George Loo

Effects of epigallocatechin gallate and quercetin on oxidative damage to cellular DNA

Phenolic phytochemicals are thought to promote optimal health, partly via their antioxidant effects in protecting cellular components against free radicals. The aims of this study were to assess the free radical-scavenging activities of several common phenolic phytochemicals, and then, the effects of the most potent phenolic phytochemicals on oxidative damage to DNA in cultured cells. Epigallocatechin gallate (EGCG) scavenged the stable free radical, alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH), most effectively, while quercetin was about half as effective. Genistein, daidzein, hesperetin, and naringenin did not scavenge DPPH appreciably. Jurkat T-lymphocytes that were pre-incubated with relatively low concentrations of either EGCG or quercetin were less susceptible to DNA damage induced by either a reactive oxygen species or a reactive nitrogen species, as evaluated by the comet assay. More specifically, control cells had a comet score of only 17+/-5, indicating minimal DNA damage. Cells challenged with 25 microM hydrogen peroxide (H(2)O(2)) or 100 microM 3-morpholinosydnonimine (SIN-1, a peroxynitrite generator) had comet scores of 188+/-6 and 125+/-12, respectively, indicating extensive DNA damage. The H(2)O(2)-induced DNA damage was inhibited with 10 microM of either EGCG (comet score: 113+/-23) or quercetin (comet score: 82+/-7). Similarly, the SIN-1-mediated DNA damage was inhibited with 10 microM of either EGCG (comet score: 79+/-13) or quercetin (comet score: 72+/-17). In contrast, noticeable DNA damage was induced in Jurkat T-lymphocytes by incubating with 10-fold higher concentrations (i.e., 100 microM) of either EGCG (comet score: 56+/-17) or quercetin (comet score: 64+/-13) by themselves. Collectively, these data suggest that low concentrations of EGCG and quercetin scavenged free radicals, thereby inhibiting oxidative damage to cellular DNA. But, high concentrations of either EGCG or quercetin alone induced cellular DNA damage.

Redox-sensitive mechanisms of phytochemical-mediated inhibition of cancer cell proliferation (Review)

Phytochemicals are potential cancer chemopreventive agents, based partly on cellular research establishing that phytochemicals inhibit the proliferation of cancer cells. To elucidate the mechanism of phytochemicals, a basic understanding is needed of what stimulates cancer cell proliferation. Cancer cells, particularly those that are highly invasive or metastatic, may require a certain level of oxidative stress to maintain a balance between undergoing either proliferation or apoptosis. They constitutively generate large but tolerable amounts of H2O2 that apparently function as signaling molecules in the mitogen-activated protein kinase pathway to constantly activate redox-sensitive transcription factors and responsive genes that are involved in the survival of cancer cells as well as their proliferation. With such a reliance of cancer cells on H2O2 it follows that if the excess H2O2 can be scavenged by phenolic phytochemicals having antioxidant activity, the oxidative stress-responsive genes can be suppressed and consequently cancer cell proliferation inhibited. On the other hand, phenolic phytochemicals and another group of phytochemicals known as isothiocyanates can induce the formation of H2O2 to achieve an intolerable level of high oxidative stress in cancer cells. As an early response, the stress genes are activated. However, when the critical threshold for cancer cells to cope with the induced oxidative stress has been reached, key cellular components such as DNA are damaged irreparably. In conjunction, genes involved in initiating cell cycle arrest and/or apoptosis are activated. Therefore, phytochemicals can either scavenge the constitutive H2O2 or paradoxically generate additional amounts of H2O2 to inhibit the proliferation of cancer cells.

Disparate effects of similar phenolic phytochemicals as inhibitors of oxidative damage to cellular DNA.

