ATP Cell Viability Research Articles

Effect of TFAM on ATP content in tachypacing primary cultured cardiomyocytes and atrial fibrillation patients.

Atrial fibrillation (AF) is one of the most common types of arrhythmia worldwide; although a number of theories have been proposed to explain the mechanisms of AF, the treatment of AF is far from satisfactory. Energy metabolism is associated with the development of AF. Mitochondrial transcription factor A (TFAM) serves a role in the maintenance and transcription of mitochondrial DNA. The present study aimed to investigate the association between TFAM and AF and the effect of TFAM on ATP content in cardiomyocytes. Left atrial appendage tissues were collected from 20 patients with normal sinus rhythm (SR) and 20 patients with AF, and the expression levels of TFAM in SR and AF tissues were evaluated. In addition, a tachypacing model of primary cultured cardiomyocytes was constructed to assess ATP content, cell viability and expression levels of TFAM, mitochondrially encoded (MT)-NADH dehydrogenase 1 (ND1), MT-cytochrome c oxidase 1 (CO1), NADH ubiquinone oxidoreductase core subunit 1 (NDUFS1) and cytochrome c oxidase subunit 6C (COX6C). Finally, the effects of overexpression and inhibition of TFAM on ATP content, cell viability and the expression levels of MT-ND1 and MT-CO1 were investigated. The expression levels of TFAM were decreased in AF tissues compared with SR tissues (P<0.05). The ATP content, cell viability and expression levels of TFAM, MT-ND1 and MT-CO1 were decreased in tachypacing cardiomyocytes compared with non-pacing cardiomyocytes (P<0.05), whereas the expression levels of NDUFS1 and COX6C were not changed (P>0.05). Overexpression of TFAM increased ATP content, cell viability and expression levels of MT-ND1 and MT-CO1 (P<0.05). The inhibition of TFAM decreased ATP content, cell viability and expression levels of MT-ND1 and MT-CO1 (P<0.05). In summary, the results of the present study demonstrated that the expression levels of TFAM were decreased in AF tissues and tachypacing cardiomyocytes and that the restoration of TFAM increased the ATP content by upregulating the expression levels of MT-ND1 and MT-CO1 in tachypacing cardiomyocytes. Thus, TFAM may be a novel beneficial target for treatment of patients with AF.

Mitochondrial transplantation-a possible therapeutic for mitochondrial dysfunction?: Mitochondrial transfer is a potential cure for many diseases but proof of efficacy and safety is still lacking.

Transplantation of functional mitochondria directly into defective cells is a novel approach that has recently caught the attention of scientists and the general public alike. Could this be too good to be true?

Evaluation of pyrrolizidine alkaloid-induced genotoxicity using metabolically competent TK6 cell lines

Pyrrolizidine alkaloid (PA)-containing plants are among the most common poisonous plants affecting humans, livestock, and wildlife worldwide. A large number of PAs are known to induce genetic damage after metabolic activation. In the present study, using a battery of fourteen newly developed TK6 cell lines, each expressing a single human cytochrome P450 (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C18, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7), we identified specific CYPs responsible for bioactivating three PAs – lasiocarpine, riddelliine, and senkirkine. Among the fourteen cell lines, cells expressing CYP3A4 showed significant increases in PA-induced cytotoxicity, evidenced by decreased ATP production and cell viability, and increased caspase 3/7 activities. LC-MS/MS analysis revealed the formation of 1-hydroxymethyl-7-hydroxy-6,7-dihydropyrrolizine (DHP), the main reactive metabolite of PAs, in CYP3A4-expressing TK6 cells. DHP was also detected in CYP3A5- and 3A7-expressing cells after PA exposure, but to a much lesser extent. Subsequently, using a high-throughput micronucleus assay, we demonstrated that PAs induced concentration-dependent increases in micronuclei and G2/M phase cell cycle arrest in three CYP3A variant-expressing TK6 cell lines. Using Western blotting, we observed that PA-induced apoptosis, cell cycle changes, and DNA damage were primarily mediated by CYP3A4. Benchmark dose (BMD) modeling demonstrated that lasiocarpine, of the three PAs, was the most potent inducer of micronuclei, with a BMD100 of 0.036 μM. These results indicate that our TK6 cell system holds promise for genotoxicity screening of compounds requiring metabolic activation, identifying specific CYPs involved in bioactivation, and discriminating the genotoxic compounds that have different chemical structures.

