ATP Cell Viability Research Articles

Miltefosine induces reproductive toxicity during sperm capacitation by altering PI3K/AKT signaling pathway

Miltefosine is the first and only drug approved for the treatment of leishmaniasis. It is also known as a PI3K/AKT signaling pathway inhibitor utilized in anti-cancer or anti-viral therapies. However, the impact of miltefosine on male fertility has not been fully understood. Therefore, this study was performed to investigate the effects of miltefosine on sperm function during capacitation. Duroc spermatozoa were exposed to 0, 2.5, 5, 10, 20, 40, and 80 μM miltefosine and induced for capacitation. Our results showed that miltefosine dramatically increased the expression of PI3K/AKT signaling pathway-associated proteins. Sperm motility, motion kinetics, capacitation, and tyrosine phosphorylation were significantly suppressed by miltefosine. However, intracellular ATP levels and cell viability were not significantly affected. Our findings suggest that miltefosine may disrupt sperm function by abnormally increasing the levels of PI3K/AKT signaling pathway-associated proteins. Therefore, the harmful effects of miltefosine on male reproduction should be considered when using this drug.

Short-term regulation of TSFM level does not alter amyloidogenesis and mitochondrial function in type-specific cells.

Mitochondrial Ts translation elongation factor (TSFM) is an enzyme that catalyzes exchange of guanine nucleotides. By forming a complex with mitochondrial Tu translation elongation factor (TUFM), TSFM participates in mitochondrial protein translation. We have previously reported that TUFM regulates translation of beta-site APP cleaving enzyme 1 (BACE1) via ROS (reactive oxygen species)-dependent mechanism, suggesting a potential role in amyloid precursor protein (APP) processing associated with Alzheimer's disease (AD), which led to the speculation that TSFM may regulate APP processing in a similar way to TUFM. Here, we report that in cultured cells, knockdown or overexpression TSFM did not change protein levels in BACE1 and APP. Besides, the levels of cytoplasmic ROS and mitochondrial superoxide, in addition to ATP level, cell viability and mitochondrial membrane potential were not significantly altered by TSFM knockdown in the short term. Further transcriptome analysis revealed that expression of majority of mitochondrial genes were not remarkably changed by TSFM silencing. The possibility of TSFM involved in cardiomyopathy and cancer development was uncovered using bioinformatics analysis. Collectively, short-term regulation of TSFM level in cultured cells does not cause a significant change in proteins involved in APP processing, levels in ROS and ATP associated with mitochondrial function. Whereas our study could contribute to comprehend certain clinical features of TSFM mutations, the roles of TSFM in cardiomyopathy and cancer development might deserve further investigation.

Genetic and pharmacologic p32-inhibition rescue CHCHD2-linked Parkinson's disease phenotypes in vivo and in cell models.

Mutations in CHCHD2 have been linked to Parkinson's disease, however, their exact pathophysiologic roles are unclear. The p32 protein has been suggested to interact with CHCHD2, however, the physiological functions of such interaction in the context of PD have not been clarified. Interaction between CHCHD2 and p32 was confirmed by co-immunoprecipitation experiments. We studied the effect of p32-knockdown in the transgenic Drosophila and Hela cells expressing the wild type and the pathogenic variants of hCHCHD2. We further investigated the rescue ability of a custom generated p32-inhibitor in these models as well as in the human fibroblast derived neural precursor cells and the dopaminergic neurons harboring hCHCHD2-Arg145Gln. Our results showed that wildtype and mutant hCHCHD2 could bind to p32 in vitro, supported by in vivo interaction between human CHCHD2 and Drosophila p32. Knockdown of p32 reduced mutant hCHCHD2 levelsin Drosophila and in vitro. In Drosophila hCHCHD2 models, inhibition of p32 through genetic knockdown and pharmacological treatment using a customized p32-inhibitor restored dopaminergic neuron numbers and improved mitochondrial morphology. These were correlated with improved locomotor function, reduced oxidative stress and decreased mortality. Consistently, Hela cells expressing mutant hCHCHD2 showed improved mitochondrial morphology and function after treatment with the p32-inhibitor. As compared to the isogenic control cells, large percentage of the mutant neural precursor cells and dopaminergic neurons harboring hCHCHD2-Arg145Gln contained fragmented mitochondria which was accompanied by lower ATP production and cell viability. The NPCs harboring hCHCHD2-Arg145Gln also had amarked increase in α-synuclein expression. The p32-inhibitor was able to ameliorate the mitochondrial fragmentation, restored ATP levels, increased cell viability and reduced α-synuclein level in these cells. Our study identified p32 as a modulator of CHCHD2, possibly exerting its effects by reducing the toxic mutant hCHCHD2 expression and/or mitigating the downstream effects. Inhibition of the p32 pathway can be a potential therapeutic intervention for CHCHD2-linked PD and diseases involving mitochondrial dysfunction.

