Expression of voltage-dependent anion channels in endometrial cancer and its potential prognostic significance.

The exchange of metabolites between mitochondria and cytosol occurs through pores formed by voltage-dependent anion channel proteins. Voltage-dependent anion channels appear to be master regulators of mitochondrial bioenergetics and the intracellular flow of energy. Deregulation of voltage-dependent anion channels expression is thought to be related to mitochondrial dysfunction in cancer. The aim of this study was to investigate the mRNA and protein expression levels of VDAC1, VDAC2, and VDAC3 in relation to clinicopathological characteristics of endometrial cancer as well as the prognostic significance of voltage-dependent anion channels expression for overall survival. VDAC1 and VDAC3 expressions were significantly higher in cancer compared to normal tissues. Kaplan-Meier analysis indicated that high expression of all VDAC genes or high VDAC2 protein level predicted poor overall survival. Multivariate analysis identified the VDAC1 and VDAC2 mRNA levels as well as VDAC2 protein level as independent prognostic factors. Our results suggest that increased expression of voltage-dependent anion channels correlates with tumor progression and may serve as a potential prognostic biomarker in endometrial cancer.

Silibinin inhibits migration and invasion of breast cancer MDA-MB-231 cells through induction of mitochondrial fusion.

Human triple negative breast cancer cells, MDA-MB-231, show typical epithelial to mesenchymal transition associated with cancer progression. Mitochondria play a major role in cancer progression, including metastasis. Changes in mitochondrial architecture affect cellular migration, autophagy and apoptosis. Silibinin is reported to have anti-breast cancer effect. We here report that silibinin at lower concentrations (30-90 µM) inhibits epithelial to mesenchymal transition (EMT) of MDA-MB-231, by increasing the expression of epithelial marker, E-cadherin, and decreasing the expression of mesenchymal markers, N-cadherin and vimentin. Besides, silibinin inhibition of cell migration is associated with reduction in the protein expression of matrix metalloproteinases 2 and 9 (MMP2 and MMP9) and paxillin. In addition, silibinin treatment increases mitochondrial fusion through down-regulating the expression of mitochondrial fission-associated protein dynamin-related protein 1 (DRP1) and up-regulating the expression of mitochondrial fusion-associated proteins, optic atrophy 1, mitofusin 1 and mitofusin 2. Silibinin perturbed mitochondrial biogenesis via down-regulating the levels of mitochondrial biogenesis regulators including mitochondrial transcription factor A (TFAM), peroxisome proliferator-activated receptor gamma coactivator (PGC1) and nuclear respiratory factor (NRF2). Moreover, DRP1 knockdown or silibinin inhibited cell migration, and MFN1&2 knockdown restored it. Mitochondrial fusion contributes to silibinin's negative effect on cell migration. Silibinin decreased reactive oxygen species (ROS) generation, leading to inhibition of the NLRP3 inflammasome activation. In addition, knockdown of mitofusin 1&2 (MFN 1&2) relieved silibinin-induced inhibition of NLRP3 inflammasome activation. Repression of ROS contributes to the inhibition of the expression of NLRP3, caspase-1 and IL-ß proteins as well as of cell migration. Taken together, our study provides evidence that silibinin impairs mitochondrial dynamics and biogenesis, resulting in reduced migration and invasion of the MDA-MB-231 breast cancer cells.

Coupling of import and assembly pathways in mitochondrial protein biogenesis.

Biogenesis and function of mitochondria depend on the import of about 1000 precursor proteins that are produced on cytosolic ribosomes. The translocase of the outer membrane (TOM) forms the entry gate for most proteins. After passage through the TOM channel, dedicated preprotein translocases sort the precursor proteins into the mitochondrial subcompartments. Many proteins have to be assembled into oligomeric membrane-integrated complexes in order to perform their functions. In this review, we discuss a dual role of mitochondrial preprotein translocases in protein translocation and oligomeric assembly, focusing on the biogenesis of the TOM complex and the respiratory chain. The sorting and assembly machinery (SAM) of the outer mitochondrial membrane forms a dynamic platform for coupling transport and assembly of TOM subunits. The biogenesis of the cytochrome c oxidase of the inner membrane involves a molecular circuit to adjust translation of mitochondrial-encoded core subunits to the availability of nuclear-encoded partner proteins. Thus, mitochondrial protein translocases not only import precursor proteins but can also support their assembly into functional complexes.