Highly Promising Antitumor Agent of a Novel Platinum(II) Complex Bearing a Tetradentate Chelating Ligand.

A new mononuclear cationic platinum(II) coordination compound with 6,6'-bis(NH-benzimidazol-2-yl)-2,2'-bipyridine (L) ligand having N4-tetradentate binding pocket [Pt(L)]Cl2·2H2O (Complex 1) was synthesized and characterized by FT-IR(ATR), UV-vis, 1H NMR, APCI and MALDI MS, and CHN analysis. The antigrowth effect of Complex 1 was tested in breast cancer (MDA-MB-231), lung cancer (A549), colorectal cancer (HCT-116), prostate cancer (PC-3) cell lines, and bronchial epithelial cell line (BEAS-2B) by the SRB and ATP cell viability assays. Apoptosis was detected with Annexin V, mitopotential, BCL-2 inactivation, and γH2AX assays by flow cytometry. Complex 1 was found to have cytotoxic activity of MDA-MB-231, A549, HCT-116, and PC-3 cancer cell lines in a dose-dependent manner for 48 h. Complex 1 has been found to cause cell death through different mechanisms depending on the type of cancer. The findings indicated that complex induced intrinsic apoptosis with the increased mitochondrial membrane depolarization level, Bcl-2 inactivation, and DNA damage in PC-3 and A549 cell lines.

Enhanced cytotoxicity of imidacloprid by biotransformation in isolated hepatocytes and perfused rat liver

Imidacloprid (IMD) is a neonicotinoid insecticide widely used in crops, pets, and on farm animals for pest control, which can cause hepatotoxicity in animals and humans. In a previous study using isolated rat liver mitochondria, we observed that IMD inhibited the activity of FoF1-ATP synthase. The aim of this study was to evaluate the effects of IMD on rat isolated hepatocytes and perfused rat liver, besides the influence of its biotransformation on the toxicological potential. For the latter goal, rats were pretreated with dexamethasone or phenobarbital, two classical cytochrome P-450 stimulators, before hepatocytes isolation or liver perfusion. IMD (150 and 200 μM) reduced state 3 mitochondrial respiration in digitonin-permeabilized cells that were energized with glutamate plus malate but did not dissipate the mitochondrial membrane potential. In intact (non-permeabilized) hepatocytes, the intracellular ATP concentration and cell viability were reduced when high IMD concentrations were used (1.5–3.0 mM), and only in cells isolated from dexamethasone-pretreated rats, revealing that IMD biotransformation increases its toxicity and that IMD itself affects isolated mitochondria or mitochondria in permeabilized hepatocytes in concentrations that do not affect mitochondrial function in intact hepatocytes. Coherently, in the prefused liver, IMD (150 and 250 μM) inhibited gluconeogenesis from alanine, but without affecting oxygen consumption and urea production, indicating that such effect was not of mitochondrial origin. The gluconeogenesis inhibition was incomplete and occurred only when the rats were pretreated with phenobarbital, signs that IMD biotransformation was involved in the observed effect. Our findings reveal that changes in hepatic energy metabolism may be acutely implicated in the hepatotoxicity of IMD only when animals and humans are exposed to high levels of this compound, and that IMD metabolites seem to be the main cause for its toxicity.

The ying and yang of idebenone: Not too little, not too much – cell death in NQO1 deficient cells and the mouse retina

Idebenone has recently been investigated as a drug therapy for Leber's hereditary optic neuropathy (LHON), a rare genetic mitochondrial disease that causes rapid and progressive bilateral vision loss. Although several studies have shown that idebenone can promote vision recovery in patients with LHON, the evidence for the efficacy of idebenone is still limited. Idebenone failed to demonstrate superiority over placebo in the primary end-points of the only published randomised, double-blind, placebo-controlled trial. There appears to be a patient-specific response to idebenone with high variability in therapeutic outcomes. A recent study suggested that the cytosolic enzyme NAD(P)H: quinone acceptor oxidoreductase (NQO1) is the major enzyme involved in the activation of idebenone, and the beneficial effects of idebenone are dependent on the expression of NQO1. Here, we confirm the NQO1-dependent activity of idebenone, but we also show, for the first time, that the cytotoxicity of idebenone is linked to cellular expression of NQO1. Upon idebenone administration, cells deficient in NQO1 show a marked decrease in viability in comparison to NQO1 expressing cells, with idebenone causing ROS production and deleterious effects on ATP levels and cell viability. In addition, our data highlights that only cells expressing NQO1 can significantly activate idebenone, indicating that other proposed metabolic activation pathways, such as complex II and glycerol-3-phosphate dehydrogenase, do not play a significant role in idebenone activation. Furthermore, we provide evidence of idebenone-induced toxicity in the retina ex-vivo, which can be explained by the variation of NQO1 expression between different cell types in the mouse retina. Idebenone mediated cell rescue in the rotenone ex vivo model also indicated that this drug has a narrow therapeutic window. These findings will help to guide the development of future therapies and drug delivery strategies including intra-ocular administration. The specific dependence of idebenone activity on NQO1 may also explain the variation in patient outcomes in clinical trials.

