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

Paradoxical Effect of Diphenyleneiodonium in Inducing DNA Damage and Apoptosis

Diphenyleneiodonium (DPI) is often used as a molecular tool in unravelling redox-sensitive cellular events involving NADPH oxidase. However, to better understand unexpected actions of DPI, it was ascertained if DPI affects cellular DNA. DPI induced single-strand breaks in DNA of HCT-116 cells, although this only slightly increased GADD153 expression. Nevertheless, after sustaining DNA damage, the DPI-treated cells subsequently had features characteristic of apoptosis, such as translocated membrane phospholipid and nuclei containing condensed chromatin. Paradoxically, DPI attenuated the DNA damage and overall ROS production caused by sodium deoxycholate (DOC), although DPI did not inhibit DOCinduced generation of mitochondrial O2–. Furthermore, DPI prevented the occurrence of apoptosis caused by DOC. However, other known chemical inhibitors of NADPH oxidase did not produce the same results as DPI in negating the effects of DOC. Collectively, these disparate findings suggest that DPI can act not in accord with conventional wisdom depending on the experimental conditions

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 µM of either NDGA or curcumin to allow for uptake. The phenolic phytochemical-treated cells were then oxidatively challenged with 25 µM 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, a-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

Increased GADD153 Gene Expression During Iron Chelation-Induced Apoptosis in Jurkat T-Lymphocytes.

Depriving cells of iron likely stresses them and can result in cell death. To examine the potential relationship between this form of stress and cell death, Jurkat T-lymphocytes were made iron-deficient by exposing them to the iron chelator, deferoxamine (DFO). Such treatment produced evidence of apoptosis, including cell shrinkage, membrane blebbing, chromatin condensation and fragmentation, and also formation of apoptotic bodies. Additionally, proteolytic cleavage of poly(ADP-ribose)polymerase was detected, suggesting involvement of caspases in initiating apoptosis. Indeed, a selective caspase-3 inhibitor prevented the effects of DFO. During the early induction period of apoptosis, GRP78 and HSP70 mRNA expression was not affected. In contrast, there was mainly increased mRNA expression of Growth Arrest and DNA Damage-inducible gene 153 (GADD153), which seemed to be at the level of transcription rather than mRNA stability. Furthermore, fortifying cells with antioxidants did not prevent the increased GADD 153 mRNA expression, and no evidence of single-strand breaks in DNA was found, suggesting that neither reactive oxygen species nor DNA damage was involved in triggering GADD153 gene activation. DFO also caused GADD153 protein to be expressed. Because GADD153 is recognized as a pro-apoptotic gene, these findings generate the notion that GADD153 might help mediate apoptosis in iron-deficient cells

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

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

Curcumin-induced GADD153 gene up-regulation in human colon cancer cells

Ingestion of plant products containing the phenolic phytochemical, curcumin, has been linked to lower incidences of colon cancer, suggesting that curcumin has cancer chemopreventive effects. Supporting this suggestion at the cellular level, apoptosis occurs in human colon cancer cells exposed to curcumin. However, the mechanism is unclear, prompting this investigation to further clarify the molecular effects of curcumin. HCT-116 colonocytes were incubated with 0-20 mM curcumin for 0-48 h. In concentration-dependent and time-dependent manners, curcumin induced DNA damage, resulting later in the appearance of cellular features characteristic of apoptosis. To identify a potential pro-apoptotic gene that could be responsive to the DNA damage in curcumin-treated cells, growth arrest and DNA damage-inducible gene 153 (GADD153) was considered. Curcumin increased GADD153 mRNA (and also protein) expression, which was prevented by actinomycin D and also by a broad protein kinase C inhibitor, but not by selective MAPK inhibitors. These findings suggest that curcumin-induced up-regulation of GADD153 mRNA expression was at the level of transcription, but apparently without depending on upstream MAPK. In determining the involvement of reactive oxygen species in mediating the effect of curcumin on GADD153, the antioxidants pyrrolidine dithiocarbamate and N-acetylcysteine (NAC), but neither a-tocopherol nor catalase, also blunted or prevented up-regulation of GADD153 mRNA expression caused by curcumin. Most noteworthy, when NAC was tested, it inhibited the DNA damage and apoptosis caused by curcumin. Because expression of GADD153 protein was detected before the appearance of apoptotic features, this observation raises the possibility that GADD153 protein might be important for curcumin-induced apoptosis

Upregulation of haeme oxygenase-1 by zinc in HCT-116 cells

Haeme oxygenase-1 (HO-1) is often viewed as a cytoprotective gene. Toxic heavy metals induce HO-1, but it is unclear whether particular metal micronutrients also induce HO-1. Hence, the ability of exogenously-added copper, iron and zinc to influence HO-1 expression in HCT-116 cells was evaluated. Under the chosen experimental conditions, only zinc noticeably increased the expression of HO-1 mRNA and protein. Concurrently, zinc decreased non-protein thiol levels to a certain extent, but zinc did not increase the production of reactive oxygen species (ROS). Moreover, ascorbate and Trolox did not inhibit zinc-induced HO-1 upregulation. In contrast, deferoxamine blunted the induction of HO-1 mRNA, protein, and enzymatic activity caused by zinc. Additionally, N-acetylcysteine and Tiron inhibited zinc-induced HO-1 upregulation and also nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). Collectively, these findings suggest that zinc at above normal levels upregulates HO-1 expression in HCT-116 cells in a ROS-independent manner

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 µM 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-benzene 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 µM hydrogen peroxide (H2O2), Supplemental copper but not zinc or iron prevented the potentiation of the H2O2-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

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, a,a-diphenyl-ß-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 pM hydrogen peroxide (H2O2) or 100 µM 3-morpholinosydnonimine (SIN-1, a peroxynitrite generator) had comet scores of 188 ± 6 and 125 ± 12, respectively, indicating extensive DNA damage. The H2O2-induced DNA damage was inhibited with 10 µM of either EGCG (comet score: 113 ± 23) or quercetin (comet score: 82 ± 7), Similarly, the SIN-1-mediated DNA damage was inhibited with 10 µM 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 pM) 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