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.     

IL‐17A promotes cell migration and invasion of glioblastoma cells via activation of PI3K/AKT signalling pathway

Glioblastomas (GBMs) are the most common of both benign and malignant primary brain tumours, in which the inflammatory and immunologic abnormalities are involved. Interleukin‐17A (IL‐17A) plays an important role in various inflammatory diseases and cancers. Several recent studies revealed that the expression of IL‐17A was overexpressed in human GBMs tissue. However, the accurate role of IL‐17A in GBMs remains unclear. In this study, we aimed to explore the effect of IL‐17A on cell migration and invasion of GBMs and the mechanism by which the effects occurred. We found that exogenous IL‐17A promoted significantly cell migration and invasion abilities in two GBMs cell lines (U87MG and U251) in a time‐dependent manner. In addition, the protein expressions of PI3K, Akt and MMP‐2/9 were increased in the GBMs cells challenged by IL‐17A. Furthermore, a tight junction protein ZO‐1 was down‐regulated but Twist and Bmi1 were up‐regulated. Treatment with a PI3K inhibitor (LY294002) significantly reduced the abilities of both migration and invasion in U87MG and U251 cells. LY294002 treatment also attenuated the IL‐17A causing increases of protein levels of PI3K, AKT, MMP‐2/9, Twist and the decreases of protein level of ZO‐1 in the U87MG and U251 cells. Taken together, we concluded that IL‐17A promotes the GBM cells migration and invasion via PI3K/AKT signalling pathway. IL‐17A and its related signalling pathways may be potential therapeutic targets for GBM.     

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.     

Crystal structure and functional analysis of MiD49, a receptor for the mitochondrial fission protein Drp1

Mitochondrial fission requires recruitment of dynamin‐related protein 1 (Drp1) to the mitochondrial surface, where assembly leads to activation of its GTP‐dependent scission function. MiD49 and MiD51 are two receptors on the mitochondrial outer membrane that can recruit Drp1 to facilitate mitochondrial fission. Structural studies indicated that MiD51 has a variant nucleotidyl transferase fold that binds an ADP co‐factor essential for activation of Drp1 function. MiD49 shares sequence homology with MiD51 and regulates Drp1 function. However, it is unknown if MiD49 binds an analogous co‐factor. Because MiD49 does not readily crystallize, we used structural predictions and biochemical screening to identify a surface entropy reduction mutant that facilitated crystallization. Using molecular replacement, we determined the atomic structure of MiD49 to 2.4 Å. Like MiD51, MiD49 contains a nucleotidyl transferase domain; however, the electron density provides no evidence for a small‐molecule ligand. Structural changes in the putative nucleotide‐binding pocket make MiD49 incompatible with an extended ligand like ADP, and critical nucleotide‐binding residues found in MiD51 are not conserved. MiD49 contains a surface loop that physically interacts with Drp1 and is necessary for Drp1 recruitment to the mitochondrial surface. Our results suggest a structural basis for the differential regulation of MiD51‐ versus MiD49‐mediated fission.     

MicroRNA‐29b‐3p suppresses oral squamous cell carcinoma cell migration and invasion via IL32/AKT signalling pathway

Oral squamous cell carcinoma (OSCC) is aggressive accompanied with poor prognosis. We previously isolated the most invasive cells resembling the invasive tumour front by microfluidic technology and explored their differentially expressed microRNAs (miRNAs) in our previous work. Here, we verified the miR‐29b‐3p as a guarder that suppressed migration and invasion of OSCC cells and was down‐regulated in the most invasive cells. Besides that, the invasion suppression role of miR‐29b‐3p was achieved through the IL32/AKT pathway. Thus, miR‐29b‐3p and IL32 might serve as therapeutic targets for blocking the progression and improving the outcome of OSCC.     

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.     

Production of phosphatidylinositol 5‐phosphate via PIKfyve and MTMR3 regulates cell migration

Although phosphatidylinositol 5‐phosphate (PtdIns5P) is present in many cell types and its biogenesis is increased by diverse stimuli, its precise cellular function remains elusive. Here we show that PtdIns5P levels increase when cells are stimulated to move and we find PtdIns5P to promote cell migration in tissue culture and in a Drosophila in vivo model. First, class III phosphatidylinositol 3‐kinase, which produces PtdIns3P, was shown to be involved in migration of fibroblasts. In a cell migration screen for proteins containing PtdIns3P‐binding motifs, we identified the phosphoinositide 5‐kinase PIKfyve and the phosphoinositide 3‐phosphatase MTMR3, which together constitute a phosphoinositide loop that produces PtdIns5P via PtdIns(3,5)P₂. The ability of PtdIns5P to stimulate cell migration was demonstrated directly with exogenous PtdIns5P and a PtdIns5P‐producing bacterial enzyme. Thus, the identified phosphoinositide loop defines a new role for PtdIns5P in cell migration.     