6-n-Butoxy-10-nitro-12,13-dioxa-11-azatricyclo[7.3.1.02,7]trideca-2,4,6,10-tetraene Improves the X-ray Sensitivity on Inhibiting Proliferation and Promoting Oxidative Stress and Apoptosis of Oral Cancer Cells.

This in vitro study examines the anti-oral cancer effects and mechanisms of a combined X-ray/SK2 treatment, i.e., X-ray and 6-n-butoxy-10-nitro-12,13-dioxa-11-azatricyclo[7.3.1.02,7]trideca-2,4,6,10-tetraene (SK2). ATP cell viability and flow cytometry-based cell cycle, apoptosis, oxidative stress, and DNA damage assessments were conducted. The X-ray/SK2 treatment exhibited lower viability in oral cancer (Ca9-22 and CAL 27) cells than in normal (Smulow-Glickman, S-G) cells, i.e., 32.0%, 46.1% vs. 59.0%, which showed more antiproliferative changes than with X-ray or SK2 treatment. Oral cancer cells under X-ray/SK2 treatment showed slight subG1 and G2/M increments and induced high annexin V-monitored apoptosis compared to X-ray or SK2 treatment. The X-ray/SK2 treatment showed higher caspase 3 and 8 levels for oral cancer cells than other treatments. X-ray/SK2 showed a higher caspase 9 level in CAL 27 cells than other treatments, while Ca9-22 cells showed similar levels under X-ray and/or SK2. The X-ray/SK2 treatment showed higher reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) depletion than other treatments. Meanwhile, the mitochondrial superoxide (MitoSOX) and glutathione levels in X-ray/SK2 treatment did not exhibit the highest rank compared to others. Moreover, oral cancer cells had higher γH2AX and/or 8-hydroxy-2-deoxyguanosine levels from X-ray/SK2 treatment than others. All these measurements for X-ray/SK2 in oral cancer cells were higher than in normal cells and attenuated by N-acetylcysteine. In conclusion, X-ray/SK2 treatment showed ROS-dependent enhanced antiproliferative, apoptotic, and DNA damage effects in oral cancer cells with a lower cytotoxic influence on normal cells.

Investigation of the Dual Role of Scorzonera pygmaea: Cytotoxic Activity and Antioxidant Potential

Scorzonera pygmaea, an indigenous plant of western Anatolia, belongs to the Scorzonera genus, which includes various species recognized for their edible and medicinal properties. This study is aimed at investigating the phenolic composition of S. pygmaea; evaluating its antioxidant, cytotoxic, apoptotic, and genotoxic activities; and assessing the impact of S. pygmaea on the expression of cell cycle regulatory genes. HPLC-DAD analysis identified chlorogenic acid as the predominant phenolic compound, potentially linking plant to dietary benefits. The antioxidant potential, evaluated using DPPH radical scavenging and CUPRAC assays, yielded an IC50 value of 63.53 ± 6.29 μg/ml and a Trolox equivalent of 0.061 ± 0.013 g/g, respectively. To assess antigrowth activity, the S. pygmaea extract was tested against MCF-7 and MDA-MB-231 breast cancer cell lines, along with the nonmalignant MCF-10A cell line, using sulforhodamine B and ATP cell viability assays. The results exhibited highly consistent IC50 values of 104.63, 179.27, and 90.83 μg/ml, respectively. Notably, the S. pygmaea extract induced apoptosis in all cell lines, with MDA-MB-231 cells displaying a higher percentage of early apoptotic cells under fluorescence microscopy. The comet assay revealed that S. pygmaea extract induced DNA damage in all cell lines, with MCF-7 and MCF-10A cells showing an accumulation of DNA damage over time. Gene expression analysis revealed significant alterations in cell cycle regulators, with each cell line displaying distinct and time-dependent patterns after exposure to S. pygmaea. Collectively, these findings underscore the cytotoxic, apoptotic, and genotoxic activities of S. pygmaea while simultaneously emphasizing its antioxidant potential, especially given its high chlorogenic acid content. The dual role of S. pygmaea, evidenced by its cytotoxic and antioxidant properties, suggests the potential for further exploration of its utilization within the food industry as a possible dietary resource, while necessitating further studies to fully understand its implications.