MPC1-like Is a Placental Mammal-specific Mitochondrial Pyruvate Carrier Subunit Expressed in Postmeiotic Male Germ Cells.

Selective transport of pyruvate across the inner mitochondrial membrane by the mitochondrial pyruvate carrier (MPC) is a fundamental step that couples cytosolic and mitochondrial metabolism. The recent molecular identification of the MPC complex has revealed two interacting subunits, MPC1 and MPC2. Although in yeast, an additional subunit, MPC3, can functionally replace MPC2, no alternative MPC subunits have been described in higher eukaryotes. Here, we report for the first time the existence of a novel MPC subunit termed MPC1-like (MPC1L), which is present uniquely in placental mammals. MPC1L shares high sequence, structural, and topological homology with MPC1. In addition, we provide several lines of evidence to show that MPC1L is functionally equivalent to MPC1: 1) when co-expressed with MPC2, it rescues pyruvate import in a MPC-deleted yeast strain; 2) in mammalian cells, it can associate with MPC2 to form a functional carrier as assessed by bioluminescence resonance energy transfer; 3) in MPC1 depleted mouse embryonic fibroblasts, MPC1L rescues the loss of pyruvate-driven respiration and stabilizes MPC2 expression; and 4) MPC1- and MPC1L-mediated pyruvate imports show similar efficiency. However, we show that MPC1L has a highly specific expression pattern and is localized almost exclusively in testis and more specifically in postmeiotic spermatids and sperm cells. This is in marked contrast to MPC1/MPC2, which are ubiquitously expressed throughout the organism. To date, the biological importance of this alternative MPC complex during spermatogenesis in placental mammals remains unknown. Nevertheless, these findings open up new avenues for investigating the structure-function relationship within the MPC complex.

The epigenetic control of E-box and Myc-dependent chromatin modifications regulate the licensing of lamin B2 origin during cell cycle.

Recent genome-wide mapping of the mammalian replication origins has suggested the role of transcriptional regulatory elements in origin activation. However, the nature of chromatin modifications associated with such trans-factors or epigenetic marks imprinted on cis-elements during the spatio-temporal regulation of replication initiation remains enigmatic. To unveil the molecular underpinnings, we studied the human lamin B2 origin that spatially overlaps with TIMM 13 promoter. We observed an early G(1)-specific occupancy of c-Myc that facilitated the loading of mini chromosome maintenance protein (MCM) complex during subsequent mid-G(1) phase rather stimulating TIMM 13 gene expression. Investigations on the Myc-induced downstream events suggested a direct interaction between c-Myc and histone methyltransferase mixed-lineage leukemia 1 that imparted histone H3K4me3 mark essential for both recruitment of acetylase complex HBO1 and hyperacetylation of histone H4. Contemporaneously, the nucleosome remodeling promoted the loading of MCM proteins at the origin. These chromatin modifications were under the tight control of active demethylation of E-box as evident from methylation profiling. The active demethylation was mediated by the Ten-eleven translocation (TET)-thymine DNA glycosylase-base excision repair (BER) pathway, which facilitated spatio-temporal occupancy of Myc. Intriguingly, the genome-wide 43% occurrence of E-box among the human origins could support our hypothesis that epigenetic control of E-box could be a molecular switch for the licensing of early replicating origins.

Folding and biogenesis of mitochondrial small Tim proteins.

Correct and timely folding is critical to the function of all proteins. The importance of this is illustrated in the biogenesis of the mitochondrial intermembrane space (IMS) "small Tim" proteins. Biogenesis of the small Tim proteins is regulated by dedicated systems or pathways, beginning with synthesis in the cytosol and ending with assembly of individually folded proteins into functional complexes in the mitochondrial IMS. The process is mostly centered on regulating the redox states of the conserved cysteine residues: oxidative folding is crucial for protein function in the IMS, but oxidized (disulfide bonded) proteins cannot be imported into mitochondria. How the redox-sensitive small Tim precursor proteins are maintained in a reduced, import-competent form in the cytosol is not well understood. Recent studies suggest that zinc and the cytosolic thioredoxin system play a role in the biogenesis of these proteins. In the IMS, the mitochondrial import and assembly (MIA) pathway catalyzes both import into the IMS and oxidative folding of the small Tim proteins. Finally, assembly of the small Tim complexes is a multistep process driven by electrostatic and hydrophobic interactions; however, the chaperone function of the complex might require destabilization of these interactions to accommodate the substrate. Here, we review how folding of the small Tim proteins is regulated during their biogenesis, from maintenance of the unfolded precursors in the cytosol, to their import, oxidative folding, complex assembly and function in the IMS.

