MiRNA‐30a inhibits AECs‐II apoptosis by blocking mitochondrial fission dependent on Drp‐1

Apoptosis of type II alveolar epithelial cells (AECs‐II) is a key determinant of initiation and progression of lung fibrosis. However, the mechanism of miR‐30a participation in the regulation of AECs‐II apoptosis is ambiguous. In this study, we investigated whether miR‐30a could block AECs‐II apoptosis by repressing mitochondrial fission dependent on dynamin‐related protein‐1 (Drp‐1). The levels of miR‐30a in vivo and in vitro were determined through quantitative real‐time PCR (qRT‐PCR). The inhibition of miR‐30a in AECs‐II apoptosis, mitochondrial fission and its dependence on Drp‐1, and Drp‐1 expression and translocation were detected using miR‐30a mimic, inhibitor‐transfection method (gain‐ and loss‐of‐function), or Drp‐1 siRNA technology. Results showed that miR‐30a decreased in lung fibrosis. Gain‐ and loss‐of‐function studies revealed that the up‐regulation of miR‐30a could decrease AECs‐II apoptosis, inhibit mitochondrial fission, and reduce Drp‐1 expression and translocation. MiR‐30a mimic/inhibitor and Drp‐1 siRNA co‐transfection showed that miR‐30a could inhibit the mitochondrial fission dependent on Drp‐1. This study demonstrated that miR‐30a inhibited AECs‐II apoptosis by repressing the mitochondrial fission dependent on Drp‐1, and could function as a novel therapeutic target for lung fibrosis.     

A role for septin 2 in Drp1‐mediated mitochondrial fission

Mitochondria are essential eukaryotic organelles often forming intricate networks. The overall network morphology is determined by mitochondrial fusion and fission. Among the multiple mechanisms that appear to regulate mitochondrial fission, the ER and actin have recently been shown to play an important role by mediating mitochondrial constriction and promoting the action of a key fission factor, the dynamin‐like protein Drp1. Here, we report that the cytoskeletal component septin 2 is involved in Drp1‐dependent mitochondrial fission in mammalian cells. Septin 2 localizes to a subset of mitochondrial constrictions and directly binds Drp1, as shown by immunoprecipitation of the endogenous proteins and by pulldown assays with recombinant proteins. Depletion of septin 2 reduces Drp1 recruitment to mitochondria and results in hyperfused mitochondria and delayed FCCP‐induced fission. Strikingly, septin depletion also affects mitochondrial morphology in Caenorhabditis elegans, strongly suggesting that the role of septins in mitochondrial dynamics is evolutionarily conserved.     

Astaxanthin prevents pulmonary fibrosis by promoting myofibroblast apoptosis dependent on Drp1‐mediated mitochondrial fission

Promotion of myofibroblast apoptosis is a potential therapeutic strategy for pulmonary fibrosis. This study investigated the antifibrotic effect of astaxanthin on the promotion of myofibroblast apoptosis based on dynamin‐related protein‐1 (Drp1)‐mediated mitochondrial fission in vivo and in vitro. Results showed that astaxanthin can inhibit lung parenchymal distortion and collagen deposition, as well as promote myofibroblast apoptosis. Astaxanthin demonstrated pro‐apoptotic function in myofibroblasts by contributing to mitochondrial fission, thereby leading to apoptosis by increasing the Drp1 expression and enhancing Drp1 translocation into the mitochondria. Two specific siRNAs were used to demonstrate that Drp1 is necessary to promote astaxanthin‐induced mitochondrial fission and apoptosis in myofibroblasts. Drp1‐associated genes, such as Bcl‐2‐associated X protein, cytochrome c, tumour suppressor gene p53 and p53‐up‐regulated modulator of apoptosis, were highly up‐regulated in the astaxanthin group compared with those in the sham group. This study revealed that astaxanthin can prevent pulmonary fibrosis by promoting myofibroblast apoptosis through a Drp1‐dependent molecular pathway. Furthermore, astaxanthin provides a potential therapeutic value in pulmonary fibrosis treatment.     