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.     

miR‐96 regulates migration and invasion of bladder cancer through epithelial‐mesenchymal transition in response to transforming growth factor‐β1

Bladder cancer (BC) is one of the most frequent urological malignancies, and its molecular mechanism still remains unclear. Recent studies have revealed that MicroRNA (miRNAs) acted as oncogenes or tumor suppressors in a variety of cancers. MiRNA‐96 has been reported to play a significant role in the development and progression of many cancers. In the current study, we found that transforming growth factor (TGF)‐β1 played a significant role in the progression that miR‐96 conducted. And TGF‐β1 could also regulate the expression of FOXQ1, which is the target gene of miR‐96. Furthermore, miR‐96 induced epithelial‐mesenchymal transition in BC cells, which is driven by TGF‐β1. In conclusion, our data revealed that miR‐96 regulates the progression and epithelial‐mesenchymal transition, which is driven by TGF‐β1 in BC cells; it may provide a new thought for the therapy of BC.     

Dynamin‐related protein 1‐mediated mitochondrial fission contributes to IR‐783‐induced apoptosis in human breast cancer cells

IR‐783 is a kind of heptamethine cyanine dye that exhibits imaging, cancer targeting and anticancer properties. A previous study reported that its imaging and targeting properties were related to mitochondria. However, the molecular mechanism behind the anticancer activity of IR‐783 has not been well demonstrated. In this study, we showed that IR‐783 inhibits cell viability and induces mitochondrial apoptosis in human breast cancer cells. Exposure of MDA‐MB‐231 cells to IR‐783 resulted in the loss of mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) depletion, mitochondrial permeability transition pore (mPTP) opening and cytochrome c (Cyto C) release. Furthermore, we found that IR‐783 induced dynamin‐related protein 1 (Drp1) translocation from the cytosol to the mitochondria, increased the expression of mitochondrial fission proteins mitochondrial fission factor (MFF) and fission‐1 (Fis1), and decreased the expression of mitochondrial fusion proteins mitofusin1 (Mfn1) and optic atrophy 1 (OPA1). Moreover, knockdown of Drp1 markedly blocked IR‐783‐mediated mitochondrial fission, loss of MMP, ATP depletion, mPTP opening and apoptosis. Our in vivo study confirmed that IR‐783 markedly inhibited tumour growth and induced apoptosis in an MDA‐MB‐231 xenograft model in association with the mitochondrial translocation of Drp1. Taken together, these findings suggest that IR‐783 induces apoptosis in human breast cancer cells by increasing Drp1‐mediated mitochondrial fission. Our study uncovered the molecular mechanism of the anti‐breast cancer effects of IR‐783 and provided novel perspectives for the application of IR‐783 in the treatment of breast cancer.     

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.     

Plantamajoside inhibits the proliferation and epithelial‐to‐mesenchymal transition in hepatocellular carcinoma cells via modulating hypoxia‐inducible factor‐1α‐dependent gene expression

As a potential antitumor herbal medicine, plantamajoside (PMS) benefits the treatment of many human malignances. However, the role of PMS in the progression of hepatocellular carcinoma (HCC) and the related molecular mechanisms is still unknown. Here, we proved that the cell viabilities of HepG2 cells were gradually decreased with the increasing concentrations of CoCl₂ and/or PMS via cell counting kit‐8 assay. Meanwhile, 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) and western blot assays were used to further confirm that PMS inhibited the CoCl₂‐induced cell proliferation in HepG2 cells via suppressing the Ki67 and proliferating cell nuclear antigen expressions. We also performed wound‐healing and transwell assays and demonstrated that PMS inhibited CoCl₂‐induced migration and invasion in HepG2 cells via suppressing the epithelial–mesenchymal transition (EMT) process. In addition, the use of 3‐(5′‐hydroxymethyl‐2′‐furyl)‐1‐benzylindazole further proved that PMS inhibited the malignant biological behaviors of HepG2 cells under hypoxic condition by suppressing the hypoxia‐inducible factor‐1α (HIF‐1α) expression. Besides, we further confirmed that PMS suppressed the growth and metastasis of implanted tumors in vivo. Given that PMS suppressed the proliferation and EMT induced by CoCl₂ in HCC cells via downregulating HIF‐1α signaling pathway, we provided evidence that PMS might be a novel anti‐cancer drug for HCC treatment.     

p120‐catenin differentially regulates cell migration by Rho‐dependent intracellular and secreted signals

The adherens junction protein p120‐catenin is implicated in the regulation of cadherin stability, cell migration and inflammatory responses in mammalian epithelial tissues. How these events are coordinated to promote wound repair is not understood. We show that p120 catenin regulates the intrinsic migratory properties of primary mouse keratinocytes, but also influences the migratory behavior of neighboring cells by secreted signals. These events are rooted in the ability of p120‐catenin to regulate RhoA GTPase activity, which leads to a two‐tiered control of cell migration. One restrains cell motility via an increase in actin stress fibers, reduction in integrin turnover and an increase in the robustness of focal adhesions. The other is coupled to the secretion of inflammatory cytokines including interleukin‐24, which causally enhances randomized cell movements. Taken together, our results indicate that p120‐RhoA‐GTPase‐mediated signaling can differentially regulate the migratory behavior of epidermal cells, which has potential implications for chronic wound responses and cancer.