Mitochondrial Apoptotic Cascade Research Articles

Activation of caspase-9 on the apoptosome as studied by methyl-TROSY NMR.

Mitochondrial apoptotic signaling cascades lead to the formation of the apoptosome, a 1.1-MDa heptameric protein scaffold that recruits and activates the caspase-9 protease. Once activated, caspase-9 cleaves and activates downstream effector caspases, triggering the onset of cell death through caspase-mediated proteolysis of cellular proteins. Failure to activate caspase-9 enables the evasion of programmed cell death, which occurs in various forms of cancer. Despite the critical apoptotic function of caspase-9, the structural mechanism by which it is activated on the apoptosome has remained elusive. Here, we used a combination of methyl-transverse relaxation-optimized NMR spectroscopy, protein engineering, and biochemical assays to study the activation of caspase-9 bound to the apoptosome. In the absence of peptide substrate, we observed that both caspase-9 and its isolated protease domain (PD) only very weakly dimerize with dissociation constants in the millimolar range. Methyl-NMR spectra of isotope-labeled caspase-9, within the 1.3-MDa native apoptosome complex or an engineered 480-kDa apoptosome mimic, reveal that the caspase-9 PD remains monomeric after recruitment to the scaffold. Binding to the apoptosome, therefore, organizes caspase-9 PDs so that they can rapidly and extensively dimerize only when substrate is present, providing an important layer in the regulation of caspase-9 activation. Our work highlights the unique role of NMR spectroscopy to structurally characterize protein domains that are flexibly tethered to large scaffolds, even in cases where the molecular targets are in excess of 1 MDa, as in the present example.

Membrane-bound supramolecular nanofiber network stimulates transient receptor potential channels for calcium overload cancer therapy

Calcium (Ca2+) overload therapy is an efficient cancer treatment modality that increases aberrant cytoplasmic Ca2+ accumulation and activates mitochondrial apoptotic cascades. Current strategies primarily use exogenous inorganic Ca2+ salts to generate these effects. However, off-target Ca2+ release and the resulting hypercalcemic crisis limit their applications. We here report our serendipitous finding that a self-assembling peptide of Nap-GFFpYHWYGYTPQNVI (namely N-pGE11), possessing both alkaline phosphatase (ALP)-responsive and epidermal growth factor receptors (EGFR) binding properties, can mobilize the extracellular Ca2+ reservoir for Ca2+ overload therapeutics. N-pGE11 is capable of producing the EGFR-specific nanofibers on HeLa cell membranes in an ALP-responsive manner, which then facilitates extracellular Ca2+ influx and accumulation in the cytoplasm by indirectly stimulating TRP channels. Cytoplasmic Ca2+ overload is transmitted to the mitochondria, thus generating mitochondrial stress and caspase-3-dependent apoptosis. Meanwhile, excess cytoplasmic Ca2+ is also transported into the extracellular space and then results in thorough cell calcification via the secretion of Ca2+-enriched vesicles through exocytotic pathways. Consequently, N-pGE11 effectively inhibits tumor growth with good biocompatibility, whereas its therapeutic efficacy is enhanced significantly by feeding mice a high-calcium diet.

Deficiency of RAB39B Activates ER Stress-Induced Pro-apoptotic Pathway and Causes Mitochondrial Dysfunction and Oxidative Stress in Dopaminergic Neurons by Impairing Autophagy and Upregulating α-Synuclein.

Deletion and missense or nonsense mutation of RAB39B gene cause familial Parkinson's disease (PD). We hypothesized that deletion and mutation of RAB39B gene induce degeneration of dopaminergic neurons by decreasing protein level of functional RAB39B and causing RAB39B deficiency. Cellular model of deletion or mutation of RAB39B gene-induced PD was prepared by knocking down endogenous RAB39B in human SH-SY5Y dopaminergic cells. Transfection of shRNA-induced 90% reduction in RAB39B level significantly decreased viability of SH-SY5Y dopaminergic neurons. Deficiency of RAB39B caused impairment of macroautophagy/autophagy, which led to increased protein levels of α-synuclein and phospho-α-synucleinSer129 within endoplasmic reticulum (ER) and mitochondria. RAB39B deficiency-induced increase of ER α-synuclein and phospho-α-synucleinSer129 caused activation of ER stress, unfolded protein response, and ER stress-induced pro-apoptotic cascade. Deficiency of RAB39B-induced increase of mitochondrial α-synuclein decreased mitochondrial membrane potential and increased mitochondrial superoxide. RAB39B deficiency-induced activation of ER stress pro-apoptotic pathway, mitochondrial dysfunction, and oxidative stress caused apoptotic death of SH-SY5Y dopaminergic cells by activating mitochondrial apoptotic cascade. In contrast to neuroprotective effect of wild-type RAB39B, PD mutant (T168K), (W186X), or (G192R) RAB39B did not prevent tunicamycin- or rotenone-induced increase of neurotoxic α-synuclein and activation of pro-apoptotic pathway. Our results suggest that RAB39B is required for survival and macroautophagy function of dopaminergic neurons and that deletion or PD mutation of RAB39B gene-induced RAB39B deficiency induces apoptotic death of dopaminergic neurons via impairing autophagy function and upregulating α-synuclein.