Oleuropein exhibits anticarcinogen effects against gastric cancer cell lines.

Oleuropein (OLE), the main phenolic compound of the olive fruit and leaves, has many heathful effects. Gastric cancer is the most fatal malignancy in many parts of the world and it is generally related to harmful dietetic factors. The anticarcinogenic role of OLE in gastric cancer has not been studied sufficiently yet. In this study, we aimed to research the cytotoxic, genotoxic and apoptotic effects of OLE on gastric adenocancer (AGS) cells in vitro. A standard cell line derived from gastric adeno cancer (AGS) cells was employed, and its performance following a 24-hour exposure to OLE at various doses was examined. The ATP cell viability assay, 2',7'-dichlorodihydrofluorescein-diacetate assay (H2DCF-DA) and alkaline single cell gel electrophoresis assay (Comet Assay) were used to study the cytotoxicity, production of reactive oxygen species (ROS) and genotoxicity respectively. The induction of apoptosis was discovered using flow cytometry. OLE reduced AGS cells viability about 60% at maximum concentration (500 µmol/L) and also resulted in approximately 100% DNA damage and about 40% apoptosis with necrosis in AGS cells depending on the increased doses. Cell viability was also significantly decreased in relation to increased intracellular reactive oxygen species (ROS) levels (p < 0.05 - 0.001). Oleuropein has shown significant anticarcinogen effects against gastric adenocancer (AGS) cells in vitro. Oleuropein, a nutrient rich in olive and olive oil, seems to be both protective and therapeutic against gastric cancer and may be a new chemotherapeutic agent in the future.

Heavy metals do not induce ROS production by mitochondrial respirasome

Since the discovery of the respirasome constituted by complexes I, III2, and IV, its precise participation in mitochondrial bioenergetics is poorly understood. We previously determined a higher NADH:DBQ oxidoreductase activity coupled to a lower ROS production by the respirasome than the free complex I. Toxicological studies suggest that respiratory complexes are heavy metals target during mitochondrial intoxication increasing ROS production, reducing ATP synthesis and cell viability; however, the inhibition of respiratory complexes activities by heavy metals is still unknown. Here we showed a putative deactivation of the respirasomal-complex I by seven of the most toxicologically relevant heavy metals, without increasing the ROS production. Contrastingly, the free complex I was more resistant to heavy metals but was 30 times more ROS-producing. These results underlie the preventive role of the respirasome in mitochondrial electron leak and ROS production and recall its disassembled in some pathologies which involve mitochondrial damage and oxidative stress.

Chlorogenic acid induces endoplasmic reticulum stress in Botrytis cinerea and inhibits gray mold on strawberry

Chlorogenic acid (CGA) is a plant-derived compound with antifungal activity. However, the antifungal mechanism of CGA remains not fully revealed. The antifungal effect of CGA against Botrytis cinerea was assessed in the present study, and its mode of action was studied. Results demonstrated that CGA suppressed the growth of B. cinerea, as well as the development of gray mold (B. cinerea) on strawberry. Further analysis indicated that CGA induced endoplasmic reticulum (ER) stress in hyphae of B. cinerea resulting in an efflux of calcium (Ca2+) from the ER to the cytoplasm and then into mitochondria. The Ca2+ overload in mitochondria elicited the Ca2+-calmodulin-calcineurin cascade, resulting in a superoxide burst in the mitochondria. Finally, a decrease in ATP content and cell viability was observed in B. cinerea. This is the first report showing that the inhibitory effect of CGA towards B. cinerea involves an ER stress, followed by a Ca2+ release from the ER to the mitochondria, resulting in a superoxide burst and subsequent oxidative injury that reduces cell viability. Thus, CGA can be considered as an effectively substance to control gray mold on strawberry.

