cytochrome-c-t and pterostilbene

In the present study, neuroblastoma (SH-SY5Y) cells were used to investigate the mechanisms mediating the potential protective effects of pterostilbene (PTE) against mitochondrial metabolic impairment and oxidative stress induced by hyperglycemia for mimicking the diabetic encephalopathy. High glucose medium (100mM) decreased cellular viability after 24h incubation which was evidenced by: (i) reduced mitochondrial complex I and III activities; (ii) reduced mitochondrial cytochrome C; (iii) increased reactive oxygen species (ROS) generation; (iv) decreased mitochondrial membrane potential (ΔΨm); and (v) increased lactate dehydrogenase (LDH) levels. PTE (2.5, 5, and 10μM for 24h) was nontoxic and induced the nuclear transition of Nrf2. Pretreatment of PTE (2.5, 5, and 10μM for 2h) displayed a dose-dependently neuroprotective effect, as indicated by significantly prevented high glucose-induced loss of cellular viability, generation of ROS, reduced mitochondrial complex I and III activities, reduced mitochondrial cytochrome C, decreased ΔΨm, and increased LDH levels. Moreover, the levels of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and glutathione S-transferase (GST) were elevated after PTE treatment. In addition, the elevation of nuclear Nrf2 by PTE treatment (10μM for 2h) was abolished by Nrf2 siRNA. Importantly, Nrf2 siRNA induced the opposite changes in mitochondrial complex I and III activities, mitochondrial cytochrome C, reactive species generation, ΔΨm, and LDH. Overall, the present findings were the first to show that pterostilbene attenuated high glucose-induced central nervous system injury in vitro through the activation of Nrf2 signaling, displaying protective effects against mitochondrial dysfunction-derived oxidative stress.

Topics: Cell Line, Tumor; Cytochromes c; Electron Transport Complex I; Electron Transport Complex III; Glucose; Hippocampus; Humans; L-Lactate Dehydrogenase; Membrane Potential, Mitochondrial; Mitochondria; Neurons; Neuroprotective Agents; NF-E2-Related Factor 2; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species; Stilbenes

The ability of a breast cancer cell to evade apoptosis has a key role in tumor progression and sensitivity to treatment. High levels of Bcl-2-associated X protein (Bax) in tumor cells have been found to promote apoptosis and sensitize cells to anti-cancer therapies. Bcl-2-associated X protein redistribution to the mitochondrial membrane results in the release of proapoptotic factors including cytochrome C, second-mitochondrial-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low PI (Smac/DIABLO), and Ca(2+). We aimed to explore this pathway in cancerous breast cell lines treated with the naturally occurring antioxidant 3,5-dimethoxy-4-hydroxystilbene (pterostilbene).. We used whole cell lysates +/- Bax SiRNA from the cell lines MCF-7 and MDA-MB-231 in an enzyme-linked immunosorbent assay to quantify Bax, cytochrome C, Smac/DIABLO expression, and manganese superoxide dismutase (MnSOD) activity after treatment with pterostilbene. We quantified cell death using histone-related DNA complexes from cytosolic and mitochondrial fractions and used methylthiazol tetrazolium assay to analyze cell proliferation, in the presence of Bax-silencing or scrambled RNA. We measured changes in cytosolic calcium using the ratiometric calcium-sensitive dye fura-2-AM using an inverted ratiometric monochromator microscope.. Treatment of MCF-7 and MDA-MB-231 (MDA) cells with pterostilbene caused concentration-dependent increases in intracellular Bax at all doses tested. RNA silencing of Bax resulted in reduced rates of apoptosis in both cells types and increased cell survival when treated with pterostilbene. We observed an increase in cytochrome C in MDA cells after treatment with pterostilbene. The MCF-7 cells showed a net increase in cytosolic cytochrome C, with a corresponding reduction in mitochondrial cytochrome C after treatment with 50 and 75 μmol/L pterostilbene. We observed this again in Smac/DIABLO expression in both cell types. In MCF-7 cells, pterostilbene treatment caused an increase in cytosolic but a decrease in mitochondrial Smac/DIABLO protein concentrations. Pterostilbene significantly increase MnSOD activity in MDA-MB-231 cells. Finally, pterostilbene resulted in significant increases in cytosolic calcium concentrations.. The natural dietary compound pterostilbene has an anti-proliferative effect and induces apoptosis in breast cancer cells in vitro via Bax activation and overexpression, resulting in increased MnSOD, Smac/DIABLO, and cytochrome C activity and cytosolic Ca(2+) overload.