Phenolic phytochemicals are natural plant substances whose cellular effects have not been completely determined. Nordihydroguaiaretic acid (NDGA) and curcumin are two phenolic phytochemicals with similar molecular structures, suggesting that they possess comparable chemical properties particularly in terms of antioxidant activity. To examine this possibility in a cellular system, this study evaluated the capacities of NDGA and curcumin to function as antioxidants in inhibiting oxidative damage to DNA. Jurkat T-lymphocytes were pre-incubated for 30 min with 0-25 microM of either NDGA or curcumin to allow for uptake. The phenolic phytochemical-treated cells were then oxidatively challenged with 25 microM hydrogen peroxide (H2O2). Afterwards, cells were subjected to alkaline micro-gel electrophoresis (i.e. comet assay) to assess the extent of single-strand breaks in DNA. In a concentration-dependent manner, NDGA inhibited H2O2-induced DNA damage, whereas curcumin did not. In fact, incubating Jurkat T-lymphocytes with curcumin alone actually induced DNA damage. This effect of curcumin on DNA did not appear to reflect the DNA fragmentation associated with apoptosis because there was no proteolytic cleavage of poly-(ADP-ribose)-polymerase, which is considered an early marker of apoptosis. Curcumin-induced damage to DNA was prevented by pre-treatment of the cells with the lipophilic antioxidant, alpha-tocopherol, suggesting that curcumin damaged DNA through oxygen radicals. Therefore, it is concluded that NDGA has antioxidant activity but curcumin has prooxidant activity in cultured cells based on their opposite effects on DNA.

Effect of copper deficiency on oxidative DNA damage in Jurkat T-lymphocytes

The micronutrient copper is a catalytic cofactor for copper, zinc superoxide dismutase and ceruloplasmin, which are two important antioxidant enzymes. As such, a lack of copper may promote oxidative stress and damage. The purpose of this study was to determine the effect of copper deficiency on oxidative damage to DNA in Jurkat T-lymphocytes. To induce copper deficiency, cells were incubated for 48 h with 5-20 microM 2,3,2-tetraamine (2,3,2-tet), a high affinity copper chelator. Such treatment did not affect cell proliferation/viability, as assessed by measuring mitochondrial reduction of WST-1 reagent (4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-ben zen e disulfonate). Furthermore, the induction of copper deficiency did not promote oxidative DNA damage as evaluated by the comet assay. Comet scores were 15 +/- 0 and 16 +/- 1 for control and copper-deficient cells, respectively. However, the copper-deficient cells sustained greater oxidative DNA damage than the control cells (comet scores of 175 +/- 15 and 50 +/- 10, respectively) when both were oxidatively challenged with 50 microM hydrogen peroxide (H(2)O(2)). Supplemental copper but not zinc or iron prevented the potentiation of the H(2)O(2)-induced oxidative DNA damage caused by 2,3,2-tet. These data suggest that copper deficiency compromises the antioxidant defense system of cells, thereby increasing their susceptibility to oxidative DNA damage.

Melatonin inhibits oxidative modification of human low-density lipoprotein

ABSTRACT: An important property of melatonin is that it is a free‐radical scavenger or antioxidant. Since free radicals can induce oxidative modification of low‐density lipoprotein (LDL), a process believed to be involved in atherogenesis, we were prompted to evaluate the capacity of melatonin to prevent oxidative modification of LDL. To induce oxidation, human LDL (0.4 mg protein/ml) was incubated at 37°C with either 10 μM cupric chloride or 10 mM 2,2′‐azo‐bis‐(2‐amidinopropane) dihydrochloride (AAPH) for 3 hr or 24 hr, respectively. Several assays were then performed to unequivocally determine the extent of LDL oxidation. Compared to native LDL, oxidized LDL had increased agarose gel electrophoretic mobility and weaker immunoreactivity with a murine monoclonal antibody to human apolipoprotein B‐100. Measurement of thiobarbituric acid‐reactive substances (TBARS) revealed that native LDL contained 1.8 ± 0.6 nmoles TBARS/mg protein, whereas copper‐oxidized LDL contained 53 ± 4 nmoles TBARS/mg protein. However, when present during incubation, melatonin (0.125–4 mM) inhibited in a concentration‐dependent manner the increase in electrophoretic mobility, decrease in immunoreactivity of LDL, and increase in formation of TBARS caused by either copper or AAPH. In a fourth assay, phospholipid analysis of LDL was performed. Native LDL contained 420 ± 9 nmoles phosphatidylcholine (PC)/mg LDL protein and 30 ± 20 nmoles lysophosphatidylcholine (LysPC)/mg LDL protein. LDL incubated with copper had a decreased PC content (276 ± 48 nmoles PC/mg LDL protein) and increased LysPC content (76 ± 22 nmoles LysPC/mg LDL protein). But when present during the incubation of LDL with copper, melatonin attenuated in a concentration‐dependent manner the degradation of PC to LysPC. Therefore, we conclude that melatonin can inhibit oxidative modification of LDL in vitro.