Inhibiting Very Long Chain Acyl-CoA Dehydrogenase (VLCAD) Induces Selective Leukemia Cell Death

Targeting cell metabolism has emerged as a viable treatment strategy in acute myeloid leukemia (AML), a malignant hematological disease characterized by poor patient outcomes and limited chemotherapeutic options. Compared to the normal hematopoietic population, leukemia cells exhibit an altered phenotype characterized by increased mitochondrial mass as well as a greater reliance on oxidative phosphorylation and fatty acid oxidation (FAO) for survival. Mitochondrial FAO is a four-reaction process that catabolizes fatty acids to acetyl-CoA, generating reductive equivalents for the electron transport chain (ETC) and anaplerotic intermediates for the TCA cycle. Clinically approved FAO inhibitors, such as trimetazidine and ranolazine, are tissue-specific, targeting FAO in some tissues but affecting additional pathways in others. To better understand the potential clinical utility of targeting FAO, we systematically tested a panel of clinical and pre-clinical FAO inhibitors, reasoning that the most potent FAO inhibitor would lead to a novel anti-AML target. Avocadyne was the most potent anti-AML compound, inducing leukemic cell death (EC50: 2.5 µM) and suppressing clonogenic growth of primary samples, while sparing normal hematopoietic cells. Further, avocadyne (100mg/kg twice weekly for 5 weeks) reduced patient-derived AML cell engraftment in the bone marrow of immune deficient mice. As a component of avocatin-B, a mixture of two fatty alcohols previously determined to accumulate in the mitochondria, we confirmed that avocadyne inhibited long chain FAO using radiolabeled studies and high resolution respirometry. To identify a molecular target, avocadyne treated cells were immunoprecipitated with antibodies against each intramitochondrial enzyme involved in long chain FAO (e.g., very long acyl-CoA dehydrogenase (VLCAD; step 1) and the alpha and beta subunits of the mitochondrial trifunctional protein: HADHA, HADHB; steps 2-4). Immunoblotting and LC/MS analysis confirmed that avocadyne co-eluted with VLCAD, but not with HADHA or HADHB, confirming a direct physical interaction between avocadyne and VLCAD. VLCAD introduces a double bond to a fully saturated long chain fatty acid and reduces electron transfer flavoprotein (ETF)-bound FAD, transferring these electrons to the ETC. Using fluorescence spectrophotometry and respirometry, avocadyne directly inhibited VLCAD activity, resulting in reduced ETF-supported respiration. The activity of MCAD, an acyl-CoA dehydrogenase catalyzing medium chain fats, was not affected, suggesting avocadyne inhibits long chain FAO exclusively. Further, profiling of acyl-carnitines following avocadyne treatment also showed a pattern characteristic of long chain FAO inhibition at VLCAD. To further understand how VLCAD modulated avocadyne sensitivity, avocadyne-resistant and VLCAD knockdown cells were generated. Lentiviral knockdown of VLCAD sensitized leukemic cells to avocadyne-induced FAO inhibition and death. In contrast, cells resistant to avocadyne had increased protein expression of VLCAD but no change in other long chain FAO enzymes. With increased VLCAD-supported ETF respiration, higher concentrations of avocadyne were required to induce cell death, compared to the parental line. These results show that VLCAD expression modulated leukemic sensitivity to avocadyne. Inhibiting FAO at VLCAD triggered an adaptive metabolic switch towards glycolysis, characterized by increased extracellular acidification. This compensatory increase in glycolysis was ultimately insufficient to prevent the depletion of TCA metabolites and ATP, leading to leukemic death. In contrast, following avocadyne treatment, normal umbilical cord blood-derived mononuclear cells increased glycolytic as well as pyruvate dehydrogenase activity and had no decrease in ATP levels, cell viability, or clonogenic growth. Together, these results highlight, for the first time, VLCAD as a novel anti-AML target and further suggest the clinical utility of FAO inhibition as a potential anti-AML strategy. Disclosures Minden: Trillium Therapetuics: Other: licensing agreement. Schimmer:Medivir Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Consultancy; Novartis Pharmaceuticals: Consultancy; Otsuka Pharmaceuticals: Consultancy.