Vildagliptin improves high glucose‐induced endothelial mitochondrial dysfunction via inhibiting mitochondrial fission

The dipeptidyl peptidase 4 inhibitor vildagliptin (VLD), a widely used anti‐diabetic drug, exerts favourable effects on vascular endothelium in diabetes. We determined for the first time the improving effects of VLD on mitochondrial dysfunction in diabetic mice and human umbilical vein endothelial cells (HUVECs) cultured under hyperglycaemic conditions, and further explored the mechanism behind the anti‐diabetic activity. Mitochondrial ROS (mtROS) production was detected by fluorescent microscope and flow cytometry. Mitochondrial DNA damage and ATP synthesis were analysed by real time PCR and ATPlite assay, respectively. Mitochondrial network stained with MitoTracker Red to identify mitochondrial fragmentation was visualized under confocal microscopy. The expression levels of dynamin‐related proteins (Drp1 and Fis1) were determined by immunoblotting. We found that VLD significantly reduced mtROS production and mitochondrial DNA damage, but enhanced ATP synthesis in endothelium under diabetic conditions. Moreover, VLD reduced the expression of Drp1 and Fis1, blocked Drp1 translocation into mitochondria, and blunted mitochondrial fragmentation induced by hyperglycaemia. As a result, mitochondrial dysfunction was alleviated and mitochondrial morphology was restored by VLD. Additionally, VLD promoted the phosphorylation of AMPK and its target acetyl‐CoA carboxylase in the setting of high glucose, and AMPK activation led to a decreased expression and activation of Drp1. In conclusion, VLD improves endothelial mitochondrial dysfunction in diabetes, possibly through inhibiting Drp1‐mediated mitochondrial fission in an AMPK‐dependent manner.     

Rotaviral nonstructural protein 4 triggers dynamin‐related protein 1‐dependent mitochondrial fragmentation during infection

Dynamic equilibrium between mitochondrial fission and mitochondrial fusion serves as an important quality control system within cells ensuring cellular vitality and homeostasis. Viruses often target mitochondrial dynamics as a part of their obligatory cellular reprogramming. The present study was undertaken to assess the status and regulation of mitochondrial dynamics during rotavirus infection. Distinct fragmentation of mitochondrial syncytia was observed during late hours of RV (SA11, Wa, A5‐13) infection. RV nonstructural protein 4 (NSP4) was identified as the viral trigger for disrupted mitochondrial morphology. Severance of mitochondrial interconnections was found to be a dynamin‐related protein 1 (Drp1)‐dependent process resulting synergistically from augmented mitochondrial fission and attenuated mitochondrial fusion. Cyclin‐dependent kinase 1 was subsequently identified as the cellular kinase responsible for fission‐active Ser616 phosphorylation of Drp1. In addition to its positive role in mitochondrial fission, Drp1 also resulted in mitochondrial translocation of E3‐ubiquitin ligase Parkin leading to degradation of mitochondrial fusion protein Mitofusin 1. Interestingly, RV‐NSP4 was found to interact with and be involved in recruiting fission‐active pool of Serine 616 phosphoDrp1 (Ser616 pDrp1) to mitochondria independent of accessory adaptors Mitochondrial fission factor and Fission protein 1 (Fis1). Inhibition of either Drp1 or Ser616 pDrp1 resulted in significant decrease in RV‐NSP4‐induced intrinsic apoptotic pathway. Overall, this study underscores an efficient strategy utilised by RV to couple apoptosis to mitochondrial fission facilitating dissemination of viral progeny.     

