Redox regulation of protein kinase C by selenometabolites and selenoprotein thioredoxin reductase limits cancer prevention by selenium.

The cancer-preventive mechanism of selenium should address the way low concentrations of selenometabolites react with cellular targets without being diffused from the sites of generation, the way selenium selectively kills tumor cells, and the intriguing U-shaped curve that is seen with dietary supplementation of selenium and cancer prevention. Protein kinase C (PKC), a receptor for tumor promoters, is well suited for this mechanism. Due to the catalytic redox cycle, low concentrations of methylselenol, a postulated active metabolite of selenium, react with the tumor-promoting lipid hydroperoxide bound to PKC to form methylseleninic acid (MSA), which selectively reacts with thiol residues present within the vicinity of the PKC catalytic domain to inactivate it. Given that lipid hydroperoxide levels are high in promoting cells, PKC inactivation selectively leads to death in these cells. A biphasic effect of MSA in inducing cell death was observed in certain prostate cancer cell lines; lower concentrations of MSA induced cell death, while higher concentrations failed to do so. Lower concentrations of selenium inactivate more sensitive antiapoptotic isoenzymes of PKC (ε and α), sparing less sensitive proapoptotic isoenzymes (PKCδ and PKCζ). Higher concentrations of selenium also inactivate proapoptotic isoenzymes and consequently make tumor cells resistant to apoptosis. Due to a high-affinity binding of thioredoxin to the PKC catalytic domain, this thiol oxidation is explicitly reversed by thioredoxin reductase (TXNRD), a selenoprotein. Therefore, overexpression of TXNRD in advanced tumor cells could make them resistant to selenium-induced death. Conceivably, this mechanism, at least in part, explains why selenium prevents cancer only in certain cases.

Imbalance in Protein Thiol Redox Regulation and Cancer-Preventive Efficacy of Selenium.

Although several experimental studies showed cancer-preventive efficacy of supplemental dietary selenium, human clinical trials questioned this efficacy. Identifying its molecular targets and mechanism is important in understanding this discrepancy. Methylselenol, the active metabolite of selenium, reacts with lipid hydroperoxides bound to protein kinase C (PKC) and is oxidized to methylseleninic acid (MSA). This locally generated MSA selectively inactivates PKC by oxidizing its critical cysteine sulfhydryls. The peroxidatic redox cycle occurring in this process may explain how extremely low concentrations of selenium catalytically modify specific membrane-bound proteins compartmentally separated from glutathione and selectively induce cytotoxicity in promoting cells. Mammalian thioredoxin reductase (TR) is itself a selenoenzyme with a catalytic selenocysteine residue. Together with thioredoxin (Trx), it catalyzes reduction of selenite and selenocystine by NADPH generating selenide which in the presence of oxygen redox cycles producing reactive oxygen species. Trx binds with high affinity to PKC and reverses PKC inactivation. Therefore, established tumor cells overexpressing TR and Trx may escape the cancer-preventive actions of selenium. This suggests that in some cases, certain selenoproteins may counteract selenometabolite actions. Lower concentrations of selenium readily inactivate antiapoptotic PKC isoenzymes e and a which have a cluster of vicinal thiols, thereby inducing apoptosis. Higher concentrations of selenium also inactivate proapoptotic enzymes such as proteolytically activated PKCd fragment, holo-PKCz, caspase-3, and c-Jun N-terminal kinase, which all have a limited number of critical cysteine residues and make tumor cells resistant to selenium-induced apoptosis. This may explain the intriguing U-shaped curve that is seen with dietary selenium intake and the extent of cancer prevention.

Polyphenols from green tea prevent antineuritogenic action of Nogo-A via 67-kDa laminin receptor and hydrogen peroxide.

Axonal regeneration after injury to the CNS is hampered by myelin-derived inhibitors, such as Nogo-A. Natural products, such as green tea, which are neuroprotective and safe for long-term therapy, would complement ongoing various pharmacological approaches. In this study, using nerve growth factor-differentiated neuronal-like Neuroscreen-1 cells, we show that extremely low concentrations of unfractionated green tea polyphenol mixture (GTPP) and its active ingredient, epigallocatechin-3-gallate (EGCG), prevent both the neurite outgrowth-inhibiting activity and growth cone-collapsing activity of Nogo-66 (C-terminal domain of Nogo-A). Furthermore, a synergistic interaction was observed among GTPP constituents. This preventive effect was dependent on 67-kDa laminin receptor (67LR) to which EGCG binds with high affinity. The antioxidants N-acetylcysteine and cell-permeable catalase abolished this preventive effect of GTPP and EGCG, suggesting the involvement of sublethal levels of H2 O2 in this process. Accordingly, exogenous sublethal concentrations of H2 O2 , added as a bolus dose (5 µM) or more effectively through a steady-state generation (1-2 µM), mimicked GTPP in counteracting the action of Nogo-66. Exogenous H2 O2 mediated this action by bypassing the requirement of 67LR. Taken together, these results show for the first time that GTPP and EGCG, acting through 67LR and elevating intracellular sublethal levels of H2 O2 , inhibit the antineuritogenic action of Nogo-A. Currently, several agents are being evaluated for overcoming axonal growth inhibitors to promote functional recovery after stroke and spinal cord injury. Epigallocatechin-3-gallate (EGCG), present in green tea polyphenol mixture (GTPP), prevents antineuritogenic activity of Nogo-A, a myelin-derived axonal growth inhibitor. The preventive action of EGCG involves the cell-surface-associated 67-kDa laminin receptor and H2 O2 . GTPP may complement ongoing efforts to treat neuronal injuries.>

