Nuclear interaction between ADR-induced p65 and p53 mediates cardiac injury in iNOS (-/-) mice.

Adriamycin (ADR) treatment causes an imbalance in the levels of nitric oxide ((•)NO) and superoxide (O2(•-)) production leading to cardiac injury. Previously we demonstrated that mice lacking inducible nitric oxide synthase (iNOS) have increased oxidative stress and mitochondrial injury. The molecular events leading to increased mitochondrial injury in iNOS deficient mice is unknown. ADR in the absence of iNOS preferentially activates a proapoptotic pathway without a concurrent increase in prosurvival pathways. Treatment with ADR leads to an increase in DNA binding activity of nuclear factor kappa B (NFκB) and p53 in wildtype mice. Following ADR treatment, p53, but not NFκB DNA binding activity, as well as the level of Bax, a p53 target gene, was increased in iNOS (-/-) mice. This apoptotic signaling effect in iNOS (-/-) is alleviated by overexpression of manganese superoxide dismutase (MnSOD). Increases in NFκB and p53 in ADR-treated wildtype mice did not lead to increases in target genes such as MnSOD, bcl-xL, or Bax. Moreover, co-immunoprecipitation analysis revealed that p65, a prominent member of the NFκB family, interacts with p53 in the nucleus. These results suggest that NFκB and p53 may counter act one another's actions in ADR-treated wildtype (WT) mice. Further, these results identify a novel mechanism by which oxidative stress may regulate transcription of proapoptotic genes.

Increasing discordant antioxidant protein levels and enzymatic activities contribute to increasing redox imbalance observed during human prostate cancer progression.

A metabolomics study demonstrated a decrease in glutathione and an increase in cysteine (Cys) levels in human prostate cancer (PCa) tissues as Gleason scores increased, indicating redox imbalance with PCa progression. These results were extended in the present study by analyzing the redox state of the protein thioredoxin 1 (Trx1) and sulfinylation (SO3) of peroxiredoxins (Prxs) (PrxSO3) in PCa tissues and cell lines. Lysates of paired human PCa tissues with varying degrees of aggressiveness and adjacent benign (BN) tissues were used for analysis. Redox Western blot analysis of Trx1 demonstrated low levels of reduced and high levels of oxidized Trx1 (functional and nonfunctional, respectively) in high-grade PCa (Gleason scores 4+4 to 4+5) in comparison to intermediate-grade PCa (Gleason scores 3+3 to 3+4) or BN tissues. PrxSO3 were increased in high-grade PCa. Oxidized Trx1 and PrxSO3 are indicators of oxidative stress. To study whether redox imbalance may potentially affect enzyme activities of antioxidant proteins (APs), we determined the levels of selected APs in PCa tissues by Western blot analysis and found that mitochondrial manganese superoxide dismutase (MnSOD), Prx3, and Trx1 were increased in high-grade PCa tissues compared with BN tissues. Enzyme activities of MnSOD in high-grade PCa tissues were significantly increased but at a lower magnitude compared with the levels of MnSOD protein (0.5-fold vs 2-fold increase). Trx1 activity was not changed in high-grade PCa tissues despite a large increase in Trx1 protein expression. Further studies demonstrated a significant increase in posttranslational modifications of tyrosine and lysine residues in MnSOD protein and oxidation of Cys at the active site (Cys32 and Cys35) and the regulatory site (Cys62 and Cys69) of Trx1 in high-grade PCa compared to BN tissues. These discordant changes between protein levels and enzyme activities are consistent with protein inactivation by redox imbalance and/or posttranslational modifications. In contrast, the protein level and activity of extracellular superoxide dismutase were significantly decreased in high-grade PCa compared with adjacent BN tissues. Results from cell lines mirror those from PCa tissues. Knowledge of redox-state profiles in specific cancers may help to predict the behavior and response of each cancer to chemotherapeutic drugs and radiation.

Redox imbalance and biochemical changes in cancer.

For this article, we explore a hypothesis involving the possible role of reduction/oxidation (redox) state in cancer. We hypothesize that many modifications in cellular macromolecules, observed in cancer progression, may be caused by redox imbalance. Recent biochemical data suggest that human prostate cancer cell lines show a redox imbalance (oxidizing) compared with benign primary prostate epithelial cells; the degree of oxidation varied with aggressive behavior of each cell line. Our recent data suggest that human breast cancer tissues show a redox imbalance (reducing) compared with benign adjacent breast tissues. Accumulating data summarized in this article suggest that redox imbalance may regulate gene expression and alter protein stability by posttranslational modifications, in turn modulating existing cellular programs. Despite significant improvements in cancer therapeutics, resistance occurs, and redox imbalance may play a role in this process. Studies show that some cancer therapeutic agents increase generation of reactive oxygen/nitrogen species and antioxidant enzymes, which may alter total antioxidant capacity, cause cellular adaptation, and result in reduced effectiveness of treatment modalities. Approaches involving modulations of intra- and extracellular redox states, in combination with other therapies, may lead to new treatment options, especially for patients who are resistant to standard treatments.

