EPR Investigations to Study the Impact of Mito-Metformin on the Mitochondrial Function of Prostate Cancer Cells

Background: Mito-metformin10 (MM10), synthesized by attaching a triphenylphosphonium cationic moiety via a 10-carbon aliphatic side chain to metformin, is a mitochondria-targeted analog of metformin that was recently demonstrated to alter mitochondrial function and proliferation in pancreatic ductal adenocarcinoma. Here, we hypothesized that this compound may decrease the oxygen consumption rate (OCR) in prostate cancer cells, increase the level of mitochondrial ROS, alleviate tumor hypoxia, and radiosensitize tumors. Methods: OCR and mitochondrial superoxide production were assessed by EPR (9 GHz) in vitro in PC-3 and DU-145 prostate cancer cells. Reduced and oxidized glutathione were assessed before and after MM10 exposure. Tumor oxygenation was measured in vivo using 1 GHz EPR oximetry in PC-3 tumor model. Tumors were irradiated at the time of maximal reoxygenation. Results: 24-hours exposure to MM10 significantly decreased the OCR of PC-3 and DU-145 cancer cells. An increase in mitochondrial superoxide levels was observed in PC-3 but not in DU-145 cancer cells, an observation consistent with the differences observed in glutathione levels in both cancer cell lines. In vivo, the tumor oxygenation significantly increased in the PC-3 model (daily injection of 2 mg/kg MM10) 48 and 72 h after initiation of the treatment. Despite the significant effect on tumor hypoxia, MM10 combined to irradiation did not increase the tumor growth delay compared to the irradiation alone. Conclusions: MM10 altered the OCR in prostate cancer cells. The effect of MM10 on the superoxide level was dependent on the antioxidant capacity of cell line. In vivo, MM10 alleviated tumor hypoxia, yet without consequence in terms of response to irradiation.     

Photodynamically induced effects in colon carcinoma cells (WiDr) by endogenous photosensitizers generated by incubation with 5-aminolaevulinic acid.

Human adenocarcinoma cells of the line WiDr have been treated with 2 mM 5-aminolaevulinic acid (5-ALA) in the presence of 10% foetal calf serum. The treatment induces a linear accumulation of protoporphyrin IX (PpIX) for at least 7.5 h. After 7.5 h of incubation about 45% of the PpIX accumulated is cell-bound, while the rest is found in the medium (25%) or lost from the cells during washing with phosphate-buffered saline (30%). Exposure to white light at an intensity of 30 W/m2 for 18 min results in 95% reduction of clonogenicity in cells treated with 2 mM 5-ALA for 3.5 h. The enzymatic activities of enzymes located in cytosol (glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase) and lysosomes (acid phosphatase and beta-glucuronidase) are not influenced by a 5-ALA and light treatment inactivating about 35% of the cells. The MTT assay, which reflects mitochondrial dehydrogenase activity, but not succinate dehydrogenase, is partly inhibited by the same treatment. Treatment with 5-ALA in the absence of light increases O2 consumption by a factor of two, while the O2 consumption is inhibited when 5-ALA treatment is combined with exposure to light. In addition, 5-ALA and light exposure enhance accumulation of rhodamine 123 by 40% and reduce the intracellular ATP level by 25%. Confocal laser scanning microscopical analysis indicates granular perinuclear localization of the PpIX formed by 5-ALA treatment. In conclusion, photodynamic treatment using 5-ALA as a prodrug induces damage to mitochondrial function without inhibiting lysosomal and cytosolic marker enzymes.     

Tetrabromobisphenol Exposure Impairs Bovine Oocyte Maturation by Inducing Mitochondrial Dysfunction