Unfolded Protein Response is Involved in Trans-Platinum (II) Complex-Induced Apoptosis in Prostate Cancer Cells via ROS Accumulation.

Prostate cancer is one of the most common cancer types and it is the sixth leading cause of cancer-related death in men worldwide. Even though novel treatment modalities have been developed, it still a lifethreatening disease. Therefore novel compounds are needed to improve the overall survival. In our study, it was aimed to evaluate the anti-cancer activity of newly synthesized Platinum (II) [Pt(II)] complex on DU145, LNCaP and PC-3 prostate cancer cell lines. The cytotoxic activity of Pt(II) complex was tested by SRB and ATP cell viability assays. To detect the mode of cell death; fluorescent staining, flow cytometry and western blot analyses were performed. The Pt(II) complex treatment resulted in a decrease in cell viability and increasing levels of apoptotic markers (pyknotic nuclei, annexin-V, caspase 3/7 activity) and a decrease in mitochondrial membrane potential in a dose dependent manner. Among cell types, tested PC-3 cells were found to be more sensitive to Pt(II) complex, demonstrating elevation of DNA damage in this cell line. In addition, Pt(II) complex induced Endoplasmic Reticulum (ER) stress by triggering ROS generation. More importantly, pre-treatment with NAC alleviated Pt(II) complex-mediated ER stress and cell death in PC-3. These findings suggest an upstream role of ROS production in Pt(II) complex-induced ER stressmediated apoptotic cell death. Considering the ROS-mediated apoptosis inducing the effect of Pt(II) complex, it warrants further evaluation as a novel metal-containing anticancer drug candidate.

Idarubicin-bromelain combination sensitizes cancer cells to conventional chemotherapy.

Objective(s):The primary cytotoxic effects of anticancer drugs like idarubicin, a chemotherapeutic agent, are not limited to neoplastic cells; they also produce similar effects in normal cells. In this study, we hypothesized that the combination of idarubicin-bromelain could make cancer cells more susceptible to cytotoxicity and genotoxicity.Materials and Methods:To test our hypothesis, the optimal concentrations of idarubicin and bromelain were combined and incubated in the HL-60 cancer cell line and normal human mononuclear leukocytes (PBMC) for 24, 48, and 72 hr. Cytotoxicity and genotoxicity were evaluated by measurement of ATP cell viability test, DNA damage, Caspase-3, Acridine orange/ethidium bromide (AO/EB), and DAPI fluorescent dyes in both cell types.Results:The combination of idarubicin-bromelain significantly reduced cell proliferation in the more potent HL-60 compared to PBMC in all incubation times (P<0.05). DNA damage and Caspase-3 levels (except for 24 hr) were also higher in the HL-60 cell line in comparison with PBMC and were statistically significant (P<0.05). The percentages of apoptotic images obtained by DAPI and AO / EB morphological examination were increased in both cells, depending on the combination dose.Conclusion:Based on these results, it can be concluded that idarubicin combined with bromelain produces more cytotoxic effects in low concentrations in comparison with when it was used per se in the HL-60 cells. Conversely, it was found that this combination in PBMC caused less cytotoxicity and less genotoxicity. Taken together, it can be said that this new combination makes cancer cells more sensitive to conventional therapy.

Utilization of18F-Fluorodeoxyglucose–Positron Emission Tomography To Understand the Mechanism of Nicotinamide Phosphoribosyltransferase Inhibitors In Vivo