Knockdown of Mtfp1 can minimize doxorubicin cardiotoxicity by inhibiting Dnm1l‐mediated mitochondrial fission

The long‐term usage of doxorubicin (DOX) is largely limited due to the development of severe cardiomyopathy. Many studies indicate that DOX‐induced cardiac injury is related to reactive oxygen species generation and ultimate activation of apoptosis. The role of novel mitochondrial fission protein 1 (Mtfp1) in DOX‐induced cardiotoxicity remains elusive. Here, we report the pro‐mitochondrial fission and pro‐apoptotic roles of Mtfp1 in DOX‐induced cardiotoxicity. DOX up‐regulates the Mtfp1 expression in HL‐1 cardiac myocytes. Knockdown of Mtfp1 prevents cardiac myocyte from undergoing mitochondrial fission, and subsequently reduces the DOX‐induced apoptosis by preventing dynamin 1‐like (Dnm1l) accumulation in mitochondria. In contrast, when Mtfp1 is overexpressed, a suboptimal dose of DOX can induce a significant percentage of cells to undergo mitochondrial fission and apoptosis. These data suggest that knocking down of Mtfp1 can minimize the cardiomyocytes loss in DOX‐induced cardiotoxicity. Thus, the regulation of Mtfp1 expression could be a novel therapeutic approach in chemotherapy‐induced cardiotoxicity.     

A novel autophagy/mitophagy inhibitor liensinine sensitizes breast cancer cells to chemotherapy through DNM1L-mediated mitochondrial fission

Autophagy inhibition has been widely accepted as a promising therapeutic strategy in cancer, while the lack of effective and specific autophagy inhibitors hinders its application. Here we found that liensinine, a major isoquinoline alkaloid, inhibits late-stage autophagy/mitophagy through blocking autophagosome-lysosome fusion. This effect is likely achieved via inhibiting the recruitment of RAB7A to lysosomes but not to autophagosomes. We further investigated the effects of autophagy inhibition by liensinine on the therapeutic efficacy of chemotherapeutic drugs and found that cotreatment of liensinine markedly decreased the viability and increased apoptosis in breast cancer cells treated with various chemotherapeutic agents. Mechanistically, we found that inhibition of autophagy/mitophagy by liensinine enhanced doxorubicin-mediated apoptosis by triggering mitochondrial fission, which resulted from dephosphorylation and mitochondrial translocation of DNM1L. However, blocking autophagosome/mitophagosome formation by pharmacological or genetic approaches markedly attenuated mitochondrial fission and apoptosis in cells with combinatatorial treatment. Moreover, liensinine was synergized with doxorubicin to inhibit tumor growth in MDA-MB-231 xenograft in vivo. Our findings suggest that liensinine could potentially be further developed as a novel autophagy/mitophagy inhibitor, and a combination of liensinine with classical chemotherapeutic drugs could represent a novel therapeutic strategy for treatment of breast cancer.     

Seleno-short-chain chitosan induces apoptosis in human breast cancer cells through mitochondrial apoptosis pathway in vitro

Seleno-short-chain chitosan (SSCC) was a synthesized chitosan derivative with the molecular weight of 4826.986 Da. The study is aimed to investigate cytotoxicity of SSCC on human breast cancer MCF-7 and BT-20 cells and explore apoptosis-related mechanism in vitro. The MTT (3- [4,5-Dimethylthiazol-2-yl]-2, 5-diphenylterazolium bromide) assay showed that SSCC exhibited significantly cytotoxic effects on MCF-7 and BT-20 cells in a dose- and time-dependent manner, and the effective inhibitory concentration was 100 μg/ml and 200 μg/ml, respectively. Apoptosis assay of these two kinds of cells was determined by Hoechst 33,342/PI and Annexin V-FITC/PI double staining. The cell cycle assay showed that SSCC triggered S and G2/M phase cell cycle arrest in MCF-7 cells and S phase cell cycle arrest in BT-20 cells in a time-dependent manner. Further studies demonstrated that SSCC led to the generation of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (MMP) in these two kinds of cells. N- acetyl-L cysteine (NAC), as a radical scavenger, significantly inhibited the generation of ROS and decreased the apoptosis of MCF-7 and BT-20 cells. Moreover, the expression of mitochondrial apoptosis-related proteins was detected by western blot assay. SSCC up-regulated the expression of Bax, down-regulated the expression of Bcl-2, subsequently increased the release of cytochrome c from mitochondria to cytoplasm, and activated the cleavage of caspase-9 and ?3, which finally induced apoptosis in MCF-7 and BT-20 cells in vitro. Consequently, these data indicated that SSCC could induce apoptosis of MCF-7and BT-20 cells in vitro by mitochondrial pathway.     