Inner mitochondrial translocase Tim50 interacts with 3ß-hydroxysteroid dehydrogenase type 2 to regulate adrenal and gonadal steroidogenesis.

In the adrenals, testes, and ovaries, 3ß-hydroxysteroid dehydrogenase type 2 (3ßHSD2) catalyzes the conversion of pregnenolone to progesterone and dehydroepiandrostenedione to androstenedione. Alterations in this pathway can have deleterious effects, including sexual development impairment, spontaneous abortion, and breast cancer. 3ßHSD2, synthesized in the cytosol, is imported into the inner mitochondrial membrane (IMM) by translocases. Steroidogenesis requires that 3ßHSD2 acts as both a dehydrogenase and isomerase. To achieve this dual functionality, 3ßHSD2 must undergo a conformational change; however, what triggers that change remains unknown. We propose that 3ßHSD2 associates with IMM or outer mitochondrial membrane translocases facing the intermembrane space (IMS) and that this interaction promotes the conformational change needed for full activity. Fractionation assays demonstrate that 3ßHSD2 associated with the IMM but did not integrate into the membrane. Through mass spectrometry and Western blotting of mitochondrial complexes and density gradient ultracentrifugation, we show that that 3ßHSD2 formed a transient association with the translocases Tim50 and Tom22 and with Tim23. This association occurred primarily through the interaction of Tim50 with the N terminus of 3ßHSD2 and contributed to enzymatic activity. Tim50 knockdown inhibited catalysis of dehydroepiandrostenedione to androstenedione and pregnenolone to progesterone. Although Tim50 knockdown decreased 3ßHSD2 expression, restoration of expression via proteasome and protease inhibition did not rescue activity. In addition, protein fingerprinting and CD spectroscopy reveal the flexibility of 3ßHSD2, a necessary characteristic for forming multiple associations. In summary, Tim50 regulates 3ßHSD2 expression and activity, representing a new role for translocases in steroidogenesis.

Identification of mitochondrial thiamin diphosphate carriers from Arabidopsis and maize.

It is currently held that thiamin is made in chloroplasts and converted in the cytosol to the active cofactor thiamin diphosphate (ThDP), and that mitochondria and plastids import ThDP. The organellar transporters that mediate ThDP import in plants have not been identified. Comparative genomic analysis indicated that two members of the mitochondrial carrier family (MCF) in Arabidopsis (At5g48970 and At3g21390) and two in maize (GRMZM2G118515 and GRMZM2G124911) are related to the ThDP carriers of animals and Saccharomyces cerevisiae. Expression of each of these plant proteins in a S. cerevisiae ThDP carrier (TPC1) null mutant complemented the growth defect on fermentable carbon sources and restored the level of mitochondrial ThDP and the activity of the mitochondrial ThDP-dependent enzyme acetolactate synthase. The plant proteins were targeted to mitochondria as judged by dual import assays with purified pea mitochondria and chloroplasts, and by microscopic analysis of the subcellular localization of green fluorescent protein fusions in transiently transformed tobacco suspension cells. Both maize genes were shown to be expressed throughout the plant, which is consistent with the known ubiquity of mitochondrial ThDP-dependent enzymes. Collectively, these data establish that plants have mitochondrially located MCF carriers for ThDP, and indicate that these carriers are highly evolutionarily conserved. Our data provide a firm basis to propagate the functional annotation of mitochondrial ThDP carriers to other angiosperm genomes.

MICU1-dependent mitochondrial calcium uptake regulates lung alveolar type 2 cell plasticity and lung regeneration.