Cancer cells are highly dependent on NAD+/NADH produced via the nicotinamide salvage pathway. The rate-limiting enzyme in this pathway is the nicotinamide phosphoribosyltransferase (NAMPT), which we have targeted with novel NAMPT inhibitors. NAMPT inhibition elicits depletion of total cellular NAD+ levels and ultimately cytotoxicity via depletion of cellular ATP levels. 18F-fluorodeoxyglucose- positron emission tomography (FDG-PET) is a translational imaging tool to assess glucose utilization in tumors and normal tissue. We used FDG-PET to understand the timing of ATP depletion in vivo and better understand the pharmacology of NAMPT inhibitors. Because of the intimate relationship between cellular ATP levels and cell viability, we developed an in-depth understanding of our NAMPT inhibitor pharmacology and the relationship with changes in tumor FDG uptake. Taken together, we show that FDG-PET could be used as a biomarker in clinical studies to understand dose and provide proof of mechanism for NAMPT inhibitors. SIGNIFICANCE STATEMENT: Our imaging data suggest that tumor 18F-fluorodeoxyglucose uptake can provide insight into the ATP status inside the tumor after nicotinamide phosphoribosyltransferase (NAMPT) therapy, with a novel NAMPT inhibitor. Such an approach could be used clinically as a pharmacodynamic biomarker to help understand the implications of dose, schedule, rescue strategy, or other clinical biomarkers.

An Optimum Dose of Olive Leaf Extract Improves Insulin Receptor Substrate-1, Tyrosine Kinase, and Glucose Transporters, While High Doses Have Genotoxic and Apoptotic Effects

Type 2 diabetes is the most common type of diabetes. Conventionally many drugs are used for the treatment of diabetes such as biguanides, sulfonylureas, meglitinides, etc. But the desired effective treatment is still not to be achieved. So researches are going on for the development of effective alternative therapy against diabetes. Olive leaves are traditionally used in the treatment of the disease. However, studies on its mechanism of action are not yet enough. The aim of this study was to investigate whether olive leaf extract (OLE) improves insulin receptor substrate-1 (IRS-1), tyrosine kinase (TK), GLUT-2, and GLUT-4. Oleuropein levels were analyzed from OLE obtained by using four different solvents, and the highest content of methanol extract was selected for the study. Different concentrations of OLE (2.5 to 320 μg/mL) were incubated with hepatocellular carcinoma (HepG2) cells for 24 hours. After incubation, cell viability was assessed based on luminometric ATP cell viability assay kit. Intracellular reactive oxygen species (ROS) generating level was detected using 2,7dichlorodihydrofluorescein-diacetate (H2DCF-DA) fluorescent probes. Apoptosis was evaluated by acridine orange/ethidium bromide double staining method. Genotoxicity was evaluated by alkaline single cell gel electrophoresis assay (Comet Assay). Protein expression levels of IRS-1, TK, GLUT-2, and GLUT-4 were analyzed by western blotting technique from the obtained cell lysates. Although an optimum doses of OLE (10 μg/mL) maximally increased cell proliferation, decreased ROS generation improved IRS-1, TK, GLUT-2, and GLUT-4 protein expression levels (about fivefold), higher doses (10 to 320 μg/mL) markedly decreased the cell viability, increased DNA damage, apoptosis and ROS generation in a concentration-dependent manner. OLE can be used in the treatment of type 2 diabetes. However, in order to find the most effective and non-toxic concentration, dose optimization is required.

ATP Content and Cell Viability as Indicators for Cryostress Across the Diversity of Life.

In many natural environments, organisms get exposed to low temperature and/or to strong temperature shifts. Also, standard preservation protocols for live cells or tissues involve ultradeep freezing in or above liquid nitrogen (-196°C or -150°C, respectively). To which extent these conditions cause cold- or cryostress has rarely been investigated systematically. Using ATP content as an indicator of the physiological state of cells, we found that representatives of bacteria, fungi, algae, plant tissue, as well as plant and human cell lines exhibited similar responses during freezing and thawing. Compared to optimum growth conditions, the cellular ATP content of most model organisms decreased significantly upon treatment with cryoprotectant and cooling to up to -196°C. After thawing and a longer period of regeneration, the initial ATP content was restored or even exceeded the initial ATP levels. To assess the implications of cellular ATP concentration for the physiology of cryostress, cell viability was determined in parallel using independent approaches. A significantly positive correlation of ATP content and viability was detected only in the cryosensitive algae Chlamydomonas reinhardtii SAG 11-32b and Chlorella variabilis NC64A, and in plant cell lines of Solanum tuberosum. When comparing mesophilic with psychrophilic bacteria of the same genera, and cryosensitive with cryotolerant algae, ATP levels of actively growing cells were generally higher in the psychrophilic and cryotolerant representatives. During exposure to ultralow temperatures, however, psychrophilic and cryotolerant species showed a decline in ATP content similar to their mesophilic or cryosensitive counterparts. Nevertheless, psychrophilic and cryotolerant species attained better culturability after freezing. Cellular ATP concentrations and viability measurements thus monitor different features of live cells during their exposure to ultralow temperatures and cryostress.

