Charles A. Kuszynski

Free radicals and grape seed proanthocyanidin extract: importance in human health and disease prevention.

Free radicals have been implicated in over a hundred disease conditions in humans, including arthritis, hemorrhagic shock, atherosclerosis, advancing age, ischemia and reperfusion injury of many organs, Alzheimer and Parkinsons disease, gastrointestinal dysfunctions, tumor promotion and carcinogenesis, and AIDS. Antioxidants are potent scavengers of free radicals and serve as inhibitors of neoplastic processes. A large number of synthetic and natural antioxidants have been demonstrated to induce beneficial effects on human health and disease prevention. However, the structure-activity relationship, bioavailability and therapeutic efficacy of the antioxidants differ extensively. Oligomeric proanthocyanidins, naturally occurring antioxidants widely available in fruits, vegetables, nuts, seeds, flowers and bark, have been reported to possess a broad spectrum of biological, pharmacological and therapeutic activities against free radicals and oxidative stress. We have assessed the concentration- or dose-dependent free radical scavenging ability of a novel IH636 grape seed proanthocyanidin extract (GSPE) both in vitro and in vivo models, and compared the free radical scavenging ability of GSPE with vitamins C, E and beta-carotene. These experiments demonstrated that GSPE is highly bioavailable and provides significantly greater protection against free radicals and free radical-induced lipid peroxidation and DNA damage than vitamins C, E and beta-carotene. GSPE was also shown to demonstrate cytotoxicity towards human breast, lung and gastric adenocarcinoma cells, while enhancing the growth and viability of normal human gastric mucosal cells. The comparative protective effects of GSPE, vitamins C and E were examined on tobacco-induced oxidative stress and apoptotic cell death in human oral keratinocytes. Oxidative tissue damage was determined by lipid peroxidation and DNA fragmentation, while apoptotic cell death was assessed by flow cytometry. GSPE provided significantly better protection as compared to vitamins C and E, singly and in combination. GSPE also demonstrated excellent protection against acetaminophen overdose-induced liver and kidney damage by regulating bcl-X(L) gene, DNA damage and presumably by reducing oxidative stress. GSPE demonstrated excellent protection against myocardial ischemia-reperfusion injury and myocardial infarction in rats. GSPE was also shown to upregulate bcl(2) gene and downregulate the oncogene c-myc. Topical application of GSPE enhances sun protection factor in human volunteers, as well as supplementation of GSPE ameliorates chronic pancreatitis in humans. These results demonstrate that GSPE provides excellent protection against oxidative stress and free radical-mediated tissue injury.

The cytotoxic effects of a novel IH636 grape seed proanthocyanidin extract on cultured human cancer cells

Grape seed proanthocyanidins are natural antioxidants which possess a broad spectrum of chemoprotective properties against free radicals and oxidative stress. In this study, we have assessed the cytotoxicity of a novel IH636 grape seed proanthocyanidin extract (GSPE) against MCF-7 human breast cancer cells, A-427 human lung cancer cells, CRL-1739 human gastric adenocarcinoma cells and K562 chronic myelogenous leukemic cells at 25 and 50 mg/lit concentrations for 0-72 h using cytomorphology and MTT cytotoxicity assay. In addition, we compared the effects on normal human gastric mucosal cells and normal J774A.1 murine macrophage cells with the effects on the cancer cell lines. Concentration- and time-dependent cytotoxic effects of GSPE were observed on the MCF-7 breast cancer, A-427 lung cancer and gastric adenocarcinoma cells. Following incubation of the MCF-7 cells with 25 mg/lit of the GSPE approximately 6.5, 30 and 43% inhibitions in cell growth were observed at 24, 48 and 72 h of incubation, respectively, while incubation of the MCF-7 cells with 50 mg/lit of the GSPE resulted in 11, 35 and 47% inhibition in cell growth at these same points, respectively. Similar results were observed in the A-427 and gastric adenocarcinoma cells. GSPE exhibited no cytotoxicity toward the neoplastic K562 myelogenous leukemic cells. However, GSPE enhanced the growth and viability of the normal human gastric mucosal cells and J774A.1 murine macrophage cells. These data demonstrate that GSPE exhibited cytotoxicity towards some cancer cells, while enhancing the growth and viability of the normal cells which were examined.