Lung alveolar type 2 (AT2) cells are progenitors for alveolar type 1 (AT1) cells. Although many factors regulate AT2 cell plasticity, the role of mitochondrial calcium (mCa2+) uptake in controlling AT2 cells remains unclear. We previously identified that the miR-302 family supports lung epithelial progenitor cell proliferation and less differentiated phenotypes during development. Here, we report that a sustained elevation of miR-302 in adult AT2 cells decreases AT2-to-AT1 cell differentiation during the Streptococcus pneumoniae-induced lung injury repair. We identified that miR-302 targets and represses the expression of mitochondrial Ca2+ uptake 1 (MICU1), which regulates mCa2+ uptake through the mCa2+ uniporter channel by acting as a gatekeeper at low cytosolic Ca2+ levels. Our results reveal a marked increase in MICU1 protein expression and decreased mCa2+ uptake during AT2-to-AT1 cell differentiation in the adult lung. Deletion of Micu1 in AT2 cells reduces AT2-to-AT1 cell differentiation during steady-state tissue maintenance and alveolar epithelial regeneration after bacterial pneumonia. These studies indicate that mCa2+ uptake is extensively modulated during AT2-to-AT1 cell differentiation and that MICU1-dependent mCa2+ uniporter channel gating is a prominent mechanism modulating AT2-to-AT1 cell differentiation.

A new Caucasian case of neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD): a clinical, molecular, and functional study.

Citrin is the liver-specific isoform of the mitochondrial aspartate/glutamate carrier (AGC2). AGC2 deficiency is an autosomal recessive disorder with two age related phenotypes: neonatal intrahepatic cholestasis (NICCD, OMIM#605814) and adult-onset type II citrullinemia (CTLN2, OMIM#603471). NICCD arises within the first few weeks of life resulting in prolonged cholestasis and metabolic abnormalities including aminoacidemia and galactosuria. Usually symptoms disappear within the first year of life, thus making a diagnosis difficult after this time. In this study we report a new Caucasian case of NICCD, a seven week old Romanian boy with prolonged jaundice. Sequencing of the AGC2 gene showed a novel homozygous missense double-nucleotide (doublet) mutation, which produces the change of the glycine at position 437 into glutamate. Functional studies, carried out on the recombinant mutant protein, for the first time demonstrated, that NICCD is caused by a reduced transport activity of AGC2. The presence of AGC2 deficiency in other ethnic groups besides Asian population suggests further consideration for NICCD diagnosis of any neonate with an unexplained cholestasis; a prompt diagnosis is crucial to resolve the metabolic decompensation with an appropriate dietary treatment.

Translocase of outer mitochondrial membrane 70 expression is induced by hepatitis C virus and is related to the apoptotic response.

The localization of hepatitis C virus (HCV) proteins in cells leads to several problems. The translocase of outer mitochondrial membrane 70 (TOM70) is a mitochondrial import receptor. In this study, TOM70 expression was induced by HCV infection. TOM70 overexpression induced resistance to tumor necrosis factor-alpha (TNF-α)-mediated apoptosis but not to Fas-induced apoptosis in HepG2 cells. TOM70 was found to be induced by the HCV non-structural protein (NS)3/4A protein, and silencing of TOM70 decreased the levels of the NS3 and Mcl-1 proteins. These results indicate that TOM70 can directly interact with the NS3 protein. In hepatoma cells, silencing of TOM70 induced apoptosis and increased caspase-3/7 activity but did not modify caspase-8 and caspase-9 activity. TOM70 silencing-induced apoptosis was impaired in HCV NS3/4A protein-expressing cells. Thus, this study revealed a novel finding, that is, TOM70 is linked with the NS3 protein and the apoptotic response.

Altered L-Arginine Metabolic Pathways in Gastric Cancer: Potential Therapeutic Targets and Biomarkers.