Hesperetin induces apoptosis in A549 cells via the Hsp70‑mediated activation of Bax.

Hesperetin, a predominant flavonoid found in citrus fruits, has received considerable attention for its potential anticancer activity through the reduction of cell viability and the induction of apoptosis. Several effector mechanisms have been demonstrated underlying the antitumor properties of hesperetin but its specific mechanisms have not yet been fully elucidated. In the present study, how hesperetin affects the proliferation of A549 cells and the related cell proliferation regulatory mechanisms, were inevstigated. To elucidate the mechanisms underlying the effects of hesperetin on A549 cells, MTT assay, colony formation assay, flow cytometry, immunoblotting, reverse transcription‑quantitative PCR and JC‑1 staining were performed. The data revealed that hesperetin inhibited cell proliferation and induced apoptosis in these cells. Hesperetin also decreased the level of heat shock protein70 (Hsp70), a negative regulator of the mitochondrial apoptosis pathway, often overexpressed in various cancer cells and suspected to contribute to tumor development. Hesperetin‑induced Hsp70 suppression was associated with reduced cytosolic Bax and increased mitochondrial Bax levels, leading to the enhancement of the mitochondrial apoptotic cascade. The Hsp70 overexpression‑induced reduction in the level of hesperetin‑induced apoptosis provides evidence to hesperetin‑induced apoptosis being mediated by affecting Hsp70. Furthermore, it was demonstrated that hesperetin reduced Hsp70 expression by inducing a proteasome‑mediated degradation via the upregulation of E3‑ligase, C‑terminus of Hsp70‑interacting protein (CHIP). The present study highlighted the importance of the Bax activation‑triggered mitochondria‑mediated pathway for hesperetin‑induced apoptosis and demonstrated a novel mechanism of how Hsp70 played a critical role in the negative regulation of this apoptotic network in cancer cells.

Pelargonidin ameliorates reserpine-induced neuronal mitochondrial dysfunction and apoptotic cascade: a comparative in vivo study

Background Targeting the neuronal mitochondria as a possible intervention to guard against neurodegenerative disorder progression has been investigated in the current work via the administration of pelargonidin (PEL) to rats intoxicated by the mitochondrial toxin reserpine. The main criteria for choosing PEL were its reported antioxidant, anti-apoptotic and anti-inflammatory activities. Methods Male albino Wistar rats were randomized into five experimental groups; normal control, reserpinized to induce mitochondrial failure, standard PARP-1-inhibitor 1,5-isoquinolinediol (DIQ)-treated reserpinized, PEL-treated reserpinized, and GSK-3β inhibitor (AR-A 014418) -treated reserpinized. Results PEL administration reversed the reserpine-induced abnormal behaviors marked by decreased catalepsy time. In addition, PEL restored brain glutathione with a reduction in nitric oxide content as compared to the reserpine-challenged group. Meanwhile, it improved neuronal mitochondrial function by the elevation of complex I activity associated with a low ADP/ATP ratio. Likely through its anti-inflammatory effect, PEL reduced the elevation of serum interleukin-1ß level and inhibited serum lactate dehydrogenase activity. These findings are aligned with the reduced expression of cleaved PARP and cleaved caspase-3 proteins, indicating PEL’s suppressive effect on the intrinsic apoptotic pathway. Those biochemical findings were confirmed through comparable histopathological tissue examination among the experimental groups. Conclusions In conclusion, PEL is a promising candidate for future use in the management of mitochondria-associated neuronal complications via controlling the ongoing inflammatory and degeneration cascades.