Rapid immunologic reconstitution following transplantation with mobilized peripheral blood stem cells as compared to bone marrow

A majority of patients with intermediate or high-grade non-Hodgkin’s lymphoma (NHL) who are treated with high-dose chemotherapy (HDT) and hematopoietic stem cell transplantation subsequently relapse. Until recently, transplantation was associated with high morbidity and mortality and the focus was on improving the safety of this procedure. However, the use of growth factors and other supportive measures has successfully reduced treatment mortality to less than 5%. Therefore, new strategies need to be developed to eliminate the growth of any occult tumor cells reinfused with the stem cell products and the tumor cells remaining in the patient. One approach is to improve the immune function of the patients by a more rapid immune reconstitution and augmentation of effector cell function. We report studies comparing immune recovery following HDT and autologous peripheral stem cell transplantation (PSCT) as compared to autologous bone marrow transplantation (ABMT). These studies examined patients with intermediate and high-grade non-Hodgkin’s lymphoma (NHL) who were treated with HDT and PSCT (n = 56) or ABMT (n = 60). The PSCT patients had a significantly faster recovery of circulating monocytes (CD14+ cells), natural killer ((NK) CD56+) cells, T helper (CD4+) cells, TCRγ/δ cells, and naive T lymphocytes (CD45RA+). Following ABMT there was a significantly more rapid increase in the frequency of T suppressor/effector (CD8+) cells, B (CD19+) cells, CD34+ cells, polymorphonuclear leukocytes (PMN) and memory T lymphocytes (CD45RO+). The CD4:CD8 and CD45RA:CD45RO ratios were consistently higher in the PSCT group as compared to ABMT suggesting an improved ratio of T helper to T effector/suppressor cells and naive T cells. The differences in cellular phenotype translated into improved T cell function (PHA mitogenesis) and T cell help (pokeweed mitogenesis). In addition, there was an accelerated reconstitution of NK cell activity following PSCT as compared to ABMT. The more rapid reconstitution of NK and T cells in patients rescued with PSCT as compared to ABMT may contribute to an improved clinical outcome. Further, patients receiving a PSCT may be more responsive to adjuvant immunotherapy following transplantation.

Cadmium- and chromium-induced oxidative stress, DNA damage, and apoptotic cell death in cultured human chronic myelogenous leukemic K562 cells, promyelocytic leukemic HL-60 cells, and normal human peripheral blood mononuclear cells.

Sodium dichromate [Cr(VI)] and cadmium chloride [Cd(II)] are both cytotoxic and mutagenic. This study examined the toxic and apoptotic potentials of these two cations on three cell types in vitro, namely, human chronic myelogenous leukemic (CML) K562 cells, promyelocytic leukemic HL‐60 cells, and normal human peripheral blood mononuclear cells. The cells were incubated with 0–100 μM concentrations of the two cations for 0, 24, or 48 hours at 37°C. Both Cr(VI) and Cd(II) induced changes in intracellular oxidized states of cells, which were detected using laser scanning confocal microscopy. Cell cycle modulation and apoptosis of the K562 cells by Cr(VI) and Cd(II) were determined by flow cytometry. Significant decreases in the G2/M phase were observed in the Cr(VI) and Cd(II) treated CML cells compared with untreated cells. At 12.5 μM, Cr(VI) induced greater apoptosis in K562 cells as compared with Cd(II). In the K562 cells, 2.2‐ and 3.0‐fold increases in DNA fragmentation were observed following incubation with 12.5 and 25 μM Cr(VI), respectively, and 1.2‐ and 1.7‐fold increases in DNA fragmentation were observed with Cd(II). Furthermore, approximately 2.7‐ and 4.9‐fold increases in cytochrome c reduction were observed following incubation with 12.5 and 25 μM Cr(VI), respectively, and 1.6‐ and 3.3‐fold increases in cytochrome c reduction were observed with Cd(II), demonstrating enhanced production of superoxide anion. Approximately 3.1 to 6‐fold increases in hydroxyl radical production were observed following incubation of the K562 cells with these cations at 12.5 and 25 μM concentrations. These results in K562 cells were compared with promyelocytic leukemic HL‐60 cells and normal human peripheral blood mononuclear cells. More pronounced effects were observed on K562 and HL‐60 cells, and much lesser effects were observed on normal human peripheral blood mononuclear cells. The results demonstrate that both cations are toxic, producing oxidative tissue damage and apoptosis. Furthermore, more drastic effects were observed on K562 and HL‐60 cells as compared with normal human peripheral blood mononuclear cells.