There is a pressing need for molecular targets and biomarkers in gastric cancer (GC). We aimed at identifying aberrations in L-arginine metabolism with therapeutic and diagnostic potential. Systemic metabolites were quantified using mass spectrometry in 293 individuals and enzymes' gene expression was quantified in 29 paired tumor-normal samples using qPCR and referred to cancer pathology and molecular landscape. Patients with cancer or benign disorders had reduced systemic arginine, citrulline, and ornithine and elevated symmetric dimethylarginine and dimethylamine. Citrulline and ornithine depletion was accentuated in metastasizing cancers. Metabolite diagnostic panel had 91% accuracy in detecting cancer and 70% accuracy in differentiating cancer from benign disorders. Gastric tumors had upregulated NOS2 and downregulated ASL, PRMT2, ORNT1, and DDAH1 expression. NOS2 upregulation was less and ASL downregulation was more pronounced in metastatic cancers. Tumor ASL and PRMT2 expression was inversely related to local advancement. Enzyme up- or downregulation was greater or significant solely in cardia subtype. Metabolic reprogramming in GC includes aberrant L-arginine metabolism, reflecting GC subtype and pathology, and is manifested by altered interplay of its intermediates and enzymes. Exploiting L-arginine metabolic pathways for diagnostic and therapeutic purposes is warranted. Functional studies on ASL, PRMT2, and ORNT1 in GC are needed.

Expression of TOMM34 and Its Clinicopathological Correlations in Urothelial Carcinoma of the Bladder.

The substantial difference between normal cells and cancer cells in terms of their energy metabolism in mitochondria provides an interesting basis for the development of novel therapeutic agents targeting energy machinery of tumour cells. TOMM34 is one of the Tom (translocase of the outer membrane of mitochondria) family that was found to be overexpressed in colorectal, hepatocellular, lung and early invasive breast carcinomas. The expression profile of mitochondrial translocases in bladder cancer compared to normal urinary bladder tissues has not been investigated yet. Therefore, the aim of the current study is to investigate the expression pattern of TOMM34 in bladder cancer tissues and explore its correlation with the clinico-pathological parameters of those cases. Sixty patients who underwent either transurethral resection or radical cystectomy for bladder cancer were included in this study with revision of all their clinicopathological data and tumor slides. Ten histologically normal urothelial biopsies were also included. Immunohistochemical staining for TOMM34 was done and semi-quantitatively scored using the modified H-score. All relations were analysed using established statistical methodologies. TOMM34 overexpression was significantly associated with high tumour stage, muscle invasion and high grade. Significant positive association was observed between TOMM34 expression and poor outcome in terms of shorter disease-specific survival. This study suggests TOMM34 as a biomarker of progression and poor prognosis in urothelial cell carcinoma patients. Furthermore, we suggest a role played by mitochondrial machinery in urothelial cell carcinoma progression, which is a potential target for the newly-discovered vaccine therapy for urothelial cell carcinoma.

Mitochondrial pyruvate carrier abundance mediates pathological cardiac hypertrophy.

Cardiomyocytes rely on metabolic substrates, not only to fuel cardiac output, but also for growth and remodelling during stress. Here we show that mitochondrial pyruvate carrier (MPC) abundance mediates pathological cardiac hypertrophy. MPC abundance was reduced in failing hypertrophic human hearts, as well as in the myocardium of mice induced to fail by angiotensin II or through transverse aortic constriction. Constitutive knockout of cardiomyocyte MPC1/2 in mice resulted in cardiac hypertrophy and reduced survival, while tamoxifen-induced cardiomyocyte-specific reduction of MPC1/2 to the attenuated levels observed during pressure overload was sufficient to induce hypertrophy with impaired cardiac function. Failing hearts from cardiomyocyte-restricted knockout mice displayed increased abundance of anabolic metabolites, including amino acids and pentose phosphate pathway intermediates and reducing cofactors. These hearts showed a concomitant decrease in carbon flux into mitochondrial tricarboxylic acid cycle intermediates, as corroborated by complementary 1,2-[13C2]glucose tracer studies. In contrast, inducible cardiomyocyte overexpression of MPC1/2 resulted in increased tricarboxylic acid cycle intermediates, and sustained carrier expression during transverse aortic constriction protected against cardiac hypertrophy and failure. Collectively, our findings demonstrate that loss of the MPC1/2 causally mediates adverse cardiac remodelling.

The mitochondrial morphology protein Mdm10 functions in assembly of the preprotein translocase of the outer membrane.