Tetrabromobisphenol (TBBPA) is the most widely used brominated flame retardant in the world and displays toxicity to humans and animals. However, few studies have focused on its impact on oocyte maturation. Here, TBBPA was added to the culture medium of bovine cumulus–oocyte complexes (COCs) to examine its effect on oocytes. We found that TBBPA exposure displayed an adverse influence on oocyte maturation and subsequent embryonic development. The results of this study showed that TBBPA exposure induced oocyte meiotic failure by disturbing the polar-body extrusion of oocytes and the expansion of cumulus cells. We further found that TBBPA exposure led to defective spindle assembly and chromosome alignment. Meanwhile, TBBPA induced oxidative stress and early apoptosis by mediating the expression of superoxide dismutase 2 (SOD2). TBBPA exposure also caused mitochondrial dysfunction, displaying a decrease in mitochondrial membrane potential, mitochondrial content, mtDNA copy number, and ATP levels, which are regulated by the expression of pyruvate dehydrogenase kinase 3 (PDK3). In addition, the developmental competence of oocytes and the quality of blastocysts were also reduced after TBBPA treatment. These results demonstrated that TBBPA exposure impaired oocyte maturation and developmental competence by disrupting both nuclear and cytoplasmic maturation of the oocyte, which might have been caused by oxidative stress induced by mitochondrial dysfunction.     

Antioxidant adaptive response of human mononuclear cells to UV-B: effect of lipoic acid

Supplementation of human mononuclear cells with 3 and 6 mM of lipoic acid produces an inhibition of the antioxidant adaptive response triggered by treatment with UV-B light (0.30 W/m2 for 15 min). Supplementation with 1.5 mM of lipoic acid gives no conclusive results. The adaptive response is characterized by an increase in the activities of superoxide dismutase, catalase, glutathione peroxidase and DT-diaphorase. Catalase (5.5 +/- 0.6 pmol/mg prot) increases its activity by up to 22 +/- 3 pmol/mg prot, after irradiation with UV-B. Supplementation with 3 and 6 mM of lipoic acid completely inhibits the adaptive response. The activities of the membrane-bound mitochondrial enzymes succinate dehydrogenase and cytochrome oxidase do not increase after UV-B exposure. Moreover, their activities are found to decrease and the addition of lipoic acid does not prevent this effect. The inhibition of the antioxidant response by lipoic acid in human cells appears as indirect evidence of the existence of oxidative stress in the development of this response. As lipoic acid behaves as an effective antioxidant, it seems that its action decreases the intracellular oxidative signals necessary to develop the adaptive response in human mononuclear cells.     

Evaluation of mitochondrial respiratory chain activity in wound healing by low-level laser therapy

Laser therapy is used in many biomedical sciences to promote tissue regeneration. Many studies involving low-level laser therapy have shown that the healing process is enhanced by such therapy. In this work, we evaluated mitochondrial respiratory chain complexes II and IV and succinate dehydrogenase activities in wounds after irradiation with low-level laser. The animals were divided into two groups: group 1, the animals had no local nor systemic treatment and were considered as control wounds; group 2, the wounds were treated immediately after they were made and every day after with a low-level laser (AsGa, wavelength of 904 nm) for 10 days. The results showed that low-level laser therapy improved wound healing. Besides, our results showed that low-level laser therapy significantly increased the activities of complexes II and IV but did not affect succinate dehydrogenase activity. These findings are in accordance to other works, where cytochrome c oxidase (complex IV) seems to be activated by low-level laser therapy. Besides, we showed, for the first time, that complex II activity was also activated. More studies are being carried out in order to evaluate other mitochondrial enzymes activities after different doses and irradiation time of low-level laser.     

Melatonin Reverses the Warburg-Type Metabolism and Reduces Mitochondrial Membrane Potential of Ovarian Cancer Cells Independent of MT1 Receptor Activation

Ovarian cancer (OC) is the most lethal gynecologic malignancy, and melatonin has shown various antitumor properties. Herein, we investigated the influence of melatonin therapy on energy metabolism and mitochondrial integrity in SKOV-3 cells and tested whether its effects depended on MT1 receptor activation. SKOV-3 cells were exposed to different melatonin concentrations, and experimental groups were divided as to the presence of MT1 receptors (melatonin groups) or receptor absence by RNAi silencing (siRNA MT1+melatonin). Intracellular melatonin levels increased after treatment with melatonin independent of the MT1. The mitochondrial membrane potential of SKOV-3 cells decreased in the group treated with the highest melatonin concentration. Melatonin reduced cellular glucose consumption, while MT1 knockdown increased its consumption. Interconversion of lactate to pyruvate increased after treatment with melatonin and was remarkable in siRNA MT1 groups. Moreover, lactate dehydrogenase activity decreased with melatonin and increased after MT1 silencing at all concentrations. The UCSC XenaBrowser tool showed a positive correlation between the human ASMTL gene and the ATP synthase genes, succinate dehydrogenase gene (SDHD), and pyruvate dehydrogenase genes (PDHA and PDHB). We conclude that melatonin changes the glycolytic phenotype and mitochondrial integrity of SKOV-3 cells independent of the MT1 receptor, thus decreasing the survival advantage of OC cells.     