Abstract 326: A novel antimetabolite, FF-10502-01 exhibits potent antitumor activity via inhibition of both DNA replication and DNA damage repair in solid tumor cells

Abstract Introduction FF10502-01 is a synthetic pyrimidine nucleoside analogue that is structurally similar to gemcitabine (GEM) with a substitution of sulfur for oxygen in the pentose ring. FF-10502-01 demonstrated potent anti-tumor activity in a number of preclinical studies. In both Capan-1 and SUIT-2 pancreatic cancer xenograft models, FF-10502-01 achieved superior tumor growth suppression and survival prolongation, respectively, compared to GEM, with less toxicity at clinically relevant doses. In GEM-resistant pancreatic PDX models, FF-10502-01 showed greater efficacy and tolerability than GEM. A Phase 1 trial of FF-10502-01 in patients with advanced cancer has been conducted in United States with positive results, and a Phase 2a trial is scheduled for 2018. Methods To determine the putative differences between FF-10502-01 and GEM activity the following studies were undertaken. The inhibitory activity for polymerase alpha and beta was measured using a DNA synthesis assay in vitro. Purified human polymerases and tri-phosphates of test drugs were incubated for 30 minutes at 37 °C in the presence of [methyl-3H]dTTP, and radioactivity was measured by liquid scintillation counting. SUIT-2 cells were cultured in serum-free medium for 72 hours to establish a dormant cell model. The test drugs, with DNA damage inducers, were then added as combination treatment. After 72 hours, the cell growth inhibition was evaluated using an ATP cell viability assay. DNA damage in the dormant cell model was evaluated by alkaline comet assay. Results To explain why FF-10502-01 was more potent than GEM in vivo, we investigated possible differences in their mechanisms of action (MOA). Both FF-10502-01 and GEM inhibited DNA replication in vitro and in vivo. On the other hand, only FF-10502-01 showed potent cytotoxic activity against serum starvation-induced dormant SUIT-2 cells in combination with DNA damage inducers (DDIs: H2O2, cisplatin, or temozolomide). Similar results were obtained in cholangiocarcinoma and ovarian cancer cell lines. In vitro enzymatic assays showed that FF-10502-01 was far more potent than GEM in inhibiting DNA polymerase-beta, which plays an essential role in repairing oxidative DNA damage via the base-excision repair pathway. The inhibitory activity of DNA repair may explain the differential effect of FF-10502-01 in solid tumors in which reactive oxygen species are generated by tumor specific microenvironment. Conclusions Unlike GEM, FF-10502 is unique in preventing DNA repair via polymerase-beta inhibition when combined with DDIs, suggesting a dual MOA for FF-10502 in solid tumors compared to GEM. Citation Format: Kazunori Saeki, Rena Uematsu, Kaoru Morimura, Takayuki Yamada, Hiroyuki Iwamura, Shinichi Watanabe, Timothy Madden, Shinji Hagiwara. A novel antimetabolite, FF-10502-01 exhibits potent antitumor activity via inhibition of both DNA replication and DNA damage repair in solid tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 326.

In vitro effects of two major phenolic compounds from the family Lamiaceae plants on the human gastric carcinoma cells.

Phenolic compounds of essential oils from the family Lamiaceae are commonly used substances in the food industry because of their flavouring, antimicrobial and antioxidant properties. In this context, it has become important to have healthy and safe products for consumers who are exposed to these phenolic compounds. The present study was aimed to investigate the toxic effects of carvacrol, thymol and their mixture on human gastric carcinoma (AGS) cells. Cells were analyzed after 24 h of exposure to different concentrations of carvacrol, thymol and their mixture by the ATP cell viability, 2',7' dichlorodihydrofluorescein diacetate (H2DCF-DA), reducte glutatione/oxide glutathione((GSH)/GSSG-Glo) and comet assays. Apoptosis induction was studied by acridine orange/ethidium bromide staining and western blotting. Carvacrol, thymol and their mixture induced cytotoxicity, genotoxicity, apoptosis, increased reactive oxygen species (ROS) and decreased GSH levels after 24 h of their exposure in a dose-dependent manner. A close negative relationship was found between cell viability and ROS generation. We examined dose-dependent cytotoxic effects of carvacrol, thymol and their mixture in human AGS cells. Increased intracellular ROS causes oxidative stress in cells. The results indicated that these compounds should be used carefully in the food industry.