KEAP1 is a redox sensitive target that arbitrates the opposing radiosensitive effects of parthenolide in normal and cancer cells.

Elevated oxidative stress is observed more frequently in cancer cells than in normal cells. It is therefore expected that additional exposure to a low level of reactive oxygen species (ROS) will push cancer cells toward death, whereas normal cells might maintain redox homeostasis through adaptive antioxidant responses. We previously showed that parthenolide enhances ROS production in prostate cancer cells through activation of NADPH oxidase. The present study identifies KEAP1 as the downstream redox target that contributes to parthenolide's radiosensitization effect in prostate cancer cells. In vivo, parthenolide increases radiosensitivity of mouse xenograft tumors but protects normal prostate and bladder tissues against radiation-induced injury. Mechanistically, parthenolide increases the level of cellular ROS and causes oxidation of thioredoxin (TrX) in prostate cancer cells, leading to a TrX-dependent increase in a reduced state of KEAP1, which in turn leads to KEAP1-mediated PGAM5 and Bcl-xL (BCL2L1) degradation. In contrast, parthenolide increases oxidation of KEAP1 in normal prostate epithelial cells, leading to increased Nrf2 (NFE2L2) levels and subsequent Nrf2-dependent expression of antioxidant enzymes. These results reveal a novel redox-mediated modification of KEAP1 in controlling the differential effect of parthenolide on tumor and normal cell radiosensitivity. Furthermore, they show it is possible to develop a tumor-specific radiosensitizing agent with radioprotective properties in normal cells.

Regulation of prostate cancer cell invasion by modulation of extra- and intracellular redox balance.

Recent metabolic profiles of human prostate cancer tissues showed a significant increase in cysteine (Cys) and a significant decrease in reduced glutathione (GSH) during cancer progression from low- to high-grade Gleason scores. Cys is primarily localized extracellularly, whereas GSH is present mostly inside the cell. We hypothesized that extra- or intracellular redox state alterations differentially regulate cell invasion in PC3 prostate carcinoma cells versus PrEC normal prostate epithelial cells. Cells were exposed to media with calculated Cys/CySS redox potentials (E(h)CySS) ranging from -60 to -180mV. After 3h exposure to a reducing extracellular redox state (E(h)CySS=-180mV), matrix metalloprotease (MMP), gelatinase, and NADPH oxidase activities increased, correlating with increases in cell invasion, cell migration, and extracellular hydrogen peroxide levels in PC3 cells but not PrECs. Knockdown of NADPH oxidase or MMP with silencing RNAs during cultivation with E(h)CySS=-180mV medium significantly decreased PC3 cell invasion. Modulation of extra- and intracellular redox states by exposure of PC3 cells to Cys/CySS-free medium (approx E(h)CySS=-87mV) containing 500µMN-acetylcysteine resulted in a more reducing intracellular redox state and a significant decrease in cell invasive ability. The decrease in PC3 cell invasion induced by these conditions correlated with a decrease in MMP activity. Our studies demonstrated that an extracellular redox state that was more reducing than a physiologic microenvironment redox state increased PC3 cancer cell invasive ability, whereas an intracellular redox environmental that was more reducing than an intracellular physiologic redox state inhibited PC3 cell invasive ability.

Distinct redox profiles of selected human prostate carcinoma cell lines: implications for rational design of redox therapy.

The effects of several cancer chemotherapeutic drugs and radiation are mediated, at least in part, by oxidative stress. To better understand this process, we analyzed certain biochemical properties affecting reduction-oxidation (redox) balance in normal prostate epithelial cells and several prostate cancer cell lines. Highly aggressive androgen-independent prostate cancer PC3 cells demonstrated significantly higher levels of total antioxidant capacity (AC) and intra- and extracellular glutathione (GSH)/glutathione disulfide (GSSG) ratios when compared with normal prostate epithelial PrEC cells. WPE1-NB26 cells, a prostate cancer cell line derived from immortalized RWPE1 human prostate epithelial cells, demonstrated significantly higher levels of total AC and intra- and extracellular GSH/GSSG ratios, but lower levels of intracellular reactive oxygen/nitrogen species and lipid peroxidation compared with RWPE1 cells. LNCaP-C4-2 cells, a more aggressive prostate cancer derived from less aggressive androgen-responsive LNCaP cells, exhibited higher levels of AC and extracellular GSH/GSSG ratio when compared to LNCaP cells. Specific cell types showed distinct cytotoxic responses to redox-modulating compounds. WPE1-NB26 cells were more sensitive to phenethyl isothiocyanate and tumor necrosis factor (TNF) than RWPE1 cells, while PC3 cells were more sensitive to TNF than PrEC cells. These results are consistent with the hypothesis that cancer cell redox state may modulate responses to redox-modulating therapeutic regimens.