The biogenesis of mitochondrial outer membrane proteins involves the general translocase of the outer membrane (TOM complex) and the sorting and assembly machinery (SAM complex). The two known subunits of the SAM complex, Mas37 and Sam50, are required for assembly of the abundant outer membrane proteins porin and Tom40. We have identified an unexpected subunit of the SAM complex, Mdm10, which is involved in maintenance of mitochondrial morphology. Mitochondria lacking Mdm10 are selectively impaired in the final steps of the assembly pathway of Tom40, including the association of Tom40 with the receptor Tom22 and small Tom proteins, while the biogenesis of porin is not affected. Yeast mutants of TOM40, MAS37, and SAM50 also show aberrant mitochondrial morphology. We conclude that Mdm10 plays a specific role in the biogenesis of the TOM complex, indicating a connection between the mitochondrial protein assembly apparatus and the machinery for maintenance of mitochondrial morphology.

Nuclear immunoreactivity of BLM-s, a proapoptotic BCL-2 family member, is specifically detected in salivary adenoid cystic carcinoma.

Tumor cells frequently evade apoptosis triggered by cellular stress via aberrant regulation of the BCL-2 family members, which are key players in regulating cell death under physiological and pathological situations. Previously, we have identified a novel BH3-only protein of the BCL-2 family, BLM-s (BCL-2-like molecule, short form), that modulates apoptosis of postmitotic immature neurons during corticohistogenesis. Whether BLM-s expression correlates with any subtype of human tumors has not been investigated. Here, via BLM-s immunohistochemistry performed in various kinds of human tumors, we demonstrate that BLM-s is specifically expressed in tumors derived from salivary gland (specificity, 0.76 [95% confidence interval, or CI], 0.65-0.85]; sensitivity, 1 [95% CI, 0.99-1]). Stratification of BLM-s immunointensity and its subcellular localization in correlation with salivary gland tumor subtype shows a statistically significant increase in proportion and in intensity of nuclear staining for adenoid cystic carcinoma (ACC; specificity, 0.92 [95% CI, 0.88-0.95]; sensitivity, 0.82 [95% CI, 0.66-0.92]), a locally aggressive head and neck malignancy. Comparison among salivary ACC in correlation with MYB/MYBL fluorescence in situ hybridization, c-KIT immunohistochemistry, and BLM-s immunohistochemistry shows that BLM-s' nuclear immunoreactivity has lower false-negative detection rate (18.5% compared with 26.3% [MYB/MYBL fluorescence in situ hybridization] and 34.2% [c-KIT], respectively). Intriguingly, ACC derived from other cell origins such as breast shows negative BLM-s immunoreactivity. We thus propose that nuclear localization of BLM-s detected by immunohistochemistry could be potentially used as an ancillary diagnostic marker for ACC originating from the salivary gland, especially when the biopsy specimen is small with an unknown tumor origin.

PGC1α promotes cholangiocarcinoma metastasis by upregulating PDHA1 and MPC1 expression to reverse the Warburg effect.

PGC1α acts as a central regulator of mitochondrial metabolism, whose role in cancer progression has been highlighted but remains largely undefined. Especially, it is completely unknown about the effect of PGC1α on cholangiocarcinoma (CCA). Here we showed that PGC1α overexpression had no impact on CCA growth despite the decreased expression of PGC1α in CCA compared with adjacent normal tissue. Instead, PGC1α overexpression-promoted CCA metastasis both in vitro and in vivo. Mechanistically, for the first time, we illuminated that PGC1α reversed the Warburg effect by upregulating the expression of pyruvate dehydrogenase E1 alpha 1 subunit and mitochondrial pyruvate carrier 1 to increase pyruvate flux into the mitochondria for oxidation, whereas simultaneously promoting mitochondrial biogenesis and fusion to mediate the metabolic switch to oxidative phosphorylation. On the one hand, enhanced mitochondrial oxidation metabolism correlated with elevated reactive oxygen species (ROS) production; on the other hand, increased PGC1α expression upregulated the expression levels of mRNA for several ROS-detoxifying enzymes. To this end, the ROS levels, which were elevated but below a critical threshold, did not inhibit CCA cells proliferation. And the moderately increased ROS facilitated metastatic dissemination of CCA cells, which can be abrogated by antioxidants. Our study suggests the potential utility of developing the PGC1α-targeted therapies or blocking PGC1α signaling axis for inhibiting CCA metastasis.

Dihydroartemisinin inhibits the viability of cervical cancer cells by upregulating caveolin 1 and mitochondrial carrier homolog 2: Involvement of p53 activation and NAD(P)H:quinone oxidoreductase 1 downregulation.