Topics: Antioxidants; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Breast Neoplasms; Calcium; Cell Line, Tumor; Cytochromes c; Female; Humans; Intracellular Signaling Peptides and Proteins; Mitochondria; Mitochondrial Proteins; Stilbenes; Superoxide Dismutase

It has been previously shown that the naturally occurring antioxidant (-)-epigallocatechin-3-gallate (EGCG), found in green tea, and pterostilbene, a stilbenoid derived from blueberries, inhibit pancreatic cancer in vitro when used individually. We hypothesized that the combination of EGCG and pterostilbene would reveal additive effects in vitro.. Using the pancreatic cancer cell lines MIA PaCa-2 and PANC-1, efficacy and synergism were evaluated for cell proliferation and viability (3-(4,5-dimethyltiazol-2-y1)-2,5-diphenltetrazolium bromide assays, cell cycle analysis) and mitochondrial apoptosis (mitochondrial depolarization, cytochrome C release, caspase-3/7 activity, cell death detection using enzyme-linked immunosorbent assay).. Cell proliferation assays revealed significant additive antiproliferative effects with pterostilbene and EGCG in both cell lines at the later, 72-h, point (P < 0.05). MIA underwent S-phase arrest with the combination (10-12% increase); however, cell cycle arrest was not observed in PANC. The combination induced mitochondrial depolarization and upregulated cytochrome C (P < 0.05) in MIA, but these effects were not observed in PANC. EGCG increased caspase-3/7 in MIA; however, the combination did not significantly increase the activity in either cell line (P < 0.05). Apoptosis was only observed in PANC (P < 0.05). The reduction in proliferation in MIA in the 3-(4,5-dimethyltiazol-2-y1)-2,5-diphenltetrazolium bromide assays with the combination indicated that cell death occurs, possibly through another mechanism.. Our results are encouraging regarding the future use of EGCG and pterostilbene to improve traditional pancreatic cancer therapies. In conclusion, EGCG and pterostilbene have additive, antiproliferative effects in vitro and alter the apoptotic mechanisms in both cell lines by modulation at different points in the mechanism.

Topics: Anticarcinogenic Agents; Carcinoma; Caspases; Catechin; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cytochromes c; DNA Fragmentation; Drug Evaluation, Preclinical; Humans; Pancreatic Neoplasms; Stilbenes

Pterostilbene, an active constituent of blueberries, is known to possess anti-inflammatory activity and also induces apoptosis in various types of cancer cells. Here, the effects of pterostilbene on cell viability in human gastric carcinoma AGS cells were investigated. This study demonstrated that pterostilbene was able to inhibit cell proliferation and induce apoptosis in a concentration- and time-dependent manner. Pterostilbene-induced cell death was characterized with changes in nuclear morphology, DNA fragmentation, and cell morphology. The molecular mechanism of pterostilbene-induced apoptosis was also investigated. The results show the caspase-2, -3, -8, and -9 are all activated by pterostilbene, together with cleavage of the downstream caspase-3 target DNA fragmentation factor (DFF-45) and poly(ADP-riobse) polymerase. Moreover, the results indicate that the Bcl-family of proteins, the mitochondrial pathway, and activation of the caspase cascade are responsible for pterostilbene-induced apoptosis. Pterostilbene markedly enhanced the expression of growth arrest DNA damage-inducible gene 45 and 153 (GADD45 and GADD153) in a time-dependent manner. Flow cytometric analysis indicated that pterostilbene blocked cell cycle progression at G1 phase in a dose- and time-dependent manner. Pterostilbene increased the p53, p21, p27, and p16 proteins and decreased levels of cyclin A, cyclin E, cyclin-dependent kinase 2 (Cdk2), Cdk4, and Cdk6, but the expression of cyclin D1 was not affected. Over a 24 h exposure to pterostilbene, the degree of phosphorylation of Rb was decreased after 6 h. In summary, pterostilbene induced apoptosis in AGS cells through activating the caspase cascade via the mitochondrial and Fas/FasL pathway, GADD expression, and by modifying cell cycle progress and changes in several cycle-regulating proteins. The induction of apoptosis by pterostilbene may provide a pivotal mechanism of the antitumor effects and for treatment of human gastric cancer.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Blueberry Plants; Cell Cycle; Cell Division; Cell Line, Tumor; Cytochromes c; Humans; Male; Mitochondria; Stilbenes; Stomach Neoplasms