Cyfluthrin exposure during pregnancy causes neurotoxicity in offspring-Ca2+ overload via IP3R-GRP75-VDAC1 pathway.

Cyfluthrin (Cy) is a widely used pyrethroid insecticide. There is growing evidence that Cy can cause damage to the nervous, reproductive, and immune systems, but there is limited evidence on the potential effects of maternal Cy exposure on offspring. A model of maternal Cy exposure was used to assess its neurobehavioral effects on young-adult offspring. We found that gestational Cy exposure affected pregnancy outcomes and fetal development, and that offspring showed impairments in anxiety as well as learning and memory, accompanied by impairments in hippocampal synaptic ultrastructure and synaptic plasticity. In addition, the IP3R-GRP75-VDAC1 apoptogenic pathway was also upregulated, and in vitro models showed that inhibition of this pathway alleviated neuronal apoptosis as well as synaptic plasticity damage. In conclusion, maternal Cy exposure during pregnancy can cause neurobehavioral abnormalities and synaptic damage in offspring, which may be related to neuronal apoptosis induced by activation of the IP3R-GRP75-VDAC1 pathway in the hippocampus of offspring. Our findings provide clues to understand the neurotoxicity mechanism of maternal Cy exposure to offspring during pregnancy.

Hierridin B Isolated from a Marine Cyanobacterium Alters VDAC1, Mitochondrial Activity, and Cell Cycle Genes on HT-29 Colon Adenocarcinoma Cells.

BACKGROUND: Hierridin B was isolated from a marine cyanobacterium Cyanobium sp. strain and induced cytotoxicity selectively in HT-29 adenocarcinoma cells. The underlying molecular mechanism was not yet elucidated. METHODS: HT-29 cells were exposed to the IC50 concentration of hierridin B (100.2 µM) for 48 h. Non-targeted proteomics was performed using 2D gel electrophoresis and MALDI-TOF/TOF mass spectrometry. The mRNA expression of apoptotic and cell cycle genes were analyzed by real-time PCR. Automated quantification of 160 cytoplasm and mitochondrial parameter was done by fluorescence microscopy using CellProfiler software. RESULTS: Proteomics identified 21 significant different proteins, which belonged to protein folding/synthesis and cell structure amongst others. Increase of VDAC1 protein responsible for formation of mitochondrial channels was confirmed by mRNA expression. A 10-fold decrease of cytoskeleton proteins (STMN1, TBCA) provided a link to alterations of the cell cycle. CCNB1 and CCNE mRNA were decreased two-fold, and P21CIP increased 10-fold, indicative of cell cycle arrest. Morphological analysis of mitochondrial parameter confirmed a reduced mitochondrial activity. CONCLUSION: Hierridin B is a potential anticancer compound that targets mitochondrial activity and function.

Protein coingestion with alcohol following strenuous exercise attenuates alcohol-induced intramyocellular apoptosis and inhibition of autophagy.

Alcohol ingestion decreases postexercise rates of muscle protein synthesis, but the mechanism(s) (e.g., increased protein breakdown) underlying this observation is unknown. Autophagy is an intracellular "recycling" system required for homeostatic substrate and organelle turnover; its dysregulation may provoke apoptosis and lead to muscle atrophy. We investigated the acute effects of alcohol ingestion on autophagic cell signaling responses to a bout of concurrent (combined resistance- and endurance-based) exercise. In a randomized crossover design, eight physically active males completed three experimental trials of concurrent exercise with either postexercise ingestion of alcohol and carbohydrate (12 ± 2 standard drinks; ALC-CHO), energy-matched alcohol and protein (ALC-PRO), or protein (PRO) only. Muscle biopsies were taken at rest and 2 and 8 h postexercise. Select autophagy-related gene (Atg) proteins decreased compared with rest with ALC-CHO (P < 0.05) but not ALC-PRO. There were parallel increases (P < 0.05) in p62 and PINK1 commensurate with a reduction in BNIP3 content, indicating a diminished capacity for mitochondria-specific autophagy (mitophagy) when alcohol and carbohydrate were coingested. DNA fragmentation increased in both alcohol conditions (P < 0.05); however, nuclear AIF accumulation preceded this apoptotic response with ALC-CHO only (P < 0.05). In contrast, increases in the nuclear content of p53, TFEB, and PGC-1α in ALC-PRO were accompanied by markers of mitochondrial biogenesis at the transcriptional (Tfam, SCO2, and NRF-1) and translational (COX-IV, ATPAF1, and VDAC1) level (P < 0.05). We conclude that alcohol ingestion following exercise triggers apoptosis, whereas the anabolic properties of protein coingestion may stimulate mitochondrial biogenesis to protect cellular homeostasis.

mTOR-targeted therapy: differential perturbation to mitochondrial membrane potential and permeability transition pore plays a role in therapeutic response.