Dose-dependent proliferative and cytotoxic effects of melatonin on human epidermoid carcinoma and normal skin fibroblast cells

New in vitro studies have demonstrated that N-acetyl-5-methoxytryptamine (Melatonin) has cytotoxic and apoptotic effects on various cell types although most of the previous investigations document that it is a potent antioxidant. However, the precise molecular mechanism(s) of its effects are not fully elucidated. In this study, we examined dose-dependent cytotoxic, genotoxic, apoptotic and reactive oxygen species (ROS) generating effects of melatonin in human epidermoid carcinoma cells (A-431) and human normal skin fibroblastic cells (CCD-1079Sk). The cells were incubated with different doses of melatonin (0.031–5 mM) for 24 h. Cell viability was assessed based on luminometric ATP cell viability assay. Intracellular ROS was detected using 2,7-dichlorodihydrofluorescein-diacetate (H2DCF-DA) fluorescent probes. Genotoxicity was evaluated by alkaline single cell gel electrophoresis assay (Comet Assay). Apoptosis was evaluated by western blotting, DAPI staining, acridine orange/ethidium bromide and Annexin V‐FITC/propidium iodide double staining methods Mitochondrial membrane potentials were measured by flow cytometry. Although lower doses of melatonin (0.031–0.06 mM) increased cell proliferation and decreased ROS generation, higher doses (0.125–5 mM) markedly inhibited the cell viability, induced DNA damage, apoptosis and ROS generation. Cytotoxic, genotoxic, apoptotic and ROS generating effects were significantly higher in cancer cells than those observed in normal cells. Melatonin-induced cell death, and ROS generating activity were effectively inhibited by N-acetyl-l-cysteine (NAC) In conclusion, at low doses, melatonin has proliferative effects on both cancer and normal cells, whereas high concentrations have cytotoxic effects. Cytotoxic, genotoxic and apoptotic effects at higher doses of melatonin may be due to its ROS production capacity.

Usnea filipendula Induces Apoptosis in Human Colon Cancer Cell Lines

Lichens are complex organisms living in a symbiotic relationship with fungi and algae have recently received special interest in cancer research. The cytotoxic activities of Usnea filipendula Stirt. lichen extract was investigated on colon cancer cell lines, HCT-15 and HT-29. Sulforhodamine B and ATP cell viability tests were used to monitor cytotoxic activity. The mode of cell death (apoptosis/necrosis) was determined using caspase-cleaved cytokeratin 18 (M30), caspase-3/7 activity and fluorescence staining techniques that included, Annexin-V, Hoechst 33342 and propidium iodide. Usnea filipendula showed dose and time-dependent antiproliferative effect in HCT-15 and HT-29 cells. The IC50 values in HCT-15 and HT-29 cells were 17.92 and 41.87 µg/ml, respectively. The extract induced apoptosis in both cell lines especially in HCT-15 cells in which caspase-3/7 activity was increased. Usnea filipendula was cytotoxic to colon cancer HCT-15 and HT-29 cell lines by inducing early or late apoptosis as evidenced by translocation of phosphatidylserine, pyknotic nuclei and nuclear condensation. Further studies would help to understand the full potential of Usnea filipendula as a novel anticancer therapy.

Regulation of bioenergetics through dual inhibition of aldehyde dehydrogenase and mitochondrial complex I suppresses glioblastoma tumorspheres.

Targeted approaches for treating glioblastoma (GBM) attempted to date have consistently failed, highlighting the imperative for treatment strategies that operate on different mechanistic principles. Bioenergetics deprivation has emerged as an effective therapeutic approach for various tumors. We have previously found that cancer cells preferentially utilize cytosolic NADH supplied by aldehyde dehydrogenase (ALDH) for ATP production through oxidative phosphorylation (OxPhos). This study is aimed at examining therapeutic responses and underlying mechanisms of dual inhibition of ALDH and OxPhos against GBM. For inhibition of ALDH and OxPhos, the corresponding inhibitors, gossypol and phenformin were used. Biological functions, including ATP levels, stemness, invasiveness, and viability, were evaluated in GBM tumorspheres (TSs). Gene expression profiles were analyzed using microarray data. In vivo anticancer efficacy was examined in a mouse orthotopic xenograft model. Combined treatment of GBM TSs with gossypol and phenformin significantly reduced ATP levels, stemness, invasiveness, and cell viability. Consistently, this therapy substantially decreased expression of genes associated with stemness, mesenchymal transition, and invasion in GBM TSs. Supplementation of ATP using malate abrogated these effects, whereas knockdown of ALDH1L1 mimicked them, suggesting that disruption of ALDH-mediated ATP production is a key mechanism of this therapeutic combination. In vivo efficacy confirmed remarkable therapeutic responses to combined treatment with gossypol and phenformin. Our findings suggest that dual inhibition of tumor bioenergetics is a novel and effective strategy for the treatment of GBM.