Persistent expression of hepatitis C virus non-structural proteins leads to increased autophagy and mitochondrial injury in human hepatoma cells.

HCV infection is a major cause of chronic liver disease and liver cancer in the United States. To address the pathogenesis caused by HCV infection, recent studies have focused on the direct cytopathic effects of individual HCV proteins, with the objective of identifying their specific roles in the overall pathogenesis. However, this approach precludes examination of the possible interactions between different HCV proteins and organelles. To obtain a better understanding of the various cytopathic effects of and cellular responses to HCV proteins, we used human hepatoma cells constitutively replicating HCV RNA encoding either the full-length polyprotein or the non-structural proteins, or cells constitutively expressing the structural protein core, to model the state of persistent HCV infection and examined the combination of various HCV proteins in cellular pathogenesis. Increased reactive oxygen species (ROS) generation in the mitochondria, mitochondrial injury and degeneration, and increased lipid accumulation were common among all HCV protein-expressing cells regardless of whether they expressed the structural or non-structural proteins. Expression of the non-structural proteins also led to increased oxidative stress in the cytosol, membrane blebbing in the endoplasmic reticulum, and accumulation of autophagocytic vacuoles. Alterations of cellular redox state, on the other hand, significantly changed the level of autophagy, suggesting a direct link between oxidative stress and HCV-mediated activation of autophagy. With the wide-spread cytopathic effects, cells with the full-length HCV polyprotein showed a modest antioxidant response and exhibited a significant increase in population doubling time and a concomitant decrease in cyclin D1. In contrast, cells expressing the non-structural proteins were able to launch a vigorous antioxidant response with up-regulation of antioxidant enzymes. The population doubling time and cyclin D1 level were also comparable to that of control cells. Finally, the cytopathic effects of core protein appeared to focus on the mitochondria without remarkable disturbances in the cytosol.

Manganese superoxide dismutase is a p53-regulated gene that switches cancers between early and advanced stages.

Manganese superoxide dismutase (MnSOD) plays a critical role in the survival of aerobic life, and its aberrant expression has been implicated in carcinogenesis and tumor resistance to therapy. However, despite extensive studies in MnSOD regulation and its role in cancer, when and how the alteration of MnSOD expression occurs during the process of tumor development in vivo are unknown. Here, we generated transgenic mice expressing a luciferase reporter gene under the control of human MnSOD promoter-enhancer elements and investigated the changes of MnSOD transcription using the 7,12-dimethylbenz(α)anthracene (DMBA)/12-O-tetradecanoylphorbol-l3-acetate (TPA) multistage skin carcinogenesis model. The results show that MnSOD expression was suppressed at a very early stage but increased at late stages of skin carcinogenesis. The suppression and subsequent restoration of MnSOD expression were mediated by two transcription-factors, Sp1 and p53. Exposure to DMBA and TPA activated p53 and decreased MnSOD expression via p53-mediated suppression of Sp1 binding to the MnSOD promoter in normal-appearing skin and benign papillomas. In squamous cell carcinomas, Sp1 binding increased because of the loss of functional p53. We used chromatin immunoprecipitation, electrophoretic mobility shift assay, and both knockdown and overexpression of Sp1 and p53 to verify their roles in the expression of MnSOD at each stage of cancer development. The results identify MnSOD as a p53-regulated gene that switches between early and advanced stages of cancer. These findings also provide strong support for the development of means to reactivate p53 for the prevention of tumor progression.

Thioredoxin 1 as a subcellular biomarker of redox imbalance in human prostate cancer progression.

We determined protein levels and subcellular distribution of thioredoxin 1 (Trx1) in human prostate tissues using tissue microarrays and analyzed redox changes in Trx1 in the nucleus and cytoplasm in cell culture models with a redox Western blot technique. We demonstrated increased nuclear Trx1 levels in high- versus low-grade human prostate cancers. Despite increased protein levels, the oxidized forms of nuclear Trx1 were higher in prostate cancer cell lines compared to their benign counterparts, suggesting that nuclear redox imbalance occurred selectively in cancer cells. A growth-stimulating dose of androgen caused transient oxidation of Trx1 in androgen-responsive prostate cancer cells only, suggesting a loss of both androgen- and redox-signaling mechanisms during cancer progression. Androgen-independent PC3 cells showed a significant increase in nuclear and cytoplasmic Trx1 protein levels, but a significant decrease in total Trx activity. Trx1 redox state and activity correlated with the sensitivity of prostate cancer cells to pro-oxidant agents, and downregulation of Trx1 sensitized cancer cells to these agents. Our findings suggest that loss of Trx function because of oxidation and corresponding redox imbalance may play important roles in prostate cancer progression and response to therapies; and Trx1 may serve as a biomarker of subcellular redox imbalance in prostate cancer.