While cancer cell mitochondria mediate actions of many successful chemotherapeutics, little is known about mitochondrial response in mTOR-targeted anticancer therapy. We have studied mitochondrial dynamics in relation to growth suppression employing an allosteric inhibitor rapalog, a highly selective mTOR kinase inhibitor (mTOR-KI) and mTOR-ShRNA. Global targeting of mTOR increased mitochondrial membrane potential (mΔψ) and inhibited mitochondrial permeability transition pore (mPTP). Importantly, these mTOR-KI-provoked anti-survival and pro-survival effects were differentially manifested in diverse cancer cells according to intrinsic susceptibility to mTOR-targeting. The most-sensitive cells including those possessing hyperactive PI3K/AKT/mTOR and/or growth factor-dependence (LNCap, MDA361 and MG63) all displayed a dramatic increase in mΔψ, whereas the mΔψ increase was not evident in majority of resistant cancer cells. Upon mTOR-KI treatment, the resistant cells including those harboring K-Ras- or B-Raf mutation (MDA231, HT29 and HCT116) all displayed a markedly reduced mPTP opening, which paralleled a sustained AKT-hexokinase 2 (HK2) survival signaling and persistent phosphorylation (inactivation) of GSK3ß. Further studies demonstrated that the mTOR-KI-provoked mPTP closure in resistant cells was mediated through an enhanced binding of HK2 to the mitochondrial voltage-dependent anion channel (VDAC), a molecular mechanism known to promote mPTP closure and cell survival. Detaching HK2 from VDAC by an HK2-displacing peptide or methyl jasmonate specifically blocked the mTOR-KI-provoked mPTP closure and potentiated growth suppression in resistant cells. Thus, mTOR-inhibition can exert complex and differential perturbation to mitochondrial dynamics in cancer cells, which likely influence therapeutic outcome of mTOR-targeted therapy.

[Interference of hexavalent chromium on VDAC1 mRNA expression or ATP level and their potential association].

OBJECTIVE: To explore the interference of hexavalent chromium-Cr(VI) on voltage-dependent anion channel (VDAC1) mRNA expression and intracellular adenosine triphosphate (ATP) level in cells and their potential association. METHODS: Cultured L-02 hepatocytes were treated with 2, 4, 8, 16 and 32 micromol/L of Cr(VI) for 12, 24 and 36 hours (h). The expression of VDAC1 mRNA was measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR), whereas the levels of intracellular ATP was determined by an ATP-specific bioluminescence assay. RESULTS: (1) The expression of VDAC1 mRNA in cells of treated groups was less than that of control group at 12h, and then showed slight increase at 24h, and increased significantly after 36 h of treatment. (2) After being treated by Cr(VI) for 12 h, ATP level in cells increased, especially in higher dose groups, and the ATP level then decreased significantly at 24h except in the lowest dosage (2 micromol/L) group, and increased again at 36h in 2 micromol/L and 4 micromol/L groups, but the ATP level in 8, 16 and 32 micromol/L groups remained less than that in the control group. (3) Correlation analysis showed a moderate negative relationship between VDAC1 mRNA expression and intracellular ATP level during Cr (VI) treatment (r = -0.604, P < 0.05). CONCLUSION: The interference of Cr(VI) on ATP level was associated with abnormal expression of VDAC1 mRNA, over-expression of VDAC1 mRNA may be one of molecular mechanisms for the decrease of ATP level induced by heavy metal-Cr(VI).

Mitochondrial VDAC1: function in cell life and death and a target for cancer therapy.