Green tea catechins potentiate the neuritogenic action of brain-derived neurotrophic factor: role of 67-kDa laminin receptor and hydrogen peroxide.

Delivery of optimal amounts of brain-derived neurotrophic factor (BDNF) to regions of the brain affected by neurodegenerative diseases is a daunting task. Using natural products with neuroprotective properties, such as green tea polyphenols, would be a highly useful complementary approach for inexpensive long-term treatment of these diseases. In this study, we used PC12(TrkB) cells which ectopically express TrkB, a high affinity receptor for BDNF. They differentiate and induce neurite outgrowth in response to BDNF. Using this model, we show for the first time that treatment with extremely low concentrations (<0.1 µg/ml) of unfractionated green tea polyphenols (GTPP) and low concentrations (<0.5 µM) of their active ingredient, epigallocatechin-3-gallate (EGCG), potentiated the neuritogenic ability of a low concentration (2 ng/ml) of BDNF. A synergistic interaction was observed between GTPP constituents, where epigallocatechin and epicatechin, both individually lacking this activity, promoted the action of EGCG. GTPP-induced potentiation of BDNF action required the cell-surface associated 67 kDa laminin receptor (67LR) to which EGCG binds with high affinity. A cell-permeable catalase abolished GTPP/EGCG-induced potentiation of BDNF action, suggesting the possible involvement of H2O2 in the potentiation. Consistently, exogenous sublethal concentrations of H2O2, added as a bolus dose (5 µM) or more effectively through a steady-state generation (1 µM), potentiated BDNF action. Collectively, these results suggest that EGCG, dependent on 67 LR and H2O2, potentiates the neuritogenic action of BDNF. Intriguingly, this effect requires only submicromolar concentrations of EGCG. This is significant as extremely low concentrations of polyphenols are believed to reach the brain after drinking green tea.

Methods for studying oxidative regulation of protein kinase C.

The protein kinase C (PKC) family of isoenzymes may be a crucial player in transducing H2O2-induced signaling in a wide variety of physiological and pathophysiological processes. PKCs contain unique structural features that make them highly susceptible to oxidative modification. Depending on the site of oxidation and the extent to which it is modified, PKC can be either activated or inactivated by H2O2. The N-terminal regulatory domain contains zinc-binding, cysteine-rich motifs that are readily oxidized by H2O2. When oxidized, the autoinhibitory function of the regulatory domain is compromised, and as a result, PKC is activated in a lipid cofactor-independent manner. The C-terminal catalytic domain contains several reactive cysteine residues, which when oxidized with a higher concentration of H2O2 leads to an inactivation of PKC. Here, we describe the methods used to induce oxidative modification of purified PKC isoenzymes by H2O2 and the methods to assess the extent of this modification. Protocols are given for isolating oxidatively activated PKC isoenzymes from cells treated with H2O2. Furthermore, we describe the methods used to assess indirect regulation of PKC isoenzymes by determining their cytosol to membrane or mitochondrial translocation and tyrosine phosphorylation of PKCδ in response to sublethal levels of H2O2. Finally, as an example, we describe the methods used to demonstrate the role of H2O2-mediated cell signaling of PKCɛ in green tea polyphenol-induced preconditioning against neuronal cell death caused by oxygen-glucose deprivation and reoxygenation, an in vitro model for cerebral ischemic/reperfusion injury.

Green tea polyphenols precondition against cell death induced by oxygen-glucose deprivation via stimulation of laminin receptor, generation of reactive oxygen species, and activation of protein kinase Cε.