Dichloromethane fraction of Citrus grandis induces apoptosis in a human colorectal cancer cell lines via apoptotic signaling pathway

• The incidence of colorectal cancer is rapidly growing in Korea. • Citrus grandis (L.) Osbeck has been used as a folk remedy in Jeju Island. • CDGF effectively decreased the viability of colorectal cancer cells. • CDGF changed the expression of pro-apoptotic and anti-apoptotic proteins. • CDGF promote the mitochondrial apoptotic cascade in colorectal cells. The incidence of colorectal cancer is rapidly growing in Korea, due to increasingly westernized dietary. We investigated the inhibitory effects of Citrus grandis extracts rich in polymethoxylated flavones in colorectal cancer. CGDF treatment changed the ratio of pro-apoptotic and anti-apoptotic Bcl-2 family proteins and generated ROS. CGDF treatment could promote the release of cytochrome c into the cytosol by activating the expression of caspases and decreasing the expression of IAPs. ROS generated by CGDF may initiate upstream signaling in the apoptotic signaling pathway. CGDF treatment inhibited the growth of xenograft tumors in mice. Gut microbiota analysis showed significant intestinal microbial shifts caused by CGDF ingestion, in which SCFA producers, Clostridium _IV, Oscillibacter , and Acetatifactor , were increased, while the number of potentially tumorigenic bacteria Staphylococcus was decreased. Therefore, we suggest that C . grandis peel extract and its fractions promote the mitochondrial apoptotic cascade in human colorectal cells by shifting the gut microbiota.

A new Mfn-2 related synthetic peptide promotes vascular smooth muscle cell apoptosis via regulating the mitochondrial apoptotic pathway by inhibiting Akt signaling

BackgroundRestenosis after angioplasty is a major challenge for the treatment of coronary artery diseases. Facilitation of vascular smooth muscle cell (VSMC) apoptosis may be an attractive approach to decrease the incidence of restenosis. We synthesized a 16-amino acid mitofusin-2 (Mfn-2) gene related peptide (MRSP) based on the sequence of the p21ras signature motif, the smallest functional sequence of the Mfn-2 gene with proapoptotic properties in VSMC. We investigated whether MRSP enhanced apoptotic activities to inhibit VSMC accumulation and neointimal hyperplasia in rats with carotid balloon injury.MethodsVSMCs were treated with different concentrations of MRSP, the PI3K agonist 740 Y-P and the inhibitor LY294002. Cell apoptosis and related pathway molecules were assessed. MRSP was also given to rats with carotid artery balloon injury. Neointimal hyperplasia and cell apoptotic pathways were detected.ResultsIn vitro experiments revealed that MRSP treatment significantly increased VSMC apoptosis and induced increases in procaspase-9 cleavage, caspase-3 activation, cytochrome c release from mitochondria to the cytoplasm and the Bax/Bcl-2 ratio but not caspase-8 expression, indicating that the mitochondrial apoptotic cascade was activated by MRSP, which might be attributed to suppression of the PI3K/Akt signaling pathway. We further found that the PI3K agonist 740 Y-P prevented and that the inhibitor LY294002 strengthened the proapoptotic effects of MRSP. MRSP strongly inhibited neointimal hyperplasia and VSMC accumulation, but increased VSMC apoptosis in the vascular wall after balloon injury. Moreover, MRSP substantially enhanced Bax and cleaved caspase-3 expression and decreased Bcl-2 levels in intima, accompanied by decreased levels of phosphorylated Akt and PI3K in vivo.ConclusionsTaken together, the present study showed that MRSP treatment results in a strong proapoptotic effect by activating the mitochondrial apoptotic cascade through suppression of the PI3K/Akt pathway.

A combinatorial drug screen in PDX-derived primary rhabdomyosarcoma cells identifies the NOXA - BCL-XL/MCL-1 balance as target for re-sensitization to first-line therapy in recurrent tumors.

First-line therapy for most pediatric sarcoma is based on chemotherapy in combination with radiotherapy and surgery. A significant number of patients experience drug resistance and development of relapsed tumors. Drugs that have the potential to re-sensitize relapsed tumor cells toward chemotherapy treatment are therefore of great clinical interest. Here, we used a drug profiling platform with PDX-derived primary rhabdomyosarcoma cells to screen a large drug library for compounds re-sensitizing relapse tumor cells toward standard chemotherapeutics used in rhabdomyosarcoma therapy. We identified ABT-263 (navitoclax) as most potent compound enhancing general chemosensitivity and used different pharmacologic and genetic approaches in vitro and in vivo to detect the NOXA-BCL-XL/MCL-1 balance to be involved in modulating drug response. Our data therefore suggests that players of the intrinsic mitochondrial apoptotic cascade are major targets for stimulation of response toward first-line therapies in rhabdomyosarcoma.

Pinocembrin-7-Methylether Protects SH-SY5Y Cells Against 6-Hydroxydopamine-Induced Neurotoxicity via Modulating Nrf2 Induction Through AKT and ERK Pathways.