Found at the outer mitochondrial membrane, the voltage-dependent anion channel, VDAC, assumes a crucial position in the cell, serving as the main interface between mitochondrial and cellular metabolisms by mediating transport of ions and metabolites. VDAC thus functions as a gatekeeper, controlling cross-talk between mitochondria and the rest of the cell. Moreover, its location at the boundary between the mitochondria and the cytosol enables VDAC to interact with proteins that mediate and regulate the integration of mitochondrial functions with other cellular activities. Here, we review current knowledge related to the roles played by VDAC in the regulation of cell life and cell death, with relation to cancer. The current concepts of altered metabolism in cancer cells are presented with specific emphasis on mitochondrial, more specifically VDAC1-bound hexokinase (HK), facilitating and promoting the high glycolytic tumor phenotype. In this respect, the up-regulation of HK expression in tumor cells and its binding to VDAC provide both a metabolic benefit and apoptosis-suppressive capacity that offers the cell a growth advantage and increases its resistance to chemotherapy. VDAC has also been recognized as a key protein in mitochondria-mediated apoptosis since it is the proposed target for the pro- and antiapoptotic Bcl-2-family of proteins, as well as due to its function in the release of apoptotic proteins located in the inter-membranal space. These and other functions point to VDAC1 as being a rational target for the development of a new generation of therapeutics.

Decreased expression of the voltage-dependent anion channel in differentiated PC-12 and SH-SY5Y cells following low-level Pb exposure.

Lead (Pb) has been shown to disrupt cellular energy metabolism, which may underlie the learning deficits and cognitive dysfunctions associated with environmental Pb exposure. The voltage-dependent anion channel (VDAC) plays a central role in regulating energy metabolism in neurons by maintaining cellular ATP levels and regulating calcium buffering, and studies have shown that VDAC expression is associated with learning in mice. In this study, we examined the effect of 5 and 10microM Pb on VDAC expression in vitro in order to determine whether Pb alters VDAC expression levels in neuronal cell lines. PC-12 and SH-SY5Y cells were used since they differentiate to resemble primary neuronal cells. VDAC expression levels were significantly decreased 48 h after exposure to Pb in both cell lines. In contrast, exposure to 24 h of hypoxia failed to produce a decrease in VDAC, suggesting that decreased VDAC expression is not a general cellular stress response but is a result of Pb exposure. This decreased VDAC expression was also correlated with a corresponding decrease in cellular ATP levels. Real-time reverse transcription-polymerase chain reaction demonstrated a significant decrease in messenger RNA levels for the VDAC1 isoform, indicating that Pb reduces transcription of VDAC1. These results demonstrate that exposure to 5 and 10microM Pb reduces VDAC transcription and expression and is associated with reduced cellular ATP levels.

Molecular mechanism of reduction in pregnenolone synthesis by cigarette smoke.

Steroidogenic acute regulatory protein (StAR) facilitates the movement of cholesterol from the outer to inner mitochondrial membrane for the synthesis of pregnenolone. Here, we investigated the molecular mechanism of the reduction of pregnenolone synthesis by cigarette smoke condensate (CSC). Pre-exposure or post-exposure of cells with CSC led to reduced pregnenolone synthesis, in a fashion similar to its effect on isolated mitochondria. However, there was no difference in the expression of 30 kDa StAR in cells treated with moderately concentrated CSC by either regimen. The active form of 37 kDa StAR is degraded easily suggesting that the continuous presence of CSC reduces StAR expression. Mitochondrial import of (35)S-methionine-labeled StAR followed by extraction of the StAR-mitochondrial complex with 1% digitonin showed similarly sized complexes in the CSC-treated and untreated mitochondria. Further analysis by sucrose density gradient centrifugation showed a specific complex, "complex 2", in the untreated mitochondria but absent in the CSC-treated mitochondria. Mass spectrometric analysis revealed that complex 2 is the outer mitochondrial protein, VDAC1. Knockdown of VDAC1 expression by siRNA followed by co-transfection with StAR resulted in a lack of pregnenolone synthesis and 37 kDa StAR expression with reduced expression of the intermediate, 32 kDa StAR. Taken together, these results suggest that in the absence of VDAC1, active StAR expression is reduced indicating that VDAC1 expression is essential for StAR activity. In the absence of VDAC1-StAR interaction, cholesterol cannot be transported into mitochondria; thus the interaction with VDAC1 is a mandatory step for initiating steroidogenesis.