Evaluation of Radiation Sensitivity Differences in Mouse Liver Tumor Organoids Using CRISPR/Cas9-Mediated Gene Mutation.

To assess the radiosensitivity of liver tumors harboring different genetic mutations, mouse liver tumors were generated in vivo through the hydrodynamic injection of clustered regularly interspaced short palindromic repeat/caspase 9 (CRISPR/Cas9) constructs encoding single-guide RNAs (sgRNAs) targeting Tp53, Pten, Nf1, Nf2, Tsc2, Cdkn2a, or Rb1. The plasmid vectors were delivered to the liver of adult C57BL/6 mice via hydrodynamic tail vein injection. The vectors were injected into 10 mice in each group. Organoids were generated from mouse liver tumors. The radiation response of the organoids was assessed using an ATP cell viability assay. The mean survival period of mice injected with vectors targeting Nf2 (4.8 months) was lower than that of other mice. Hematoxylin and eosin staining, immunohistochemical (IHC) staining, and target sequencing analyses revealed that mouse liver tumors harbored the expected mutations. Tumor organoids were established from mouse liver tumors. Histological evaluation revealed marked morphological similarities between the mouse liver tumors and the generated tumor organoids. Moreover, IHC staining indicated that the parental tumor protein expression pattern was maintained in the organoids. The results of the ATP cell viability assay revealed that the tumor organoids with mutated Nf2 were more resistant to high-dose radiation than those with other gene mutations. This study developed a radiation response assessment system for mouse tumors with mutant target genes using CRISPR/Cas9 and organoids. The Tp53 and Pten double mutation in combination with the Nf2 mutation increased the radiation resistance of tumors. The system used in this study can aid in elucidating the mechanism underlying differential intrinsic radiation sensitivity of individual tumors.

PBDE-47 induces impairment of mitochondrial biogenesis and subsequent neurotoxicity through miR-128-3p/PGC-1α axis.

The potential adverse effects of 2,2',4,4'-tetrabromodiphenyl ether (PBDE-47) on neurons are extensively studied, and mitochondria are identified as critical targets. This study aimed to investigate whether PBDE-47 impairs mitochondrial biogenesis via the miR-128-3p/PGC-1α axis to trigger mitochondrial dysfunction-related neuronal damage. In vitro neuroendocrine pheochromocytoma (PC12) cells and in vivo Sprague Dawley rat model were adopted. In this study, biochemical methods were used to examine mitochondrial ATP content, cell viability, and expressions of key mitochondrial biogenesis regulators, including peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF1), and mitochondrial transcription factor A (TFAM). Mimics and inhibitors of miR-128-3p were employed to explore its role in PBDE-47-induced neurotoxicity. Both in vivo and in vitro evidences suggested that PBDE-47 suppressed PGC-1α/NRF1/TFAM signaling pathways and mitochondrial DNA (mtDNA) encoding proteins synthesis. PBDE-47 also suppressed the relative mtDNA content, mRNA levels of mtDNA-encoded subunits, and mitochondrial ATP levels in vitro. Specifically, 2-(4-tert-butylphenyl) benzimidazole (ZLN005) alleviated PBDE-47-induced neuronal death through the improvement of mitochondrial function by activating PGC-1α/NRF1/TFAM signaling pathways. Mechanistically, PBDE-47 dramatically upregulated miR-128-3p expression. Furthermore, miR-128-3p inhibition enhanced PGC-1α/NRF1/TFAM signaling and abolished PBDE-47-induced impairment of mitochondrial biogenesis. In summary, this study provides in vitro evidence to reveal the role of mitochondrial biogenesis in PBDE-47-induced mitochondrial dysfunction and related neurotoxicity and suggests that miR-128-3p/PGC-1α axis may be a therapeutic target for PBDE-47 neurotoxicity.