SENP3‐mediated deSUMOylation of dynamin‐related protein 1 promotes cell death following ischaemia

Global increases in small ubiquitin‐like modifier (SUMO)‐2/3 conjugation are a neuroprotective response to severe stress but the mechanisms and specific target proteins that determine cell survival have not been identified. Here, we demonstrate that the SUMO‐2/3‐specific protease SENP3 is degraded during oxygen/glucose deprivation (OGD), an in vitro model of ischaemia, via a pathway involving the unfolded protein response (UPR) kinase PERK and the lysosomal enzyme cathepsin B. A key target for SENP3‐mediated deSUMOylation is the GTPase Drp1, which plays a major role in regulating mitochondrial fission. We show that depletion of SENP3 prolongs Drp1 SUMOylation, which suppresses Drp1‐mediated cytochrome c release and caspase‐mediated cell death. SENP3 levels recover following reoxygenation after OGD allowing deSUMOylation of Drp1, which facilitates Drp1 localization at mitochondria and promotes fragmentation and cytochrome c release. RNAi knockdown of SENP3 protects cells from reoxygenation‐induced cell death via a mechanism that requires Drp1 SUMOylation. Thus, we identify a novel adaptive pathway to extreme cell stress in which dynamic changes in SENP3 stability and regulation of Drp1 SUMOylation are crucial determinants of cell fate.     

10H‐3,6‐Diazaphenothiazines triggered the mitochondrial‐dependent and cell death receptor‐dependent apoptosis pathways and further increased the chemosensitivity of MCF‐7 breast cancer cells via inhibition of AKT1 pathways

Breast cancer is one of the leading causes of death in cancer categories, followed by lung, colorectal, and ovarian among the female gender across the world. 10H‐3,6‐diazaphenothiazine (PTZ) is a thiazine derivative compound that exhibits many pharmacological activities. Herein, we proceed to investigate the pharmacological activities of PTZ toward breast cancer MCF‐7 cells as a representative in vitro breast cancer cell model. The PTZ exhibited a proliferation inhibition (IC₅₀ = 0.895 µM) toward MCF‐7 cells. Further, cell cycle analysis illustrated that the S‐phase checkpoint was activated to achieve proliferation inhibition. In vitro cytotoxicity test on three normal cell lines (HEK293 normal kidney cells, MCF‐10A normal breast cells, and H9C2 normal heart cells) demonstrated that PTZ was more potent toward cancer cells. Increase in the levels of reactive oxygen species results in polarization of mitochondrial membrane potential (ΔΨm), together with suppression of mitochondrial thioredoxin reductase enzymatic activity suggested that PTZ induced oxidative damages toward mitochondria and contributed to improved drug efficacy toward treatment. The RT² PCR Profiler Array (human apoptosis pathways) proved that PTZ induced cell death via mitochondria‐dependent and cell death receptor‐dependent pathways, through a series of modulation of caspases, and the respective morphology of apoptosis was observed. Mechanistic studies of apoptosis suggested that PTZ inhibited AKT1 pathways resulting in enhanced drug efficacy despite it preventing invasion of cancer cells. These results showed the effectiveness of PTZ in initiation of apoptosis, programmed cell death, toward highly chemoresistant MCF‐7 cells, thus suggesting its potential as a chemotherapeutic drug.     