Unravelling the Polytoxicology of Chlorfenapyr on Non-Target HepG2 Cells: The Involvement of Mitochondria-Mediated Programmed Cell Death and DNA Damage

Chlorfenapyr (CHL) is a type of insecticide with a wide range of insecticidal activities and unique targets. The extensive use of pesticides has caused an increase in potential risks to the environment and human health. However, the potential toxicity of CHL and its mechanisms of action on humans remain unclear. Therefore, human liver cells (HepG2) were used to investigate the cytotoxic effect and mechanism of toxicity of CHL at the cellular level. The results showed that CHL induced cellular toxicity in HepG2 cells and induced mitochondrial damage associated with reactive oxygen species (ROS) accumulation and mitochondrial calcium overload, ultimately leading to apoptosis and autophagy in HepG2 cells. Typical apoptotic changes occurred, including a decline in the mitochondrial membrane potential, the promotion of Bax/Bcl-2 expression causing the release of cyt-c into the cytosol, the activation of cas-9/-3, and the cleavage of PARP. The autophagic effects included the formation of autophagic vacuoles, accumulation of Beclin-1, transformation of LC3-II, and downregulation of p62. Additionally, DNA damage and cell cycle arrest were detected in CHL-treated cells. These results show that CHL induced cytotoxicity associated with mitochondria-mediated programmed cell death (PCD) and DNA damage in HepG2 cells.     

CHANGES IN SUPEROXIDE DISMUTASE, CATALASE AND GLUCOSE-6-PHOSPHATE DEHYDROGENASE ACTIVITIES OF RABBIT ALVEOLAR MACROPHAGES: INDUCED BY POSTNATAL MATURATION AND/OR IN VITRO HYPEROXIA

Abstract— The total superoxide dismutase activity of rabbit alveolar macrophages (AM) increased twofold during the first postnatal week. This increase in superoxide dismutase activity was primarily mitochondrial and paralleled the increase in the number of mitochondria in these cells which has been previously reported. The superoxide dismutase activity of AM in culture for 18 h was significantly increased by hyperoxia; catalase activity in hyperoxic conditions was slightly adversely affected; glucose-6-phosphate dehydrogenase activity was increased but not significantly over control values. Hyperoxic cultures beyond 42 h decreased the total number of viable cells and the superoxide dismutase activity expressed per viable cell; the disappearance of catalase and glucose-6-phosphate dehydrogenase activities in this period, however, was more rapid in control alveolar macrophages than those under hyperoxia.     

The disubstituted adamantyl derivative LW1564 inhibits the growth of cancer cells by targeting mitochondrial respiration and reducing hypoxia-inducible factor (HIF)-1α accumulation

Targeting cancer metabolism has emerged as an important cancer therapeutic strategy. Here, we describe the synthesis and biological evaluation of a novel class of hypoxia-inducible factor (HIF)-1α inhibitors, disubstituted adamantyl derivatives. One such compound, LW1564, significantly suppressed HIF-1α accumulation and inhibited the growth of various cancer cell lines, including HepG2, A549, and HCT116. Measurements of the oxygen consumption rate (OCR) and ATP production rate revealed that LW1564 suppressed mitochondrial respiration, thereby increasing the intracellular oxygen concentration to stimulate HIF-1α degradation. LW1564 also significantly decreased overall ATP levels by inhibiting mitochondrial electron transport chain (ETC) complex I and downregulated mammalian target of rapamycin (mTOR) signaling by increasing the AMP/ATP ratio, which increased AMP-activated protein kinase (AMPK) phosphorylation. Consequently, LW1564 promoted the phosphorylation of acetyl-CoA carboxylase, which inhibited lipid synthesis. In addition, LW1564 significantly inhibited tumor growth in a HepG2 mouse xenograft model. Taken together, the results indicate that LW1564 inhibits the growth of cancer cells by targeting mitochondrial ETC complex I and impairing cancer cell metabolism. We, therefore, suggest that LW1564 may be a potent therapeutic agent for a subset of cancers that rely on oxidative phosphorylation for ATP generation.Subject terms: Cancer metabolism, Cancer metabolism, Drug development     

