like phagocytosis, release of mitochondrial cytochrome c, secretion of bacterial effectors, release of both apoptotic and inflammatory cytokines, and the triggering of apoptosis. Several mechanisms are involved in bacteria-induced apoptosis including, initiation of the endogenous death machinery, pore-forming proteins, and secretion of superantigens. Either small molecules or proteins may act as a binding partner responsible for forming the protein complexes and regulate enzymatic activity via protein-protein interactions. The bacteria induce apoptosis, attack the human cell and gain control over various types of cells and tissue. Since these processes are intricate in the defense mechanisms of host organisms against pathogenic bacteria and play an important function in host-pathogen interactions. In this chapter, we focus on the various bacterial-induced apoptosis mechanisms in host cells and discuss the important proteins and bacterial effectors that trigger the host cell apoptosis. The structural characterization of bacterial effector proteins and their interaction with human host cells are also considered.

Exploiting GRK2 Inhibition as a Therapeutic Option in Experimental Cancer Treatment: Role of p53-Induced Mitochondrial Apoptosis.

Simple SummaryThe involvement of GRK2 in cancer growth and an inverse correlation with p53 levels were suggested in breast cancer. Furthermore, increased GRK2 expression and activity were detected in thyroid cancer, but its effects and mechanisms of action were not investigated yet. This study aimed to explore the role of GRK2 in thyroid cancer both in vitro and in vivo and its crosstalk with p53. We demonstrated that thyroid cancer cells bearing a mutant form of p53 but not p53 null cells rely on GRK2 as a mechanism of proliferation by regulating p53 levels. Indeed, GRK2 indirectly induces p53 degradation through means of its catalytic activity. The pharmacological inhibition of the kinase effectively inhibits cancer growth by inducing p53-dependent mitochondrial pathways of apoptosis. Our results demonstrate a p53-dependent effect of GRK2 in cancer and suggest kinase inhibition as a potential therapeutic strategy for thyroid cancer.The involvement of GRK2 in cancer cell proliferation and its counter-regulation of p53 have been suggested in breast cancer even if the underlying mechanism has not yet been elucidated. Furthermore, the possibility to pharmacologically inhibit GRK2 to delay cancer cell proliferation has never been explored. We investigated this possibility by setting up a study that combined in vitro and in vivo models to underpin the crosstalk between GRK2 and p53. To reach this aim, we took advantage of the different expression of p53 in cell lines of thyroid cancer (BHT 101 expressing p53 and FRO cells, which are p53-null) in which we overexpressed or silenced GRK2. The pharmacological inhibition of GRK2 was achieved using the specific inhibitor KRX-C7. The in vivo study was performed in Balb/c nude mice, where we treated BHT-101 or FRO-derived tumors with KRX-C7. In our in vitro model, FRO cells were unaffected by GRK2 expression levels, whereas BHT-101 cells were sensitive, thus suggesting a role for p53. The regulation of p53 by GRK2 is due to phosphorylative events in Thr-55, which induce the degradation of p53. In BHT-101 cells, the pharmacologic inhibition of GRK2 by KRX-C7 increased p53 levels and activated apoptosis through the mitochondrial release of cytochrome c. These KRX-C7-mediated events were also confirmed in cancer allograft models in nude mice. In conclusion, our data showed that GRK2 counter-regulates p53 expression in cancer cells through a kinase-dependent activity. Our results further corroborate the anti-proliferative role of GRK2 inhibitors in p53-sensitive tumors and propose GRK2 as a therapeutic target in selected cancers.

Galectin-7 overexpression destroys airway epithelial barrier in transgenic mice.

When the integrity of airway epithelium is destroyed, the ordered airway barrier no longer exists and increases sensitivity to viral infections and allergens, leading to the occurrence of airway inflammation such as asthma. Here, we found that galectin-7 transgenic(+) mice exhibited abnormal airway structures as embryos and after birth. These abnormalities included absent or substantially reduced pseudostratified columnar ciliated epithelium and increased monolayer cells with irregular arrangement and widening of intercellular spaces. Moreover, airway tissue from galectin-7 transgenic(+) mice showed evidence of impaired cell-cell junctions and decreased expression of zonula occludens-1(ZO-1) and E-cadherin. When treated with respiratory syncytial virus (RSV) or ovalbumin (OVA), galectin-7 transgenic(+) mice developed substantially increased bronchial epithelial detachment and apoptosis, airway smooth muscle and basement membrane thickening, and enhanced airway responsiveness. We found that Galectin-7 localized in the cytoplasm and nucleus of bronchial epithelial cells, and that increased apoptosis was mediated through mitochondrial release of cytochrome c and upregulated JNK1 activation and expression of caspase-3 in galectin-7 Tg(+) mice. These findings suggested that Galectin-7 causes airway structural defects and destroys airway epithelium barrier, which predispose the airways to RSV or OVA-induced epithelial apoptosis, injury, and other asthma responses.