Dihydroartemisinin (DHA) has been shown to inhibit the viability of various cancer cells. Previous studies have revealed that the mechanisms involved in the inhibitory effects of DHA are based on theactivation of p53 and the mitochondrial-related cell death pathway. However, the exact association between upstream signaling and the activation of cell death pathway remains unclear. In this study, we found that DHA treatment induced the upregulation of caveolin 1 (Cav1) and mitochondrial carrier homolog 2 (MTCH2) in HeLa cells, and this was associated with the DHA-induced inhibition of cell viability and DHA-induced apoptosis. Additionally, the overexpression of Cav1 and MTCH2 in HeLa cells enhanced the inhibitory effects of DHA on cell viability. Moreover, we also found that the upregulation of Cav1 contributed to the DHA-mediated p53 activation and the downregulation of the redox enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1), which have been reported to contribute to the activation of the cell death pathway. Of note, we also found that DHA induced the nuclear translocation and accumulation of both Cav1 and p53, indicating a novel potential mechanism, namely the regulation of p53 activation by Cav1. On the whole, our study identified Cav1 and MTCH2 as the molecular targets of DHA and revealed a new link between the upstream Cav1/MTCH2 upregulation and the downstream activation of the cell death pathway involved in the DHA-mediated inhibition of cell viability.

Mff-Dependent Mitochondrial Fission Contributes to the Pathogenesis of Cardiac Microvasculature Ischemia/Reperfusion Injury via Induction of mROS-Mediated Cardiolipin Oxidation and HK2/VDAC1 Disassociation-Involved mPTP Opening.

BACKGROUND: The cardiac microvascular system ischemia/reperfusion injury following percutaneous coronary intervention is a clinical thorny problem. This study explores the mechanisms by which ischemia/reperfusion injury induces cardiac microcirculation collapse. METHODS AND RESULTS: In wild-type mice, mitochondrial fission factor (Mff) expression increased in response to acute microvascular ischemia/reperfusion injury. Compared with wild-type mice, homozygous Mff-deficient (Mffgt) mice exhibited a smaller infarcted area, restored cardiac function, improved blood flow, and reduced microcirculation perfusion defects. Histopathology analysis demonstrated that cardiac microcirculation endothelial cells (CMECs) in Mffgt mice had an intact endothelial barrier, recovered phospho-endothelial nitric oxide synthase production, opened lumen, undivided mitochondrial structures, and less CMEC death. In vitro, Mff-deficient CMECs (derived from Mffgt mice or Mff small interfering RNA-treated) demonstrated less mitochondrial fission and mitochondrial-dependent apoptosis compared with cells derived from wild-type mice. The loss of Mff inhibited mitochondrial permeability transition pore opening via blocking the oligomerization of voltage-dependent anion channel 1 and subsequent hexokinase 2 separation from mitochondria. Moreover, Mff deficiency reduced the cyt-c leakage into the cytoplasm by alleviating cardiolipin oxidation resulting from damage to the electron transport chain complexes and mitochondrial reactive oxygen species overproduction. CONCLUSIONS: This evidence clearly illustrates that microcirculatory ischemia/reperfusion injury can be attributed to Mff-dependent mitochondrial fission via voltage-dependent anion channel 1/hexokinase 2-mediated mitochondrial permeability transition pore opening and mitochondrial reactive oxygen species/cardiolipin involved cyt-c release.

Identification of TOMM34, which shows elevated expression in the majority of human colon cancers, as a novel drug target.

In an attempt to isolate potential molecular targets for diagnosis, treatment and/or prevention of colorectal cancer (CRC), we have been analyzing expression profiles of clinical samples from CRC patients using genome-wide cDNA microarray. Among the genes up-regulated frequently in colorectal tumors, we here focused on TOMM34 (34 kDa-translocase of the outer mitochondrial membrane). Immunohistochemical staining revealed significant accumulation of TOMM34 protein in CRC tissues compared with their corresponding non-cancerous mucosae. Transfection of colon cancer HCT116 cells with short-interfering RNA (siRNA) specific to TOMM34 effectively suppressed its expression and drastically inhibited cell growth. These findings suggest that TOMM34 is involved in the growth of cancer cells, and may contribute to the development of novel anticancer drugs and/or diagnosis for CRC.