The present study aimed to evaluate the neuroprotective effects and underlying mechanisms of pinocembrin-7-methylether (PME), a natural bioflavonoid, in 6-hydroxydopamine (6-OHDA)-induced models of Parkinson's disease in vivo and in vitro. First, we found that PME decreased apoptosis in 6-OHDA-intoxicated SH-SY5Y cells. PME also blocked several 6-OHDA-induced mitochondrial apoptotic cascades, including loss of mitochondrial membrane potential, caspase 3 and PARP activation, and a decrease in the Bcl-2/Bax ratio. Also, PME suppressed 6-OHDA-induced oxidative stress while increasing antioxidant enzymatic activity. Further investigations indicated that PME significantly enhanced nuclear accumulation of Nrf2, improved ARE promoter activity, and upregulated HO-1 and NQO1 expression levels. In addition, siRNA-mediated Nrf2 knockdown abolished PME-induced anti-oxidative and anti-apoptotic effects. Interestingly, we found that PME promoted phosphorylation of AKT and ERK, whereas pharmacological inhibition of AKT or ERK pathways diminished PME-induced Nrf2 activation and protective actions. Moreover, PME attenuated 6-OHDA-induced loss of dopaminergic neurons and ameliorated locomotor deficiency in zebrafish, supporting the neuroprotective actions of PME in vivo. In summary, we found that PME conferred neuroprotection against 6-OHDA-induced neurotoxicity in PD models in vivo and in vitro. Taken together, our findings suggest that activation of Nrf2/ARE/HO-1 signaling cascades contributes to PME-induced anti-oxidative and neuroprotective actions, which are at least partially mediated by AKT and ERK pathways.

The potential mediation of nitric oxide in the activation of mitochondrion-dependent apoptosis and yak meat tenderness during postmortem aging

Nitric oxide (NO) may play a critical role in the regulation of various physiological and pathophysiological processes due to its effect on the apoptosis pathway. However, few reports have studied the effects of NO on apoptosis and yak meat tenderization. The present study was conducted to explore the molecular mechanisms of Ca2+-independent inducible NO synthase isozyme (iNOS)-derived NO in mitochondrial apoptosis and meat tenderization during postmortem aging. Results indicated that the aminoguanidi hydrochloride (AH) significantly reduced iNOS activities and NO production. Moreover, the inhibitory property of iNOS-derived NO dramatically decreased mitochondrial oxidative stress degree, which further reduced the production of mitochondrial Ca2+ and ATP. The inhibitory property of iNOS-derived NO also influenced the Bcl-2 family members, mitochondrial apoptotic cascade reaction, and meat tenderization by protecting against the degradation of myofibrils and the damage of mitochondrial microstructure. These results demonstrated that the iNOS-derived NO possessed a significant role in the mitochondrial apoptosis pathway and yak meat tenderization during aging.

Achyranthes aspera L. leaf extract induced anticancer effects on Dalton's Lymphoma via regulation of PKCα signaling pathway and mitochondrial apoptosis

Ethnopharmacological relevanceEpidemiological studies promote the inclusion of natural-products in diet due to their inhibitory effects on various types of cancer. Among them, Achyranthes aspera L. (Family Amaranthaceae) is a medicinal plant in Ayurvedic pharmacopeia, found in India, Southeast Asia, America, and Sub-Saharan Africa. It is endowed with anti-inflammatory, anti-oxidant, and anti-cancer activities. However, its potential effect on Non-Hodgkin lymphomas (NHLs), has not yet been clarified. Aim of the studyIn the present study, we aimed to investigate the effect of Achyranthes aspera L. leaf extracts on highly aggressive murine NHL called Dalton's Lymphoma (DL) in vitro and in vivo. Material and methodsGC-HRMS analysis was carried out for the identification of compounds present in A. aspera leaf extract. The cytotoxicity of various A. aspera leaf extracts was evaluated on DL cells by MTT assay. Chromatin condensation, nuclear fragmentation, and morphological changes were observed by microscopy technique. Flow cytometry was used to measure the changes in mitochondrial membrane potential (ΔΨm) and apoptosis. In addition, the expressions of apoptosis-related proteins were detected by western blotting. Meanwhile, the in vivo anti-tumor effect of leaf extract was tested in DL induced Balb/c mice. ResultGC-HRMS analysis of A. aspera methanolic leaf extract (AAML) revealed the presence of ten pharmacologically active compounds. The results showed that AAML suppressed cell proliferation, decreased mitochondrial membrane potential, changed the morphological structure, and induced apoptosis. Moreover, AAML could promote the release of cytochrome c by regulating Bcl-2 family proteins and then activated caspase-9/ -3 to triggered cell apoptosis. At the same time in DL cells treated with AAML, the protein kinase Cα (PKCα) pathway was inhibited in a concentration-dependent manner. Remarkably, in vivo, AAML mediated suppression of DL growth in Balb/c mice was accompanied by attenuation of the PKCα pathway and induction of apoptosis. Our result suggested that AAML promotes mitochondrial apoptotic cascade in DL cells by suppressing the PKCα signaling pathway. ConclusionThe study suggests that AAML could potently suppress DL progression by promoting apoptosis via mitochondrial-cascade and attenuation of the PKCα signaling pathway.