Different effects of genistein on molecular markers related to apoptosis in two phenotypically dissimilar breast cancer cell lines

The association between consumption of genistein‐containing soybean products and lower risk of breast cancer suggests a cancer chemopreventive role for genistein. Consistent with this suggestion, exposing cultured human breast cancer cells to genistein inhibits cell proliferation, although this is not completely understood. To better understand how genistein works, the ability of genistein to induce apoptosis was compared in phenotypically dissimilar MCF‐7 and MDA‐MB‐231 human breast cancer cells that express the wild‐type and mutant p53 gene, respectively. After 6 days of incubation with 50 μM genistein, MCF‐7 but not MDA‐MB‐231 cells, showed morphological signs of apoptosis. Marginal proteolytic cleavage of poly‐(ADP‐ribose)‐polymerase and significant DNA fragmentation were also detected in MCF‐7 cells. In elucidating these findings, it was determined that after 2 days of incubation with genistein, MCF‐7 but not MDA‐MB‐231 cells, had significantly higher levels of p53. Accordingly, the expression of certain proteins modulated by p53 was studied next. Levels of p21 increased in both of the genistein‐treated cell lines, suggesting that p21 gene expression was activated but in a p53‐independent manner, whereas no significant changes in levels of the pro‐apoptotic protein, Bax, were found. In MCF‐7 cells, levels of the anti‐apoptotic protein, Bcl‐2, decreased slightly at 18–24 h but then increased considerably after 48 h. Hence, the Bax:Bcl‐2 ratio initially increased but later decreased. These data suggest that at the genistein concentration tested, MCF‐7 cells in contrast to MDA‐MB‐231 cells were sensitive to the induction of apoptosis by genistein, but Bax and Bcl‐2 did not play clear roles. J. Cell. Biochem. 82: 78–88, 2001.

Induction of GADD gene expression by phenethylisothiocyanate in human colon adenocarcinoma cells

Phenethylisothiocyanate (PEITC), a potential cancer chemopreventive agent, induces colon cancer cell death, but the mechanism is not entirely clear. Therefore, the aim of this study was to further clarify the molecular effects of PEITC in causing death of human colon adenocarcinoma cells. When incubated with PEITC, HCT‐116 colonocytes showed morphological features characteristic of apoptosis, such as irregular cell shape, translocation of plasma membrane phosphatidylserine, and also chromatin condensation and fragmentation. These changes occurred after single‐strand breaks in DNA were detected, suggesting that PEITC induced irreparable DNA damage, which in turn triggered the process of apoptosis. DNA macroarray analysis of a selected small cluster of apoptosis‐related genes revealed noticeably higher expression of only GADD45, which was confirmed by gene‐specific relative RT‐PCR analysis. This led to investigation of other GADD gene members possibly affected by PEITC. Whereas GADD34 mRNA expression increased just slightly, there was an appreciable elevation of the mRNA for GADD153, which is recognized as a pro‐apoptotic gene. The effect of PEITC on GADD153 was attenuated by either actinomycin D or N‐acetylcysteine, suggesting that PEITC‐induced upregulation of GADD153 mRNA expression was partly at the level of transcriptional activation involving reactive oxygen species. Additionally, PEITC‐induced upregulation of GADD153 mRNA expression did not appear to require p53, based on the observation that PEITC also increased GADD153 mRNA expression in HCT‐15 colonocytes, which are known to express mutant p53. These findings suggest that PEITC creates an oxidative cellular environment that induces DNA damage and GADD153 gene activation, which in turn helps trigger apoptosis.

Epigallocatechin gallate protects U937 cells against nitric oxide-induced cell cycle arrest and apoptosis.