Proteomic analysis of antifungal mechanism of star anise essential oil against Aspergillus niger and its application potential in prolonging bread shelf life

In this paper, the main chemical constituents of star anise essential oil (AEO) were analyzed. Then the antifungal mechanism of AEO against Aspergillus niger was investigated by proteomics and its application potential in prolonging the shelf life of bread was analyzed. à-Guaiene was the highest content in AEO, followed by Azulene and Terpinen-4-ol. AEO can reduce the ATP content and cell viability of A. niger. A total of 283 protein remarkable expressions of A. niger after AEO treatment were found through protein omics. Further analysis showed that these differentially expressed proteins were mainly concentrated in the energy metabolism system of A. niger. AEO inhibits the growth of A. niger by blocking the synthetic pathway of NAD. In addition, A. niger activated additional energy production pathways, such as glycan metabolism, to compensate for its lack of energy after AEO treatment. Finally, shelf-life experiments showed that AEO extended the shelf life of bread to day 8. These results suggested that AEO can be used as an antifungal agent to prolong the shelf life of bread.

Cytochrome P450 3A5 polymorphism affects the metabolism of sorafenib and its toxicity for hepatocellular carcinoma cells in vitro.

Cytochrome P450 3A5 (CYP3A5) is a highly polymorphic gene and the encoded protein variants differ in catalytic activity, leading to inter-individual variation in metabolic ability. The aim of the current study was to investigate the effects of seven allelic variants on the ability of CYP3A5 to metabolize sorafenib in vitro and further explore the impacts of CYP3A5 polymorphism on the proliferation and apoptosis of hepatocellular carcinoma cell line (HepG2) induced by sorafenib. Wild-type and variant CYP3A5 enzymes were expressed in Spodoptera frugiperda insect cells using a baculovirus dual-expression system, and protein expression was checked by western blot. The enzymes were incubated with sorafenib at 37°C for 30 min, and formation of the major metabolite sorafenib N-oxide was assayed using ultra-performance liquid chromatography and tandem mass spectrometry. Intrinsic clearance values (Vmax/Km) were calculated for each enzyme. Additionally, recombinant HepG2 cells transfecting with CYP3A5 variants were used to investigate the effects of sorafenib on the proliferation of HepG2 cells. Intrinsic clearance of the six variants CYP3A5*2, CYP3A5*3A, CYP3A5*3C, CYP3A5*4, CYP3A5*5, and CYP3A5*7 was 26.41-71.04% of the wild-type (CYP3A5*1) value. In contrast, the clearance value of the variant CYP3A5*6 was significantly higher (174.74%). Additionally, the decreased ATP levels and cell viability and the increased cell apoptosis in HepG2 cells transfected with CYP3A5*2, CYP3A5*3A, CYP3A5*3C, CYP3A5*4, CYP3A5*5, and CYP3A5*7 were observed, whereas, the increased ATP levels and cell viability and the reduced cell apoptosis in HepG2 cells transfected with CYP3A5*6 were also investigated when compared to CYP3A5*1. Our results suggest that CYP3A5 polymorphism influences sorafenib metabolism and pharmacotherapeutic effect in hepatic carcinomas. These data may help explain differential response to drug therapy for hepatocellular carcinoma, and they support the need for individualized treatment.

Microplastics hamper the fertilization success of a broadcast spawning bivalve through reducing gamete collision and gamete fusion efficiency.

