Negation of the cancer-preventive actions of selenium by over-expression of protein kinase Cepsilon and selenoprotein thioredoxin reductase.

Selenium prevents cancer in some cases but fails to do so in others. Selenium's failure in this respect may be due to the development of resistance to its chemopreventive actions. Selenocompounds induce a variety of cancer-preventive actions in tumor cells, but these actions may be limited by the low concentrations of free selenocompounds able to reach cells from the plasma. Therefore, we have sought to identify the chemopreventive action requiring the lowest concentration of the redox-active form of selenium, methylseleninic acid (MSA). At submicromolar concentrations, MSA inhibited the malignant transformation of RWPE-1 prostate epithelial cells. In contrast, in already transformed prostate cancer cells, selenium in the micromolar range was required to inhibit cell growth and invasion and to induce apoptosis. The role of protein kinase C (PKC) in these cellular processes, especially the moderately selenium-sensitive PKCepsilon, was demonstrated using PKC-specific inhibitors and small interfering RNA. PKCepsilon levels inversely correlated with cellular sensitivity to MSA. An over-expression of PKCepsilon minimized MSA-induced inhibition of RWPE-1 cell transformation and induction of apoptosis. Thioredoxin reductase (TR), a selenoprotein, reversed the MSA-induced inactivation of PKC isoenzymes. High TR expression in advanced prostate cancer cells correlated with resistance to MSA. Furthermore, inhibition of TR by its specific inhibitor, auranofin, resulted in increased sensitivity of prostate cancer cells to MSA. Collectively, these results suggest that the cancer-preventive actions of selenium may be negated both by an over-expression of PKCepsilon, which is a redox-sensitive target for MSA, and by the selenoprotein TR, which reverses PKC sulfhydryl redox modification.

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.

A direct redox regulation of protein kinase C isoenzymes mediates oxidant-induced neuritogenesis in PC12 cells.

In this study, we have used the PC12 cell model to elucidate the mechanisms by which sublethal doses of oxidants induce neuritogenesis. The xanthine/xanthine oxidase (X/XO) system was used for the steady state generation of superoxide, and CoCl(2) was used as a representative transition metal redox catalyst. Upon treatment of purified protein kinase C (PKC) with these oxidants, there was an increase in its cofactor-independent activation. Redox-active cobalt competed with the redoxinert zinc present in the zinc-thiolates of the PKC regulatory domain and induced the oxidation of these cysteine-rich regions. Both CoCl(2) and X/XO induced neurite outgrowth in PC12 cells, as determined by an overexpression of neuronal marker genes. Furthermore, these oxidants induced a translocation of PKC from cytosol to membrane and subsequent conversion of PKC to a cofactor-independent form. Isoenzyme-specific PKC inhibitors demonstrated that PKCepsilon plays a crucial role in neuritogenesis. Moreover, oxidant-induced neurite outgrowth was increased with a conditional overexpression of PKCepsilon and decreased with its knock-out by small interfering RNA. Parallel with PKC activation, an increase in phosphorylation of the growth-associated neuronal protein GAP-43 at Ser(41) was observed. Additionally, there was a sustained activation of extracellular signal-regulated kinases 1 and 2, which was correlated with activating phosphorylation (Ser(133)) of cAMP-responsive element-binding protein. All of these signaling events that are causally linked to neuritogenesis were blocked by antioxidant N-acetylcysteine (both L and D-forms) and by a variety of PKC-specific inhibitors. Taken together, these results strongly suggest that sublethal doses of oxidants induce neuritogenesis via a direct redox activation of PKCepsilon.