Dynamic metabolic change of cancer cells induced by natural killer cells at the single-cell level studied by label-free mass cytometry

Natural killer cells (NK cells) are important immune cells which have attracted increasing attention in cancer immunotherapy. Due to the heterogeneity of cells, individual cancer cells show different resistance to NK cytotoxicity, which has been revealed by flow cytometry. Here we used label-free mass cytometry (CyESI-MS) as a new tool to analyze the metabolites in Human Hepatocellular Carcinoma (HepG2) cells at the single-cell level after the interaction with different numbers of NK92 MI cells. A large amount of chemical information from individual HepG2 cells was obtained showing the process of cell apoptosis induced by NK cells. Nineteen metabolites which consecutively change during cell apoptosis were revealed by calculating their average relative intensity. Four metabolic pathways were impacted during cell apoptosis which hit 4 metabolites including glutathione (GSH), creatine, glutamic acid and taurine. We found that the HepG2 cells could be divided into two phenotypes after co-culturing with NK cells according to the bimodal distribution of concentration of these 4 metabolites. The correlation between metabolites and different apoptotic pathways in the early apoptosis cell group was established by the 4 metabolites at the single-cell level. This is a new idea of using single-cell specific metabolites to reveal the metabolic heterogeneity in cell apoptosis which would be a powerful means for evaluating the cytotoxicity of NK cells.

Label-free mass cytometry is utilized to study the dynamic metabolic change during apoptosis in HepG2 cells induced by NK92 MI cells at the single-cell level. The metabolic heterogeneity of individual HepG2 cells during apoptosis was revealed.     

The effects of 1,2,3,4,6-penta-O-galloyl-beta-D-glucose on rat liver mitochondrial respiration

The inhibitory effects of pure galloylglucose (1,2,3,4,6-penta-O-galloyl-beta-D-glucose) on the respiratory chain of rat liver mitochondria were investigated. The respiratory control ratio (RCR) decreased by 50% on addition of 20 microM pentagalloylglucose to highly coupled mitochondria, but the adenosine-5'-diphosphate/oxygen (ADP/O) ratio decreased only slightly. The RCR disappeared and the ADP/O ratio could not be measured at concentrations of pentagalloylglucose above 30 microM. On the other hand, the uncoupler-induced oxygen consumption was also inhibited. These findings suggest that pentagalloylglucose at low concentrations inhibits the electron transport system to decrease the RCR, but scarcely impairs the membrane, practically retaining the coupled reaction, while at high concentrations it impairs the structural integrity of the mitochondrial membrane. Pentagalloylglucose competitively inhibited succinate dehydrogenase activity, and noncompetitively inhibited reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase and ubiquinol-1 oxidase activities of submitochondrial particles (SMP). However, it did not show significant inhibition of the cytochrome c oxidase activity of SMP. It is thus concluded that pentagalloylglucose, which is the lowest-molecular-weight component of tannic acid, exerts its effect on mitochondrial respiration and oxidative phosphorylation through action on the membrane and on succinate dehydrogenase, NADH dehydrogenase and cytochrome bc1 complex of mitochondria.     

Involvement of Heat-Shock Protein 70 and P53 Proteins in Attenuation of UVC-lnduced Apoptosis by Thermal Stress in Hepatocellular Carcinoma Cells