By employing external fertilization (broadcast spawning) as a mating strategy, the gametes and subsequent fertilization of various marine invertebrates are directly subjected to pollution. Although microplastics (MPs) are ubiquitous in marine environments, their potential effects on the fertilization of broadcast spawners remain largely unknown. Therefore in this study, the impacts of polystyrene MPs on the fertilization success of broadcast spawning bivalve (Tegillarca granosa) were investigated. In order to reveal the underlying mechanisms affecting fertilization, the sperm swimming performance, sperm ATP status, sperm viability, DNA integrity, gamete collision probability, gamete fusion efficiency, enzymatic antioxidants, and key ion transport enzyme activities were analyzed. The results showed that MPs weakened the sperm swimming performance through reducing ATP production and cell viability, thus leading to the decreased probability of gamete collision. Furthermore, MPs affected ion transport in the gametes by inducing oxidative stress, which resulted in gamete fusion failure. In conclusion, this study demonstrates that MPs could significantly decrease the fertilization success of T. granosa through reducing gamete collision and lowering gamete fusion efficiency.

Investigation of the Genotoxic, Cytotoxic, Apoptotic, and Oxidant Effects of Olive Leaf Extracts on Liver Cancer Cell Lines.

Hepatocellular carcinoma (HCC) is the seventh most common cancer and the third leading cause of tumor-related deaths worldwide. Mechanisms underlying tumor onset, progression, and metastasis in the case of HCC have not been adequately studied. In this study, we aimed to investigate the genotoxic, cytotoxic, apoptotic and oxidant effects of olive leaf extract (OLE) on HCC cells. H4IIE Rattus norvegicus hepatoma cells and Rattus norvegicus healthy liver clone-9 cells were treated with the increasing concentrations of OLEs (250-2000 ppm) in ethanol, acetone, dichloromethane, and methanol. ATP cell viability, intracellular reactive oxygen species generation levels, double staining test with acridine orange/ethidium bromide, comet assay, levels of interleukin 1-beta (IL-1β), IL-6, and tumor necrosis factor alpha were measured. Significance was determined using ANOVA test. Apoptotic, genotoxic, cytotoxic, and oxidative effects of OLEs increased with the increasing concentrations as compared to controls in H4IIE cells (p<0.001). This is the first study to show a significant and selective cytotoxic activity of OLEs in the selected H4IIE cancer cell lines. OLEs could selectively increase the apoptotic damage and show anti-proliferative and pro-apoptotic properties against the H4IIE cells. They could be recommended as potential nutraceuticals in the prevention of cancer.

Cytochrome P450 3A5 polymorphism affects the metabolism of sorafenib and its toxicity for hepatocellular carcinoma cells in vitro.

Cytochrome P450 3A5 (CYP3A5) is a highly polymorphic gene and the encoded protein variants differ in catalytic activity, leading to inter-individual variation in metabolic ability. The aim of the current study was to investigate the effects of seven allelic variants on the ability of CYP3A5 to metabolize sorafenib in vitro and further explore the impacts of CYP3A5 polymorphism on the proliferation and apoptosis of hepatocellular carcinoma cell line (HepG2) induced by sorafenib. Wild-type and variant CYP3A5 enzymes were expressed in Spodoptera frugiperda insect cells using a baculovirus dual-expression system, and protein expression was checked by western blot. The enzymes were incubated with sorafenib at 37°C for 30 min, and formation of the major metabolite sorafenib N-oxide was assayed using ultra-performance liquid chromatography and tandem mass spectrometry. Intrinsic clearance values (Vmax/Km) were calculated for each enzyme. Additionally, recombinant HepG2 cells transfecting with CYP3A5 variants were used to investigate the effects of sorafenib on the proliferation of HepG2 cells. Intrinsic clearance of the six variants CYP3A5*2, CYP3A5*3A, CYP3A5*3C, CYP3A5*4, CYP3A5*5, and CYP3A5*7 was 26.41-71.04% of the wild-type (CYP3A5*1) value. In contrast, the clearance value of the variant CYP3A5*6 was significantly higher (174.74%). Additionally, the decreased ATP levels and cell viability and the increased cell apoptosis in HepG2 cells transfected with CYP3A5*2, CYP3A5*3A, CYP3A5*3C, CYP3A5*4, CYP3A5*5, and CYP3A5*7 were observed, whereas, the increased ATP levels and cell viability and the reduced cell apoptosis in HepG2 cells transfected with CYP3A5*6 were also investigated when compared to CYP3A5*1. Our results suggest that CYP3A5 polymorphism influences sorafenib metabolism and pharmacotherapeutic effect in hepatic carcinomas. These data may help explain differential response to drug therapy for hepatocellular carcinoma, and they support the need for individualized treatment.