HIF-1-induced mitochondrial ribosome protein L52: a mechanism for breast cancer cellular adaptation and metastatic initiation in response to hypoxia.

Background: Hypoxia is a hallmark of the physical microenvironment of solid tumors. As a key factor that regulates tumor development and progression, hypoxia can reprogram the expression of multiple genes, whose biological function and molecular mechanism in cancer remain largely unclear. The mitochondrial ribosome protein family consists of nuclear-encoded mitochondrial proteins that are responsible for protein synthesis in the mitochondria.Methods: A high-throughput RNA sequencing assay was carried out to identify differentially expressed mRNAs between breast cancer tissues and adjacent normal tissues as well as breast tumors with metastasis and those without metastasis. Our clinical samples and TCGA database were analyzed to observe the clinical value of mitochondrial ribosome protein L52 (MRPL52) in human breast cancer. Potent hypoxia response elements in the promoter region of MRPL52 were identified and validated by chromatin immunoprecipitation and luciferase reporter assays. Functional experiments were performed using breast cancer cell lines with MRPL52 ectopic expression and knockdown cultured in a 20% or 1% O2 environment.Results: MRPL52 expression was upregulated in human breast cancer and was significantly associated with aggressive clinicopathological characteristics and a higher metastatic risk of breast cancer patients. We found that the overexpression of MRPL52 in breast cancer is induced by hypoxia-inducible factor-1 in response to hypoxic exposure. The role of MRPL52 in suppressing apoptosis and promoting migration and invasion of hypoxic breast cancer cells was demonstrated by our experimental evidence. Mechanistically, MRPL52 promoted PTEN-induced putative kinase 1 /Parkin-dependent mitophagy to remove oxidatively damaged mitochondria and prevent uncontrolled reactive oxygen species (ROS) generation, thus repressing activation of the mitochondrial apoptotic cascade. Additionally, MRPL52 augmented epithelial-mesenchymal transition, migration and invasion of hypoxic breast cancer cells by activating the ROS-Notch1-Snail signaling pathway. Benefited from this bidirectional regulatory mechanism, MRPL52 is responsible for maintaining ROS levels in a window that can induce tumorigenic signal transduction without causing cytotoxicity in hypoxic breast cancer cells.Conclusions: This work elucidates the molecular mechanism by which MRPL52 mediates hypoxia-induced apoptotic resistance and metastatic initiation of breast cancer, and provides new insights into the interplay between cancer and the tumor microenvironment.

Exercise‐induced Increase in Hexokinase II‐mitochondria Association Reduces Cardiac Ischemia‐Reperfusion Injury in Rats

IntroductionExercise‐training has been shown to modulate signaling pathways that contribute to improved mitochondrial function in the heart. AKT, a serine/threonine protein kinase pathway is a critical regulator of a variety of cellular functions and its activation is implicated in cardioprotection from ischemia‐reperfusion (I/R) injury. Importantly, the AKT pathway has been shown to be upregulated in exercise. However, the interplay between AKT signaling, mitochondrial function and cardio‐protection in exercise remains to be well elucidated.AIMHerein, we investigated the role of exercise‐induced AKT/Hexokinase II (HKII)‐signaling as a potential mechanism in preserving mitochondrial function and thereby, contribute to cardioprotection against I/R injury.MethodsIsolated hearts from rats, randomly assigned to either sedentary (SED) group or 6–8 weeks treadmill trained (TRN) group, were subjected to 30 min global ischemia and 5 min reperfusion. Mitochondria were isolated immediately after reperfusion and the effects of exercise on cardiac mitochondrial bioenergetics, including changes in O2 consumption rate, mitochondrial membrane potential (ΔΨm), and mitochondrial calcium retention capacity (CRC) was examined. Western blot was performed to quantify the total AKT, p‐AKT and p‐HKII. In addition, we checked the subcellular distributions of HKII and examined the regulation of apoptotic markers at the mitochondrial level. In some hearts, cardiac functional variables and infarct size were also assessed after 120 min reperfusion in SED and TRN rats.ResultsWe found that exercise increased the phosphorylation of AKT in TRN rat hearts, which was associated, concomitantly, with increased phosphorylation of HKII and increased translocation to mitochondria following IR. TRN rats also showed a decreased expression of Bax and DRP1 in mitochondrial fraction and inhibition of caspase‐3 activation. Furthermore, exercise preserved the mitochondrial bioenergetics with significantly higher respiratory control index and more normalized ΔΨm compared to SED rats (see schematic). CRC was greater in TRN rats compared to SED rats (226.6 ± 13.3 nmol /mg protein vs 173.3 ± 13.3 nmol /mg protein). These improvements in mitochondrial function in TRN rats correlated with improved cardiac function during reperfusion.ConclusionThe results indicate that AKT‐mediated HKII‐mitochondrial interaction due to exercise protects mitochondria by inhibiting mitochondrial apoptotic cascades and likely contributes to the increased cardioprotection against IR injury.Support or Funding InformationThis work was supported by NIH grant R01 HL131673‐01A1 to AKSC and WMKSchematic diagram for exercise‐induced activation of AKT‐HKII signaling pathway during IR injury (A). Time course of the mitochondrial O2 consumption, membrane potential (B) and calcium retention capacity (C) in SED and TRN rats after IR stress.Figure 1