AKAP1 mediates high glucose-induced mitochondrial fission through the phosphorylation of Drp1 in podocytes

Increasing evidence suggests that mitochondrial dysfunction plays a critical role in the development of diabetic kidney disease (DKD), however, its specific pathomechanism remains unclear. A-kinase anchoring protein (AKAP) 1 is a scaffold protein in the AKAP family that is involved in mitochondrial fission and fusion. Here, we show that rats with streptozotocin (STZ)-induced diabetes developed podocyte damage accompanied by AKAP1 overexpression and that AKAP1 closely interacted with the mitochondrial fission enzyme dynamin-related protein 1 (Drp1). At the molecular level, high glucose (HG) promoted podocyte injury and Drp1 phosphorylation at Ser637 as proven by decreased mitochondrial membrane potential, elevated reactive oxygen species generation, reduced adenosine triphosphate synthesis, and increased podocyte apoptosis. Furthermore, the AKAP1 knockdown protected HG-induced podocyte injury and suppressed HG-induced Drp1 phosphorylation at Ser637. AKAP1 overexpression aggravated HG-induced mitochondrial fragmentation and podocyte apoptosis. The coimmunoprecipitation assay showed that HG-induced Drp1 interacted with AKAP1, revealing that AKAP1 could recruit Drp1 from the cytoplasm under HG stimulation. Subsequently, we detected the effect of drp1 phosphorylation on Ser637 by transferring several different Drp1 mutants. We demonstrated that activated AKAP1 promoted Drp1 phosphorylation at Ser637, which promoted the transposition of Drp1 to the surface of the mitochondria and accounts for mitochondrial dysfunction events. These findings indicate that AKAP1 is the main pathogenic factor in the development and progression of HG-induced podocyte injury through the destruction of mitochondrial dynamic homeostasis by regulating Drp1 phosphorylation in human podocytes.     

Lysyl oxidase propeptide sensitizes pancreatic and breast cancer cells to doxorubicin‐induced apoptosis

RAS mutations or its activation by upstream receptor tyrosine kinases are frequently associated with poor response of carcinomas to chemotherapy. The 18 kDa propeptide domain of lysyl oxidase (LOX‐PP) released from the secreted precursor protein (Pro‐LOX) has been shown to inhibit RAS signaling and the transformed phenotype of breast, pancreatic, lung, and prostate cancer cells in culture, and formation of tumors by Her‐2/neu‐driven breast cancer cells in a mouse xenograft model. Here, we tested the effects of LOX‐PP on MIA PaCa‐2 pancreatic cancer cells, driven by mutant RAS. In MIA PaCa‐2 cells in culture, LOX‐PP attenuated the ERK and AKT activities and decreased the levels of the NF‐κB p65 and RelB subunits and cyclin D1, which are activated by RAS signaling. In mouse xenograft growth, LOX‐PP reduced growth of tumors by these pancreatic cancer cells, and the nuclear levels of the p65 NF‐κB subunit and cyclin D1 proteins. While biological agents attenuate tumor growth when used alone, often they have additive or synergistic effects when used in combination with chemotherapeutic agents. Thus, we next tested the hypotheses that LOX‐PP sensitizes pancreatic and breast cancer cells to the chemotherapeutic agent doxorubicin. Purified LOX‐PP enhanced the cytotoxic effects of doxorubicin in pancreatic and breast cancer cells, as judged by ATP production, Cell Death ELISA assays, caspase 3 activation, PARP cleavage, and Annexin V staining. Thus, LOX‐PP potentiates the cytotoxicity of doxorubicin on breast and pancreatic cancer cells, warranting additional studies with a broader spectrum of current cancer treatment modalities. J. Cell. Biochem. 111: 1160–1168, 2010. © 2010 Wiley‐Liss, Inc.     