Synthesis and evaluation of new pyridyl/pyrazinyl thiourea derivatives: Neuroprotection against amyloid-β-induced toxicity

Herein, we report synthesis and evaluation of new twenty six small molecules against β amyloid (Aβ)-induced opening of mitochondrial permeability transition pore (mPTP) using JC-1 assay which measures the change of mitochondrial membrane potential (ΔΨm). The neuroprotective effect of seventeen compounds against Aβ-induced mPTP opening was superior to that of the standard Cyclosporin A (CsA). Fifteen derivatives eliciting increased green to red fluorescence percentage less than 40.0% were evaluated for their impact on ATP production, cell viability and neuroprotection against Aβ-induced neuronal cell death. Among evaluated compounds, derivatives 9w, 9r and 9k had safe profile regarding ATP production and cell viability. In addition, they exhibited significant neuroprotection (69.3, 51.8 and 48.2% respectively). Molecular modeling study using CDocker algorithm predicted plausible binding modes explaining the elicited mPTP blocking activity. Hence, this study suggests compounds 9w, 9r and 9k as leads for further development of novel therapy to Alzheimer's disease.

Effects of natural phenolic compound carvacrol on the human gastric adenocarcinoma (AGS) cells in vitro.

Gastric cancer (GC) is the most common cause of morbidity and mortality because of cancer. Medicinal plants containing polyphenolic compounds have gained importance in anticancer treatment. In this context, carvacrol is a main component of many plants in the family Lamiaceae that are frequently used in folk medicine and a good candidate to investigate for GC treatment. The present study aimed to explore the cytotoxic, genotoxic, apoptotic, and reactive oxygen species (ROS)-generating effects of carvacrol on gastric adenocarcinoma in vitro. For these purposes, human gastric adenocarcinoma (AGS) cells were used and analyzed after 24 h of exposure to carvacrol with different concentrations. The cytotoxicity, ROS generation, glutathione (GSH) level, and genotoxicity were investigated by the ATP cell viability assay, 2',7'-dichlorodihydrofluorescein-diacetate assay, GSH/GSSG-Glo assay, and comet assay, respectively. Apoptosis induction was detected by acridine orange/ethidium bromide staining and western blotting at below the half-maximal growth inhibitory concentration value. Carvacrol showed cytotoxic, genotoxic, apoptotic, ROS generating, and GSH-reducing effects on AGS cells in a dose-dependent manner. There was a close negative relationship between cell viability and ROS level. Carvacrol inhibited the proliferation of AGS cells, suggesting that it could be a novel and strong anticancer agent against the human gastric adenocarcinoma. These results support the interest of natural diet components in the development of therapeutic products for diseases.

Novel 1-(7-ethoxy-1-benzofuran-2-yl) substituted chalcone derivatives: Synthesis, characterization and anticancer activity

Cancer treatment still requires new compounds to be discovered. Chalcone and its derivatives exhibit anticancer potential in different cancer cells. A new series of benzofuran substituted chalcone derivatives was synthesized by the base-catalyzed Claisen-Schmidt reaction of the 1-(7-ethoxy-1-benzofuran-2-yl) ethanone with different aromatic aldehydes to yield 1-(7-ethoxy-1-benzofuran-2-yl) substituted chalcone derivatives 3a-j. The derivatives were characterized by elemental analysis, FT-IR, 1H NMR and 13C NMR spectroscopy techniques. The anti-growth effect of chalcone compounds was tested in breast cancer (MCF-7), non-small cell lung cancer (A549) and prostate cancer (PC-3) cell lines by the SRB and ATP cell viability assays. Apoptosis was detected by mitochondrial membrane potential, Annexin V staining and caspase 3/7 activity. Formation of reactive oxygen species was determined by DCFDA. The results revealed that chalcone derivatives have anticancer activity with especially chalcone derivative 3a showing cytotoxic effects on cancer cells. In addition, chalcone derivative 3a induced apoptosis through caspase dependent pathways in prostate, lung and breast cancer cells.