Overexpression of Antiapoptotic MCL-1 Predicts Worse Overall Survival of Patients With Non-small Cell Lung Cancer

Myeloid cell leukemia-1 (MCL-1) is a member of the B-cell lymphoma-2 (Bcl-2) family of proteins, which regulate the intrinsic (mitochondrial) apoptotic cascade. MCL-1 inhibits apoptosis, which may be associated with resistance to cancer therapy. Therefore, in this study, the clinical role of MCL-1 in non-small cell lung cancer (NSCLC) was explored. This retrospective study included 80 patients with stage 1-3A NSCLC, who underwent surgery without preoperative treatment between 2010 and 2011. MCL-1 expression and Ki-67 index were determined via immunohistochemical staining. Apoptotic index (AI) was determined via terminal deoxynucleotidyl transferase dUTP nick end labeling. The receiver operating characteristic curve analysis (area under curve=0.6785) revealed that MCL-1 expression in 30.0% of the NSCLC tumor cells was a significant cut-off for predicting prognosis. Tumors were considered MCL-1-positive if staining was observed in >30% of the cells. Thirty-six tumors (45.0%) were MCL-1-positive. However, there were no significant differences between MCL-1 expression and clinical variables. AI was lower in MCL-1-positive (2.2±3.6%) than in MCL-1-negative (5.2±7.9%) tumors, although the difference was not significant (p=0.1080). The Ki-67 index was significantly higher in MCL-1-positive than in MCL-1-negative tumors (18.0% vs. 3.0%; p<0.001). Five-year survival rate was significantly worse in patients with MCL-1-positive tumors (68.3%) than in those with MCL-1-negative tumors (93.1%, p=0.0057). Univariate [hazard ratio (HR)=5.041, p=0.0013], and multivariate analyses revealed that MCL-1 expression was a significant prognostic factor (HR=3.983, p=0.0411). MCL-1 expression in NSCLC cells correlated inversely with AI and positively with Ki-67 index. MCL-1 may serve as a potential prognostic biomarker and a novel therapeutic target in NSCLC.

Antileukemic activity and mechanism of ardisianone-a crucial role of mitochondrial stress in apoptosis and necroptosis

Abstract Background Ardisianone was reported to induce apoptosis in prostate cancer cells, but necroptosis in leukemia cell lines. It is needed to clarify whether ardisianone could cause apoptosis and necroptosis in leukemia cells and investigate its underlying mechanisms. Methods Cytotoxicity was examined using the MTT assay. Cytoflowmetric analysis of JC-1 and PI staining was used to examine mitochondrial membrane potential and cell cycle progression, respectively. Apoptosis was examined by annexin V-PI staining assay. Protein expression was detected by Western blot and apoptosis antibody array chip. Immunofluorescence staining detected necroptosis effect. Results Ardisianone inhibited cell viability in HL-60, a promyelocytic leukemia cell line, with IC50 value of 1.87 µ M in a 24h exposure. Further detection showed that ardisianone induced time- and concentration-dependent apoptosis. JC-1 staining demonstrated that ardisianone caused a profound loss of mitochondrial membrane potential. Western blot also confirmed the decrease of pro-survival Bcl-2 family protein and the downregulation of necroptosis relatives (e.g., RIPK1 and RIPK3). The caspase cascade was profoundly activated by ardisianone. Notably, the specific pan-caspase inhibitor Q-VD-OPh significantly blunted ardisianone induced loss of membrane potential and apoptosis, suggesting that caspase cascade activation may further amplify mitochondrial damage and apoptotic signaling cascade. The expression of RIPK1/RIPK3/MIKL as significantly reduced by pre-treatment of necroptosis inhibitor (Necrostatin-1, Nec-1), suggesting that Nec-1 attenuates ardisianone-induced necroptosis. Conclusion The data suggest that ardisianone induces mitochondrial dysfunction and apoptosis in leukemia cells through modification of Bcl-2 family members and causes necrotic cell death.