Human MIEF1 recruits Drp1 to mitochondrial outer membranes and promotes mitochondrial fusion rather than fission

Mitochondrial morphology is controlled by two opposing processes: fusion and fission. Drp1 (dynamin-related protein 1) and hFis1 are two key players of mitochondrial fission, but how Drp1 is recruited to mitochondria and how Drp1-mediated mitochondrial fission is regulated in mammals is poorly understood. Here, we identify the vertebrate-specific protein MIEF1 (mitochondrial elongation factor 1; independently identified as MiD51), which is anchored to the outer mitochondrial membrane. Elevated MIEF1 levels induce extensive mitochondrial fusion, whereas depletion of MIEF1 causes mitochondrial fragmentation. MIEF1 interacts with and recruits Drp1 to mitochondria in a manner independent of hFis1, Mff (mitochondrial fission factor) and Mfn2 (mitofusin 2), but inhibits Drp1 activity, thus executing a negative effect on mitochondrial fission. MIEF1 also interacts with hFis1 and elevated hFis1 levels partially reverse the MIEF1-induced fusion phenotype. In addition to inhibiting Drp1, MIEF1 also actively promotes fusion, but in a manner distinct from mitofusins. In conclusion, our findings uncover a novel mechanism which controls the mitochondrial fusion-fission machinery in vertebrates. As MIEF1 is vertebrate-specific, these data also reveal important differences between yeast and vertebrates in the regulation of mitochondrial dynamics.     

Ionizing radiation accelerates Drp1-dependent mitochondrial fission, which involves delayed mitochondrial reactive oxygen species production in normal human fibroblast-like cells

Ionizing radiation is known to increase intracellular level of reactive oxygen species (ROS) through mitochondrial dysfunction. Although it has been as a basis of radiation-induced genetic instability, the mechanism involving mitochondrial dysfunction remains unclear. Here we studied the dynamics of mitochondrial structure in normal human fibroblast like cells exposed to ionizing radiation. Delayed mitochondrial production was peaked 3 days after irradiation, which was coupled with accelerated mitochondrial fission. We found that radiation exposure accumulated dynamin-related protein 1 (Drp1) to mitochondria. Knocking down of Drp1 expression prevented radiation induced acceleration of mitochondrial fission. Furthermore, knockdown of Drp1 significantly suppressed delayed production of mitochondrial . Since the loss of mitochondrial membrane potential, which was induced by radiation was prevented in cells knocking down of Drp1 expression, indicating that the excessive mitochondrial fission was involved in delayed mitochondrial dysfunction after irradiation.     

Haemin attenuates intermittent hypoxia‐induced cardiac injury via inhibiting mitochondrial fission

Obstructive sleep apnoea (OSA) characterized by intermittent hypoxia (IH) is closely associated with cardiovascular diseases. IH confers cardiac injury via accelerating cardiomyocyte apoptosis, whereas the underlying mechanism has remained largely enigmatic. This study aimed to explore the potential mechanisms involved in the IH‐induced cardiac damage performed with the IH‐exposed cell and animal models and to investigate the protective effects of haemin, a potent haeme oxygenase‐1 (HO‐1) activator, on the cardiac injury induced by IH. Neonatal rat cardiomyocyte (NRC) was treated with or without haemin before IH exposure. Eighteen male Sprague‐Dawley (SD) rats were randomized into three groups: control group, IH group (PBS, ip) and IH + haemin group (haemin, 4 mg/kg, ip). The cardiac function was determined by echocardiography. Mitochondrial fission was evaluated by Mitotracker staining. The mitochondrial dynamics‐related proteins (mitochondrial fusion protein, Mfn2; mitochondrial fission protein, Drp1) were determined by Western blot. The apoptosis of cardiomyocytes and heart sections was examined by TUNEL. IH regulated mitochondrial dynamics‐related proteins (decreased Mfn2 and increased Drp1 expressions, respectively), thereby leading to mitochondrial fragmentation and cell apoptosis in cardiomyocytes in vitro and in vivo, while haemin‐induced HO‐1 up‐regulation attenuated IH‐induced mitochondrial fragmentation and cell apoptosis. Moreover, IH resulted in left ventricular hypertrophy and impaired contractile function in vivo, while haemin ameliorated IH‐induced cardiac dysfunction. This study demonstrates that pharmacological activation of HO‐1 pathway protects against IH‐induced cardiac dysfunction and myocardial fibrosis through the inhibition of mitochondrial fission and cell apoptosis.