Induction of apoptosis is a function of external stimuli and cellular gene expression. Many cells respond to DNA damage by the induction of apoptosis, which depends on a functional p53 protein and is signaled by elevation of p53 levels. In this study, we found that a prior exposure to mild stress (42 degrees C) can protect HepG2 (p53+/+) cells from a subsequent UVC-induced apoptosis determined by DNA fragmentation and ratio of sub-G1 peak, but no heat-enhanced protection was found in Hep3B (p53-/-) cells. Although a similar inductive pattern of HSP70 protein and mRNA was detected in the two cell lines under thermal stress, the effect of thermal stress on UVC-induced apoptosis in HepG2 and Hep3B cells was obviously different. Overexpression of HSP70 by transient transfection of HSP70 expression vector in HepG2 cells significantly inhibited UVC-induced cell death; however, this inhibitory effect did not occur in transfected-Hep3B cells. Treatment of HepG2 cells with p53-specific antisense oligonucleotide could effectively block the antiapoptotic effect of thermal stress on UVC-induced apoptosis and increase of intracellular wild-type p53 protein by transfecting wtp53 expression plasmid into Hep3B cells yielded more resistance to UVC irradiation after prior thermal stress exposure. The results reveal an involvement of p53 in the antiapoptotic effect of thermal stress on UVC irradiation. Finally, a p53 protein increase was detected in UVC-treated HepG2 cells and could be coimmunoprecipitated with HSP70 after a thermal stress treatment. Prolonged p53 binding activity and enhanced expression of p53-controlled genes such as G1 arrest and DNA damage 45 and wild-type p53 activation factor 1/Cdk-interacting protein 1 by thermal stress are also observed in UVC-irradiated HepG2 cells. Based on these results, we propose that the antiapoptotic effect of thermal stress is mediated by increasing HSP70 and modulating intracellular p53 function.     

Cytotoxicity of Bacterial-Derived Toxins to Immortal Lung Epithelial and Macrophage Cells

Health risks associated with inhalation and deposition of biological materials have been a topic of great concern due to highly publicized cases of inhalation anthrax, of new regulations on the release of particulate matter, and to increased concerns on the hazards of indoor air pollution. Here, we present an evaluation of the sensitivity of two immortal cell lines (A549, human lung carcinoma epithelia) and NR8383 (rat alveolar macrophages) to a variety of bacterial-derived inhalation hazards and simulants including etoposide, gliotoxin, streptolysin O, and warfarin. The cell response is evaluated through quantification of changes in mitochondrial succinate dehydrogenase activity, release of lactate dehydrogenase, initiation of apoptosis, and through changes in morphology as determined by visible light microscopy and scanning electron microscopy. These cells display dose–response relations to each toxin, except for triton which has a step change response. The first observable responses of the epithelial cells to these compounds are changes in metabolism for one toxin (warfarin) and alterations in membrane permeability for another (gliotoxin). The other four toxins display a similar time course in response as gauged by changes in metabolism and loss of membrane integrity. Macrophages are more sensitive to most toxins; however, they display a lower level of stability. This information can be used in the design of cell-based sensors responding to these and similar hazards.     

PHOTOSENSITIZING EFFECTS OF HEMATOPORPHYRIN DERIVATIVE IMMOBILIZED ON SEPHAROSE

Abstract— The cytotoxicity that ensues following photosensitization by hematoporphyrin derivative (Hpd) is attributed to production of singlet oxygen. Many of the cellular end points reported to be affected are localized to membranes, hydrophobic environments conducive to partitioning of hydrophobic porphyrins in Hpd. In order to test the hypothesis that efficacy of Hpd-induced photosensitization is enhanced by its ability to freely enter cells or subcellular organelles, we immobilized Hpd on a sepharose support. This immobilized reagent was found to produce ¹O₂ when photoradiated, in yields similar to those observed for Hpd in solution, as evidenced by the bleaching of p -nitrosodimethylaniline in the presence of imidazole. The immobilized Hpd was capable of photosensitizing, i.e. inhibit, cytochrome c oxidase activity in intact mitochondrial membranes and in aqueous solution. However, enzymes located on the interior of mitochondrial membranes (F₀F₁ ATP synthase and succinate dehydrogenase), in the mitochondrial matrix (malate dehydrogenase), or on the inside of the plasma membrane, (Na++ K+)- ATPase, were unaffected by immobilized Hpd plus photoradiation compared to free Hpd. The results suggest that photosensitization by Hpd most likely arises from entry of the photosensitizer into the biological membrane, although proteins on the exterior membrane surface may be susceptible to damage by ¹O₂ produced in proximity to their location.     