SMOKELESS TOBACCO, OXIDATIVE STRESS, APOPTOSIS, AND ANTIOXIDANTS IN HUMAN ORAL KERATINOCYTES

We have investigated the effects of a smokeless tobacco extract (STE) on lipid peroxidation, cytochrome c reduction, DNA fragmentation and apoptotic cell death in normal human oral keratinocyte cells, and assessed the protective abilities of selected antioxidants. The cells, isolated and cultured from human oral tissues, were treated with STE (0-300 microl;g/ml) for 24 h. Superoxide anion production was determined by cytochrome c reductase. Oxidative tissue damage was determined by lipid peroxidation and DNA fragmentation, whereas apoptotic cell death was assessed by flow cytometry. STE-induced fragmentation of genomic DNA was also determined by gel electrophoresis. The comparative protective abilities of vitamin C (75 microM), vitamin E (75 microM), a combination of vitamins C & E (75 microM each), and a novel grape seed proanthocyanidin (IH636) extract (GSPE) (100 microg/ml) against STE induced oxidative stress and tissue damage were also determined. Following treatment of the cells with 300 microg STE/ml 1.5-7.6-fold increases in lipid peroxidation, cytochrome c reduction and DNA fragmentation were observed. The addition of the antioxidants to cells treated with STE provided 10-54% decreases in these parameters. Approximately 9, 29, and 35% increases in apoptotic cell death were observed following treatment with 100, 200, and 300 microg STE/ml, respectively, and 51-85% decreases in apoptotic cell death were observed with the antioxidants. The results demonstrate that STE produces oxidative tissue damage and apoptosis, which can be attenuated by antioxidants including vitamin C, vitamin E, a combination of vitamins C plus E and GSPE. GSPE exhibited better protection against STE than vitamins C and E, singly and in combination.

The Cellular and Molecular Basis of Health Benefits of Grape Seed Proanthocyanidin Extract

Red grape seed extract containing proanthocyanidins and other antioxidants are being used as nutritional supplements by many health conscious individuals. The beneficial effects of grape seed proanthocyanidins (GSPE) have been reported, however, little is known about their mechanism(s) of action. One of the beneficial effects of GSPE is chemoprevention of cellular damage. The precise mechanism by which GSPE mediates, chemoprevention is not yet understood. This report addresses this issue. We investigated the mechanisms of actions of GSPE, which ameliorates chemotherapy-induced toxic effects of Idarubicin (Ida) and 4,-hydroxyperoxycyclophosphamide (4-HC) in normal human Chang liver cells. Exposure to GSPE resulted in a significant reduction in apoptosis in response to the cytotoxicity of chemotherapeutic agents. RT-PCR analysis showed a significant increase in the anti-apoptotic gene Bcl-2 and a decrease in the cell cycle associated and proapoptotic genes, c-myc and p53 in cells treated with GSPE. These results suggest that some of the chemopreventive effects of GSPE are mediated by upregulating Bcl-2 and down regulating c-myc and p53 genes.

Porcine reproductive and respiratory syndrome virus non-structural protein 1 suppresses tumor necrosis factor-alpha promoter activation by inhibiting NF-κB and Sp1

The objective of this study was to identify porcine reproductive and respiratory syndrome virus (PRRSV)-encoded proteins that are responsible for the inhibition of TNF-α expression and the mechanism(s) involved in this phenomenon. Using a TNF-α promoter reporter system, the non-structural protein 1 (Nsp1) was found to strongly suppress the TNF-α promoter activity. Such inhibition takes place especially at the promoters proximal region. Both Nsp1α and Nsp1β, the two proteolytic fragments of Nsp1, were shown to be involved in TNF-α promoter suppression. Furthermore, using reporter plasmids specific for transcription factors (TFs) that bind to TNF-α promoter, Nsp1α and Nsp1β were demonstrated to inhibit the activity of the TFs that bind CRE-κB(3) and Sp1 elements respectively. Subsequent analyses showed that Nsp1α moderately inhibits NF-κB activation and that Nsp1β completely abrogates the Sp1 transactivation. These findings reveal one of the important mechanisms underlying the innate immune evasion by PRRSV during infection.

Chemopreventive effects of grape seed proanthocyanidin extract on Chang liver cells

In an attempt to ameliorate the chemotherapy associated normal cell toxicity, in this study a known antioxidant, grape seed proanthocyanidin extract (GSPE) using Chang liver cells has been used. Chang liver cells were treated in vitro with idarubicin (Ida) (30 nM) and 4-hydroxyperoxycyclophosphamide (4-HC) (1 microg/ml) with or without proanthocyanidin (25 microg/ml). The cells were grown in vitro and the growth rate of the cells were determined using MTT assay. The results showed that the GSPE decreased growth inhibitory effects of Ida and 4-HC on Chang liver cells in vitro. Since these chemotherapeutic agents are known to induce apoptosis in the target cells, these cells were also analyzed for presence of apoptotic cells using flow cytometry. The GSPE decreased the number of apoptotic cell population induced by either chemotherapy. In an attempt to determine the mechanisms of ameliorating effects of proanthocyanidin, the expression of apoptosis/cell cycle/growth related genes, Bcl-2, p53 and c-myc was determined in the treated and control cells using Western blotting or reverse transcriptase-polymerase chain reaction (RT-PCR) techniques. There was an increased expression of Bcl-2 in the cells treated with GSPE. However, there was a significant decrease in the expression of other cell cycle related genes such as p53 and c-myc in these cells following treatment with GSPE. Thus, these results indicate that proanthocyanidin can be a potential candidate to ameliorate the toxic effects associated with chemotherapeutic agents used in treatment of cancer.