As the development of synthetic drugs for the prevention of stroke has proven challenging, utilization of natural products capable of preconditioning neuronal cells against ischemia-induced cell death would be a highly useful complementary approach. In this study using an oxygen-glucose deprivation and reoxygenation (OGD/R) model in PC12 cells, we show that 2-day pretreatment with green tea polyphenols (GTPP) and their active ingredient, epigallocatechin-3-gallate (EGCG), protects cells from subsequent OGD/R-induced cell death. A synergistic interaction was observed between GTPP constituents, with unfractionated GTPP more potently preconditioning cells than EGCG. GTPP-induced preconditioning required the 67-kDa laminin receptor (67LR), to which EGCG binds with high affinity. 67LR also mediated the generation of reactive oxygen species (ROS) via activation of NADPH oxidase. An exogenous ROS-generating system bypassed 67LR to induce preconditioning, suggesting that sublethal levels of ROS are indeed an important mediator in GTPP-induced preconditioning. This role for ROS was further supported by the fact that antioxidants blocked GTPP-induced preconditioning. Additionally, ROS induced an activation and translocation of protein kinase C (PKC), particularly PKCε from the cytosol to the membrane/mitochondria, which was also blocked by antioxidants. The crucial role of PKC in GTPP-induced preconditioning was supported by use of its specific inhibitors. Preconditioning was increased by conditional overexpression of PKCε and decreased by its knock-out with siRNA. Collectively, these results suggest that GTPP stimulates 67LR and thereby induces NADPH oxidase-dependent generation of ROS, which in turn induces activation of PKC, particularly prosurvival isoenzyme PKCε, resulting in preconditioning against cell death induced by OGD/R.

Green tea polyphenols potentiate the action of nerve growth factor to induce neuritogenesis: possible role of reactive oxygen species.

Exogenously administered nerve growth factor (NGF) repairs injured axons, but it does not cross the blood-brain barrier. Thus, agents that could potentiate the neuritogenic ability of endogenous NGF would be of great utility in treating neurological injuries. Using the PC12 cell model, we show here that unfractionated green tea polyphenols (GTPP) at low concentrations (0.1 µg/ml) potentiate the ability of low concentrations of NGF (2 ng/ml) to induce neuritogenesis at a level comparable to that induced by optimally high concentrations of NGF (50 ng/ml) alone. In our experiments, GTPP by itself did not induce neuritogenesis or increase immunofluorescent staining for ß-tubulin III; however, it increased expression of mRNA and proteins for the neuronal markers neurofilament-L and GAP-43. Among the polyphenols present in GTPP, epigallocatechin-3-gallate (EGCG) alone appreciably potentiated NGF-induced neurite outgrowth. Although other polyphenols present in GTPP, particularly epigallocatechin and epicatechin, lack this activity, they synergistically promoted this action of EGCG. GTPP also induced an activation of extracellular signal-regulated kinases (ERKs). PD98059, an inhibitor of the ERK pathway, blocked the expression of GAP-43. K252a, an inhibitor of TrkA-associated tyrosine kinase, partially blocked the expression of these genes and ERK activation. Antioxidants, catalase (cell-permeable form), and N-acetylcysteine (both L and D-forms) inhibited these events and abolished the GTPP potentiation of NGF-induced neuritogenesis. Taken together, these results show for the first time that GTPP potentiates NGF-induced neuritogenesis, likely through the involvement of sublethal levels of reactive oxygen species, and suggest that unfractionated GTPP is more effective in this respect than its fractionated polyphenols.

Locally generated methylseleninic acid induces specific inactivation of protein kinase C isoenzymes: relevance to selenium-induced apoptosis in prostate cancer cells.

In this study, we show that methylselenol, a selenometabolite implicated in cancer prevention, did not directly inactivate protein kinase C (PKC). Nonetheless, its oxidation product, methylseleninic acid (MSA), inactivated PKC at low micromolar concentrations through a redox modification of vicinal cysteine sulfhydryls in the catalytic domain of PKC. This modification of PKC that occurred in both isolated form and in intact cells was reversed by a reductase system involving thioredoxin reductase, a selenoprotein. PKC isoenzymes exhibited variable sensitivity to MSA with Ca(2+)-dependent PKC isoenzymes (alpha, beta, and gamma) being the most susceptible, followed by isoenzymes delta and epsilon. Other enzymes tested were inactivated only with severalfold higher concentrations of MSA than those required for PKC inactivation. This specificity for PKC was further enhanced when MSA was generated within close proximity to PKC through a reaction of methylselenol with PKC-bound lipid peroxides in the membrane. The MSA-methylselenol redox cycle resulted in the catalytic oxidation of sulfhydryls even with nanomolar concentrations of selenium. MSA inhibited cell growth and induced apoptosis in DU145 prostate cancer cells at a concentration that was higher than that needed to inhibit purified PKC alpha but in a range comparable with that required for the inhibition of PKC epsilon. This MSA-induced growth inhibition and apoptosis decreased with a conditional overexpression of PKC epsilon and increased with its knock-out by small interfering RNA. Conceivably, when MSA is generated within the vicinity of PKC, it specifically inactivates PKC isoenzymes, particularly the promitogenic and prosurvival epsilon isoenzyme, and this inactivation causes growth inhibition and apoptosis.