Alterations in mitochondrial and apoptosis-regulating gene expression in photodynamic therapy-resistant variants of HT29 colon carcinoma cells

Photodynamic therapy (PDT) is a novel cancer therapy inducing irreversible photodamage to tumor tissue via photosensitizer-mediated oxidative cytotoxicity. The cellular and molecular responses associated with PDT are only partially understood. We have reported previously the generation of several photosensitizer-specific PDT-resistant cell variants of HT29 human colon adenocarcinoma cells by selecting cells from sequential PDT treatment using different photosensitizers. In this report, we describe the use of messenger RNA (mRNA) differential display to identify genes that were differentially expressed in the parental HT29 cells compared with their resistant variants. In comparison with parental HT29 cells, mRNA expression was increased in the PDT-resistant cell variants for BNIP3, estrogen receptor-binding fragment-associated gene 9, Myh-1c, cytoplasmic dynein light chain 1, small membrane protein I and differential dependent protein. In contrast, expression in the PDT-resistant variants was downregulated for NNX3, human HepG2 3' region Mbol complementary DNA, glutamate dehydrogenase, hepatoma-derived growth factor and the mitochondrial genes coding for 16S ribosomal RNA (rRNA) and nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 4. The reduction for mitochondrial 16S rRNA in the PDT-resistant variants was confirmed by Northern blotting, and the elevated expression of the proapoptotic BNIP3 in the PDT-resistant variants was confirmed by Northern and Western blotting analysis. We also examined the expression of some additional apoptosis-regulating genes using Western blotting. We show an increased expression of Bcl-2 and heat shock protein 27 and a downregulation of Bax in the PDT-resistant variants. In addition, the mutant p53 levels in the parental HT29 cells were reduced substantially in the PDT-resistant variants. We suggest that the altered expression in several mitochondrial and apoptosis-regulating genes contributes to PDT resistance.     

Photodynamic effects induced by meso-tetrakis[4-(carboxymethyleneoxy)phenyl] porphyrin on isolated Sarcoma 180 ascites mitochondria.

Using mitochondria isolated from Sarcoma 180 ascites tumour in Swiss mice as a model system, we have evaluated the ability of a novel porphyrin, meso-tetrakis[4-(carboxymethyleneoxy)phenyl]porphyrin (H₂T4CPP), to induce damage on photosensitization. Oxidative damage to mitochondria, one of the primary and crucial targets of the photodynamic effect, is assessed by measuring products of lipid peroxidation such as thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides (LOOH), besides the loss of activity of the mitochondrial marker enzyme succinate dehydrogenase (SDH). Analysis of product formation, the effect of deuteration and selective inhibition by scavengers of reactive oxygen species (ROS) show that the damage observed is due mainly to singlet oxygen (¹O₂) and to a minor extent to hydroxyl radicals (^•OH). The ¹O₂ generation and triplet lifetime of this porphyrin have also been estimated. Fluorescence spectroscopy, used to ascertain the binding of this porphyrin to the mitochondrial proteins, shows a rapid association within 0–2 h and a decline thereafter. Confocal microscopy reveals intracellular localisation of this porphyrin in cells in vitro. Our overall results suggest that the porphyrin H₂T4CPP, due to its ability to bind to mitochondrial protein components and to generate ROS upon photoexcitation, may have potential applications in photodynamic therapy.     

Determination of Apoptotic Mechanism of Action of Tetrabromobisphenol A and Tetrabromobisphenol S in Human Peripheral Blood Mononuclear Cells: A Comparative Study

Background: Tetrabromobisphenol A (TBBPA) is the most commonly used brominated flame retardant (BFR) in the industry. TBBPA has been determined in environmental samples, food, tap water, dust as well as outdoor and indoor air and in the human body. Studies have also shown the toxic potential of this substance. In search of a better and less toxic BFR, tetrabromobisphenol S (TBBPS) has been developed in order to replace TBBPA in the industry. There is a lack of data on the toxic effects of TBBPS, while no study has explored apoptotic mechanism of action of TBBPA and TBBPS in human leukocytes. Methods: The cells were separated from leucocyte-platelet buffy coat and were incubated with studied compounds in concentrations ranging from 0.01 to 50 µg/mL for 24 h. In order to explore the apoptotic mechanism of action of tested BFRs, phosphatidylserine externalization at cellular membrane (the number of apoptotic cells), cytosolic calcium ion and transmembrane mitochondrial potential levels, caspase-8, -9 and -3 activation, as well as PARP-1 cleavage, DNA fragmentation and chromatin condensation in PBMCs were determined. Results: TBBPA and TBBPS triggered apoptosis in human PBMCs as they changed all tested parameters in the incubated cells. It was also observed that the mitochondrial pathway was mainly involved in the apoptotic action of studied compounds. Conclusions: It was found that TBBPS, and more strongly TBBPA, triggered apoptosis in human PBMCs. Generally, the mitochondrial pathway was involved in the apoptotic action of tested compounds; nevertheless, TBBPS more strongly than TBBPA caused intrinsic pathway activation.     