Effect of thioltransferase (glutaredoxin) deletion on cellular sensitivity to oxidative stress and cell proliferation in lens epithelial cells of thioltransferase knockout mouse.

PURPOSE To examine the physiological function of the thioltransferase (TTase)/glutathione (GSH) system in the lens using TTase knockout mouse (TTase(-/-)) lens epithelial cells (LECs) as a model. METHODS Primary LEC cultures were obtained from wild-type (TTase(+/+)) and TTase(-/-) mice. Characterization and validation of the cells were determined by immunoblotting for TTase and alpha-crystallin proteins and by immunohistochemistry for glutathionylated proteins. Cell proliferation was examined by 3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and BrdU analysis, and cell apoptosis after H(2)O(2) stress was assessed by fluorescence-activated cell sorter analysis. Reloading of TTase protein into the TTase(-/-) cells was achieved with reagent. RESULTS Primary LEC cultures obtained from wild-type (TTase(+/+)) and TTase(-/-) mice were characterized and found to contain lens-specific alpha-crystallin protein. Western blot analysis confirmed the absence of TTase protein in the TTase(-/-) cells and its presence in the wild-type cells. TTase(-/-) LECs had significantly lower levels of glutathione (GSH) and protein thiols with extensive elevation of glutathionylated proteins, and they exhibited less resistance to oxidative stress than did TTase(+/+) cells. These cells were less viable and more apoptotic, and they had a reduced ability to remove H(2)O(2) after challenge with low levels of H(2)O(2). Reloading of purified TTase into the TTase(-/-) cells restored the antioxidant function in TTase(-/-) cells to a near normal state. CONCLUSIONS These findings confirm the importance of TTase in regulating redox homeostasis and suggest a new physiological function in controlling cell proliferation in the lens epithelial cells.

Protective effects of antioxidants against smokeless tobacco-induced oxidative stress and modulation of Bcl-2 and p53 genes in human oral keratinocytes.

The oral use of chewing tobacco has greatly increased in recent years, and this usage is associated with cancers of the mouth, lip, nasal cavities, esophagus and gut. Oral cancer accounts for 3% of all cancers in U.S.A. and is the seventh most common cancer. Previous studies in our laboratory have demonstrated the protective abilities of a novel IH636 grape seed proanthocyanidin extract (GSPE) against reactive oxygen species both in vitro and in vivo models, and provided significantly better protection as compared to vitamins C, E and β-carotene. In the recent past, we have demonstrated smokeless tobacco (STE)-induced oxidative stress, apoptotic cell death in a primary culture of normal human oral keratinocytes (NHOK), and have compared the protective abilities of vitamins C and E, singly and in combination, and GSPE in this pathobiology [Free Rad. Biol. Med., 26, 992–1000 (1999)]. In the present study, we have assessed the protective role of vitamins C and E, and GSPE against STE-induced modulation of intracellular oxidized states in NHOK cells as demonstrated by laser scanning confocal microscopy. Approximately 11%, 26%, 28% and 50% protection were observed following incubation with vitamin C, vitamin E, a combination of vitamins C plus E, and GSPE, respectively. DNA fragmentation was assessed as an index of oxidative DNA damage and similar results were observed. Furthermore, the cellular viability and functional roles of Bcl-2, p53 and c-myc genes were assessed in STE-induced oxidative stress in NHOK cells. NHOK cells were treated with STE (0–200 μg/ml) for 24h and changes in the expression of Bcl-2, p53 and c-myc genes were measured by reverse transcriptase-polymerase chain reaction (RT-PCR), and the protective effect of GSPE was assessed. Approximately a 2.0-fold increase in p53 gene expression was observed following incubation of the oral keratinocytes with 100 μg/ml of STE, beyond which the expression of p53 decreased, confirming increased apoptotic cell death with a higher concentration of STE as reported earlier. GSPE significantly modulated STE-induced changes in p53. The expression of antiapoptotic Bcl-2 gene decreased with STE treatment and the expression of Bcl-2 gene increased significantly following preincubation with GSPE. No significant change in the expression of transcription factor c-myc gene responsible for cell cycle growth was observed following incubation with STE and/or GSPE. Thus, c-myc may not be involved in STE-induced cytotoxicity towards NHOK cells. These results suggest that antioxidant protection of STE-induced cellular injury is associated with alterations in Bcl-2 and p53 expression.