Apoptosis effect of Aegiceras corniculatum on human colorectal cancer via activation of FoxO signaling pathway

Aegiceras corniculatum (L.) Blanco is known to exhibit anticancer effects against different types of cancer; however, to the best of our knowledge, the anticancer activity and underlying mechanisms of action of A. corniculatum leaf extract on colorectal cancer have not been elucidated. In the present study, colony-forming assay, western blot analysis, flow cytometry, and a xenograft model were used to investigate the effects of an n-butanol extract of A. corniculatum leaves (NACL) on colorectal cancer in vitro and in vivo. The results showed that NACL inhibits the viability and proliferation of colorectal cancer cells in a dose-dependent manner. Besides, NACL also induces cell apoptosis and cell cycle arrest by activating Forkhead box proteins and controlling the cell cycle checkpoint pathways, which are associated with the caspase-dependent mitochondrial apoptotic cascades and Bcl-2 family proteins. More importantly, the tumour sizes in HT-29 xenograft nude mice decreased after treatment with NACL in vivo. These findings indicate that A. corniculatum leaf extracts have potent anticancer activities across different colorectal and other solid tumour cell lines, via regulation of the cell cycle and apoptosis; thus, it has the potential to be developed as an anticancer agent to enhance clinical standards of care for patients with colorectal cancer.

Induction of G0/G1 Phase Cell Cycle Arrest and Apoptosis by &lt;i&gt;Aegiceras corniculatum&lt;/i&gt; in Human Colorectal Cancer via Activation of FoxO Signalling

Background: Aegiceras corniculatum (Linn.) Blanco, commonly known as black mangrove, river mangrove, kacang-kacang, and Narikandam, is a mangrove plant, found from India through Southeast Asia to southern China and Australia. Aegiceras corniculatum is known to exhibit anticancer effects against different types of cancer; however, the anticancer activity and underlying mechanisms of action of A. corniculatum leaf extract on colorectal cancer (CRC) have not been elucidated. Methods: In the present study, a colony-forming assay, western blot, flow cytometry and a xenografts model were applied to investigate the effects of n-butanol extract of A. corniculatum leaves (NACL) on CRC in vitro and in vivo. Findings: The results showed that NACL inhibits the viability and proliferation of CRC cells in a dose-dependent manner. Besides, NACL also induces cell apoptosis and cell cycle arrest by activating Forkhead box proteins and controlling the cell cycle checkpoint pathways, which are associated with the caspase-dependent mitochondrial apoptotic cascades and Bcl-2 family proteins. More importantly, the tumour sizes in HT-29 xenograft nude mice decreased after treatment with NACL in vivo. Significance: These findings demonstrate that A. corniculatum leaf extracts have potent anti-cancer activities across different colorectal and other solid tumor cell lines, via regulating cell cycle and apoptosis pathway, thus it has the potential to be developed as an anticancer therapy to enhance other standard of cares in the clinic for patients with CRC. Funding Statement: The work was supported by Guangxi scientific research and technology development plan project (No. GKH15104001-11), Guangxi Science and Technology Plan Projects (No. GKAD17195025), and Guangxi Key Laboratory Construction Project (No.17-259-20). Declaration of Interests: All authors state there are no conflicts of interest to disclose. Ethics Approval Statement: Experimental conditions and procedures involving animals were approved by the Institutional Animal Ethics Committee (IAEC) of Guangxi Medical University (Nanning, Guangxi, China). The experiments were carried out in accordance with the Laboratory Animal Use Guidelines of IAEC. Animal handling followed the National Animal Welfare Law of China.

Mitochondria-mediated apoptosis was induced by oleuropein in H1299 cells involving activation of p38 MAP kinase.