姜黄素对食管癌KYSE410细胞的生长抑制与氧化调节

通过四氮唑蓝盐化合物(MTS)和重要氧化还原酶及其代谢物的试剂盒检测等方法, 考察了姜黄素对食管癌KYSE410细胞生长以及对细胞氧化还原状态和代谢的影响. 结果表明, 姜黄素对KYSE410细胞具有较强的抑制作用, 其IC₅₀=17.9 μmol/L. 进一步研究发现, 姜黄素可引起细胞培养上层清液中超氧化物歧化酶(SOD)和丙二醛(MDA)水平发生变化. 当姜黄素浓度为40 μmol/L时, MDA水平比对照组提高了125.1%, 而SOD水平则降低了43.2%; 同时, 乳酸水平降低了44.4%, 乳酸脱氢酶的活性下降了58.2%, 丙酮酸激酶的活性升高了216.7%. 姜黄素可能通过干扰氧化还原途径, 致使发生脂质过氧化反应, 并抑制肿瘤细胞糖酵解作用, 进而抑制食管癌KYSE410细胞增殖.     

Chemical fingerprinting and the biological properties of extracts from Fomitopsis pinicola

This study investigated the in vitro antioxidant and anticancer properties of the Fomitopsis pinicola extract (EMFP). The antioxidant activity of EFMP was analysed via free radical scavenging (DPPH, ABTS and Hydroxyl radicals) assay and a protein oxidation assay. EFMP effectively scavenged free radicals and exhibited remarkable protection against protein oxidation. The proliferation of EMFP-treated HepG2 cells was remarkably decreased. EMFP effectively increased the reactive oxygen species (ROS) production, depleted the mitochondrial membrane potential (MMP) and promoted the apoptosis of HepG2 cells. In addition, EMFP increased the malondialdehyde (MDA) content and reduced the activities of SOD, CAT and GPx in HepG2 cells. Using UPLC-Triple-TOF-MS, 2 phenolic compounds and 14 triterpenes were identified. These compounds may be the primary contributors to the antioxidant and anticancer capacities of EMFP. Together, these findings highlight the possibility of exploiting EMFP for its desired pharmaceutical ingredients.     

Tiliacora triandra (Colebr.) Diels Leaf Aqueous Extract Inhibits Hepatic Glucose Production in HepG2 Cells and Type 2 Diabetic Rats

This study investigated the effects of Tiliacora triandra (Colebr.) Diels aqueous extract (TTE) on hepatic glucose production in hepatocellular carcinoma (HepG2) cells and type 2 diabetic (T2DM) conditions. HepG2 cells were pretreated with TTE and its major constituents found in TTE, epicatechin (EC) and quercetin (QC). The hepatic glucose production was determined. The in vitro data were confirmed in T2DM rats, which were supplemented daily with 1000 mg/kg body weight (BW) TTE, 30 mg/kg BW metformin or TTE combined with metformin for 12 weeks. Results demonstrate that TTE induced copper-zinc superoxide dismutase, glutathione peroxidase and catalase genes, similarly to EC and QC. TTE decreased hepatic glucose production by downregulating phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) and increasing protein kinase B and AMP-activated protein kinase phosphorylation in HepG2 cells. These results correlated with the antihyperglycemic, antitriglyceridemic, anti-insulin resistance, and antioxidant activities of TTE in T2DM rats, similar to the metformin and combination treatments. Consistently, impairment of hepatic gluconeogenesis in T2DM rats was restored after single and combined treatments by reducing PEPCK and G6Pase genes. Collectively, TTE could potentially be developed as a nutraceutical product to prevent glucose overproduction in patients with obesity, insulin resistance, and diabetes who are being treated with antidiabetic drugs.