Ingesting phenolic phytochemicals in many plant products may promote health, but the effects of phenolic phytochemicals at the cellular level have not been fully examined. Thus, it was determined if the tea phenolic phytochemical, epigallocatechin gallate (EGCG), protects U937 human pro‐monocytic cells against the nitrogen free radical, nitric oxide (•NO). Cells were incubated for 4–6 h with 500 μM S‐nitrosoglutathione (GSNO), which generates •NO, but this did not induce single‐strand breaks in DNA. Nevertheless, 82 ± 4% of GSNO‐treated cells, compared to only 39 ± 1% of untreated cells, were arrested in the G1‐phase of the cell cycle. However, dosing the GSNO‐treated cells with 9, 14, or 18 μg/ml of EGCG resulted in only 74 ± 8%, 66 ± 1%, and 43 ± 3% of the cells, respectively, in the G1‐phase. Exposing cells to GSNO also resulted in the emergence of a sub‐G1 apoptotic cell population numbering 14 ± 3%, but only 5 ± 2%, 5 ± 1%, and 2 ± 0% upon dosing of the GSNO‐treated cells with 9, 14, and 18 μg/ml of EGCG, respectively. Furthermore, exposing cells to GSNO resulted in greater cell surface binding of annexin V‐FITC, but binding was 41–89% lower in GSNO‐treated cells dosed with EGCG. Collectively, these data suggest that •NO or downstream products induced cell cycle arrest and apoptosis that was not due to single‐strand breaks in DNA, and that EGCG scavenged cytotoxic •NO or downstream products, thus reducing the number of cells in a state of cell cycle arrest or apoptosis. J. Cell. Biochem. 81: 647–658, 2001.

Increased GADD gene expression in human colon epithelial cells exposed to deoxycholate

The colonic epithelium is often exposed to high concentrations of secondary bile acids, which stresses the epithelial cells, leading potentially to activation of stress‐response genes. To examine this possibility in vitro, the purpose of this study was to determine if expression of certain growth arrest and DNA damage‐inducible genes (GADD) is upregulated in human colonic epithelial cells exposed to deoxycholate (DOC). DNA macroarray screening of a small cluster of stress/apoptosis‐related genes in DOC‐treated HCT‐116 colonocytes revealed clearly higher expression of only GADD45, which was confirmed by gene‐specific relative RT‐PCR analysis. Subsequently, it was found that DOC also increased GADD34 mRNA expression. However, mRNA expression of GADD153 was increased most markedly in DOC‐treated HCT‐116 colonocytes, which express wild‐type p53. However, the upregulation of GADD34, GADD45, and GADD153 mRNA expression apparently did not require p53, based on the finding that DOC increased expression of all three GADD genes in HCT‐15 colonocytes, which express mutant p53. In further studying GADD153 in particular, the effect of DOC on GADD153 mRNA was prevented by actinomycin‐D (Act‐D), but not by antioxidants or MAPK inhibitors. DOC also caused GADD153 protein to be expressed in close parallel with increased GADD153 mRNA expression. Induction of GADD153 protein by DOC was prevented by either anisomycin or cycloheximide. These findings suggest that DOC‐induced upregulation of GADD153 mRNA expression occurred at the level of transcription without involving reactive oxygen species and MAPK signaling, and that the expression of GADD153 protein was due also to translation of pre‐existing, and not just newly synthesized, mRNA.

Protection of human colon epithelial cells against deoxycholate by rottlerin

The bile salt, deoxycholate (DOC), can harm cells and cause disease. Hence, there is interest in identifying compounds capable of protecting cells against DOC. In HCT-116 colon epithelial cells, DOC increased generation of reactive oxygen species and caused DNA damage and apoptosis. These effects of DOC were inhibited by rottlerin, which is a phenolic compound of plant origin. In elucidating its mechansim, rottlerin prevented the release of cytochrome c from mitochondria into cytosol, and also prevented the cleavage of caspase-3. Yet, rottlerin by itself markedly decreased mitochondrial membrane potential and increased mitochondrial superoxide production, but this did not result in cytochrome c release or in caspase-3 cleavage. At a higher test concentration, two other phenolic phytochemicals, namely, quercetin and resveratrol, were each able to largely prevent the occurrence of apoptosis in cells exposed to DOC. In contrast, epigallocatechin gallate, curcumin, and genistein were ineffective.