The deleterious toxic effects of bifenthrin on male fertility

Bifenthrin (BF), a broad-spectrum synthetic pyrethroid insecticide, has been generally used to eradicate harmful insects. However, according to the U.S. EPA, BF has been classified as a “Class C” carcinogenic ingredient. Furthermore, a previous study reported that BF was considered as endocrine-disrupting chemicals and causes reproductive toxicity in mammals. Despite the various effects of BF, there is a scarcity of studies about its adverse effects on male fertility. Therefore, this study was conducted to determine the effects of BF on sperm functions at various concentrations (0.1, 1, 10, and 100 μM), including a control. Sperm motility and kinematics, capacitation status, intracellular ATP levels, cell viability, PKA activation, and protein tyrosine phosphorylation were measured. Moreover, fertilization and early embryonic development were examined through in vitro fertilization. Results showed that sperm motility and kinematic parameters were significantly decreased at a high BF concentration. Consequently, the sperm capacitation status exhibited significant alteration according to the treatment concentration. Intracellular ATP levels were significantly decreased at 10 and 100 μM treatment concentrations. Moreover, the levels of phospho-PKA substrates were significantly increased in a dose-dependent manner. In contrast, the levels of phospho-tyrosine substrates were significantly decreased at 10 and 100 μM treatment concentrations. BF treatment also diminished the rate of blastocyst formation. Altogether, our results demonstrated that BF causes detrimental effects on sperm function and can influence fertilization. Therefore, our study results might be helpful in understanding the adverse effects of BF on male fertility.

Resveratrol depolarizes the membrane potential in human granulosa cells and promotes mitochondrial biogenesis

To study the biological effects of resveratrol on the growth, electrophysiology, and mitochondrial function of human granulosa cells (h-GCs). Preclinical study. Electrophysiology laboratory and invitro fertilization unit. This study included h-GCs from seven infertile women undergoing assisted reproductive techniques. Human ovarian Granulosa Cell Tumor (GCT) cell line COV434 and h-GCs obtained after oocyte retrieval were cultured in the absence or presence of resveratrol. Granulosa cells were evaluated for cell viability and mitochondrial activity. Electrophysiological recordings and evaluation of potassium current (IKur) and Ca2+ concentration were also performed. Resveratrol induced mitochondrial activity in a bell-shaped, dose-effect-dependent manner. Specifically, resveratrol treatment (3 μM, 48 hours) increased ATP production and cell viability and promoted the induction of cellular differentiation. These biological changes were associated with mitochondrial biogenesis. Electrophysiological recordings showed that resveratrol reduced the functional expression of an ultra rapid activating, slow inactivating, delayed rectifier potassium current (IKur) that is associated with a plasma membrane depolarization and that promotes an increase in intracellular Ca2+. CONCLUSION(S): The effects of resveratrol on potassium current and mitochondrial biogenesis in h-GCs could explain the beneficial effects of this polyphenol on the physiology of the female reproductive system. These findings suggest there are therapeutic implications of resveratrol in a clinical setting.

Combinatorial Therapeutic Effect of Inhibitors of Aldehyde Dehydrogenase and Mitochondrial Complex I, and the Chemotherapeutic Drug, Temozolomide against Glioblastoma Tumorspheres.

Resident cancer cells with stem cell-like features induce drug tolerance, facilitating survival of glioblastoma (GBM). We previously showed that strategies targeting tumor bioenergetics present a novel emerging avenue for treatment of GBM. The objective of this study was to enhance the therapeutic effects of dual inhibition of tumor bioenergetics by combination of gossypol, an aldehyde dehydrogenase inhibitor, and phenformin, a biguanide compound that depletes oxidative phosphorylation, with the chemotherapeutic drug, temozolomide (TMZ), to block proliferation, stemness, and invasiveness of GBM tumorspheres (TSs). Combination therapy with gossypol, phenformin, and TMZ induced a significant reduction in ATP levels, cell viability, stemness, and invasiveness compared to TMZ monotherapy and dual therapy with gossypol and phenformin. Analysis of differentially expressed genes revealed up-regulation of genes involved in programmed cell death, autophagy, and protein metabolism and down-regulation of those associated with cell metabolism, cycle, and adhesion. Combination of TMZ with dual inhibitors of tumor bioenergetics may, therefore, present an effective strategy against GBM by enhancing therapeutic effects through multiple mechanisms of action.