Lung cancer is the main health threat in the world. Recently, oleuropein has been reported to have potent antioxidant and anticancer activities. However, the antitumor effects of oleuropein on H1299 cells are not well understood. Therefore, the purpose of this paper is tantamount to explore the effects of oleuropein on H1299 cells and its underlying mechanism that may be involved. Oleuropein treatment in H1299 cells resulted in cell cycle distribution at G2 /M arrest and apoptosis in a dose-dependent manner. Mitochondria-mediated apoptosis was verified by the increase in Bax/Bcl-2 ratio, release of cytochrome c, and activation of caspase-3 on oleuropein-induced H1299 cells. In addition, our data also demonstrated that the p38 mitogen-activated protein kinase (MAPK) signaling pathway has a critical role in oleuropein-induced apoptosis. Moreover, we used transcriptome analysis to identify differentially expressed genes (DEGs) in H1299 cells by oleuropein and SB203580 treatment. Many DEGs were annotated to metabolic pathways, cell cycle, pathways in cancer, MAPK signaling pathway by Kyoto Encyclopedia of Genes and Genomes and Gene ontology enrichment methods. Network and expression analysis found that DEGs, including RPS6A5, GADD45A, and MKP, play a key role in the p38 MAPK signaling pathway. In H1299 cells, oleuropein resulted in the expression of numerous genes related to cell signaling, metabolism pathway and directly associated with apoptosis. These results illustrated that oleuropein-induced apoptosis via mitochondrial apoptotic cascade was activated by the p38 MAPK signaling pathway in H1299 cells. Thus, oleuropein as a natural compound and therapeutic drug has potential application value in the treatment of lung cancer.

Effect of mitochondrial cytochrome c release and its redox state on the mitochondrial-dependent apoptotic cascade reaction and tenderization of yak meat during postmortem aging

We aim to investigate the effect of mitochondrial cytochrome c release and its redox state on the mitochondrial-dependent apoptotic pathway and yak meat tenderization during postmortem aging. Results indicated that the internal environment factors considerably decreased, whereas mitochondrial dysfunction increased in the early stage (0–72 h). Moreover, the release of mitochondrial cytochrome c into the cytosol, which was an apoptotic early event and the apoptosome elements in the cytoplasm changed remarkably with the increase of aging time. Meanwhile, N,N,N′,N′-tetramethylphenylene-1, 4-diamine (TMPD) inhibited the mitochondrial apoptotic process and postmortem meat tenderization by influencing the redox state of cytochrome c and apoptotic factors. These findings confirmed that the internal environment factors in the muscles provided essential conditions for mitochondrial cytochrome c, which activated the mitochondrial apoptotic pathway by forming apoptosome complexes. The reduction state of cytochrome c produced by TMPD plays a negative role in the activation of the mitochondrial apoptotic cascade reaction and meat tenderization during postmortem aging. We conclude that mitochondrial cytochrome c release and oxidized cytochrome c plays a significant role in yak meat tenderization by regulating the mitochondrial apoptotic pathway.

IGF-1 protects SH-SY5Y cells against MPP+-induced apoptosis via PI3K/PDK-1/Akt pathway.

Insulin-like growth factor (IGF)-1 is a well-known anti-apoptotic pro-survival factor and phosphatidylinositol-3-kinase (PI3K)/Akt pathway is linked to cell survival induced by IGF-1. It is also reported that Akt signaling is modulated by 3-phosphoinositide-dependent kinase-1 (PDK1). In the current study, we investigated whether the anti-apoptotic effect of IGF-1 in SH-SY5Y cells exposed to 1-methyl-4-phenylpyridinium (MPP+) is associated with the activity of PI3K/PDK1/Akt pathway. Treatment of cells with IGF-1 inhibited MPP+-induced apoptotic cell death. IGF-1-induced activation of Akt and the protective effect of IGF-1 on MPP+-induced apoptosis were abolished by chemical inhibition of PDK1 (GSK2334470) or PI3K (LY294002). The phosphorylated levels of Akt and PDK1 were significantly suppressed after MPP+ exposure, while IGF-1 treatment completely restored MPP+-induced reductions in phosphorylation. IGF-1 protected cells from MPP+ insult by suppressing intracellular reactive oxygen species (ROS) production and malondialdehyde levels and increasing superoxide dismutase activity. Mitochondrial ROS levels were also increased during MPP+ exposure, which were attenuated by IGF-1 treatment. In addition, IGF-1-treated cells showed increased activities of succinate dehydrogenase and citrate synthase, stabilization of mitochondrial transmembrane potential, increased ratio of Bcl-2 to Bax, prevention of cytochrome c release and inhibition of caspase-3 activation with PARP cleavage. Furthermore, the protective effects of IGF-1 on oxidative stress and mitochondrial dysfunction were attenuated when cells were preincubated with GSK2334470 or LY294002. Our data suggest that IGF-1 protects SH-SY5Y cells against MPP+-associated oxidative stress by preserving mitochondrial integrity and inhibiting mitochondrial apoptotic cascades via the activation of PI3K/PDK1/Akt pathway.