Investigation of cellular effects of thymoquinone on glioma cell.

Glioblastoma, as an invasive tumor, is one of the most common primary malignant brain tumors. Despite maximum aggressive treatment, patients with glioblastoma have a dismal prognosis. Thymoquinone (TQ) has been found to show anti-cancer effects on different types of cancer. There are a few in vitro studies on the effect of TQ on glial tumors. However, the molecular mechanism of TQ's anti-cancer effect has not been fully elucidated. In the present study, we aimed to investigate the genotoxic, apoptotic, and cytotoxic effects of TQ on C6 rat glioma cells.C6 glioma cells were analyzed after 24 h of exposure to different concentrations of TQ by the ATP cell viability assay for cytotoxicity, comet assay for genotoxicity, 2′,7′dichlorodihydrofluorescein diacetate (H2DCF-DA) for intracellular reactive oxygen species (iROS) generation, 3.3′dihexyloxacarbocyanine iodide (DiOC6(3)) for mitochondrial membrane potential, GSH/GSSG-Glo Assay for glutathione level and Fura-2AM for intracellular calcium levels. Apoptosis induction was studied by acridine orange/ethidium bromide double staining, flow cytometry, and western blotting analyses. Caspase-3, Caspase-9, Bax, Bcl-2, and pSTAT3 protein levels were determined by the western blotting method.Cytotoxicity was enhanced by TQ in C6 glioma cells in a concentration-dependent manner. TQ also induced DNA damage, apoptosis, and increased iROS. Also, MMP and GSH levels were decreased by TQ. It inhibited pSTAT3, resulting in apoptosis induction through the regulation of anti-apoptotic and pro-apoptotic proteins.Our results suggest that TQ would be an effective treatment in glioma. Further studies should support these findings.

Production of hydrogen sulfide by the intestinal microbiota and epithelial cells and consequences for the colonic and rectal mucosa.

Among bacterial metabolites, hydrogen sulfide (H2S) has received increasing attention. The epithelial cells of the large intestine are exposed to two sources of H2S. The main one is the luminal source that results from specific bacteria metabolic activity toward sulfur-containing substrates. The other source in colonocytes is from the intracellular production mainly through cystathionine β-synthase (CBS) activity. H2S is oxidized by the mitochondrial sulfide oxidation unit, resulting in ATP synthesis, and, thus, establishing this compound as the first mineral energy substrate in colonocytes. However, when the intracellular H2S concentration exceeds the colonocyte capacity for its oxidation, it inhibits the mitochondrial respiratory chain, thus affecting energy metabolism. Higher luminal H2S concentration affects the integrity of the mucus layer and displays proinflammatory effects. However, a low/minimal amount of endogenous H2S exerts an anti-inflammatory effect on the colon mucosa, pointing out the ambivalent effect of H2S depending on its intracellular concentration. Regarding colorectal carcinogenesis, forced CBS expression in late adenoma-like colonocytes increased their proliferative activity, bioenergetics capacity, and tumorigenicity; whereas, genetic ablation of CBS in mice resulted in a reduced number of mutagen-induced aberrant crypt foci. Activation of endogenous H2S production and low H2S extracellular concentration enhance cancerous colorectal cell proliferation. Higher exogenous H2S concentrations markedly reduce mitochondrial ATP synthesis and proliferative capacity in cancerous cells and enhance glycolysis but do not affect their ATP cell content or viability. Thus, it appears that, notably through an effect on colonocyte energy metabolism, endogenous and microbiota-derived H2S are involved in the host intestinal physiology and physiopathology.