PKCepsilon overexpression, irrespective of genetic background, sensitizes skin to UVR-induced development of squamous-cell carcinomas.

Chronic exposure to UVR is the major etiologic factor in the development of human skin cancers including squamous-cell carcinoma (SCC). We have previously shown that protein Kinase C epsilon (PKCepsilon) transgenic mice on FVB/N background, which overexpress PKCepsilon protein approximately eightfold over endogenous levels in epidermis, exhibit about threefold more sensitivity than wild-type littermates to UVR-induced development of SCC. To determine whether it is PKCepsilon and not the mouse genetic background that determines susceptibility to UVR carcinogenesis, we cross-bred PKCepsilon FVB/N transgenic mice with SKH-1 hairless mice to generate PKCepsilon-overexpressing SKH-1 hairless mice. To evaluate the susceptibility of PKCepsilon SKH-1 hairless transgenic mice to UVR carcinogenesis, the mice were exposed to UVR (1-2 KJ m(-2)) three times weekly from a bank of six kodacel-filtered FS40 sunlamps. As compared with the wild-type hairless mice, PKCepsilon overexpression in SKH-1 hairless mice decreased the latency (12 weeks), whereas it increased the incidence (twofold) and multiplicity (fourfold) of SCC. The SKH hairless transgenic mice were observed to be as sensitive as FVB/N transgenic mice to UVR-induced development of SCC and expression of proliferative markers (proliferating cell nuclear antigen, signal transducers and activators of transcription 3, and extracellular signal-regulated kinase 1/2). The results indicate that PKCepsilon level dictates susceptibility, irrespective of genetic background, to UVR carcinogenesis.

Extracellular/microenvironmental redox state.

Extracellular redox (reduction-oxidation) state is a factor that serves as an important regulator of cell-microenvironmental interactions and is determined by several known variables; including redox-modulating proteins that are located on the plasma membrane or outside of cells, extracellular thiol/disulfide couples, and reactive oxygen species (ROS)/reactive nitrogen species (RNS) that are capable of traveling across plasma membranes into the extracellular space. The extracellular redox state works in concert with the intracellular redox state to control both the influx and efflux of ROS/RNS that may serve to modulate redox signaling or to perturb normal cellular processes or both. Under physiologic conditions, the extracellular space is known to have a relatively more-oxidized redox state than the interior of the cell. During pathologic conditions, such as cancer, the extracellular redox state may be altered, causing specific proteins such as proteases, soluble factors, or the extracellular matrix to have altered functions or activities. Recent studies have strongly supported an important relation between the extracellular redox state and cancer cell aggressiveness. The purpose of this review is to identify redox buffer networks in extracellular spaces and to emphasize the possible roles of the extracellular redox state in cancer, knowledge that may contribute to potential therapeutic interventions.

RelB enhances prostate cancer growth: implications for the role of the nuclear factor-kappaB alternative pathway in tumorigenicity.

The nuclear factor-kappaB (NF-kappaB) classic pathway is thought to be critical for tumorigenesis, but little is known about the role of the NF-kappaB alternative pathway in cancer development. Recently, high constitutive nuclear levels of RelB have been observed in human prostate cancer specimens with high Gleason scores. Here, we used four complementary approaches to test whether RelB contributes to tumorigenicity of prostate cancer. Inhibiting RelB in aggressive androgen-independent PC-3 cells by stable or conditional expression of a dominant-negative p100 mutant significantly reduced the incidence and growth rate of tumors. The decrease in tumorigenicity coincided with a reduction in the NF-kappaB target interleukin-8 (IL-8). Consistently, down-regulation of RelB by small interfering RNA targeting also reduced tumor growth and decreased levels of IL-8. Conversely, stable expression of RelB in androgen-responsive LNCaP tumors increased the circulating IL-8 levels. Taken together, these results reveal a tumor-supportive role of RelB, implicate the NF-kappaB alternative pathway as a potential target for preventing prostate cancer, and suggest the use of IL-8 as a marker for prostate cancer prognosis.

Mrp1 localization and function in cardiac mitochondria after doxorubicin.

Multidrug resistance-associated protein 1 (Mrp1; Abcc1) is expressed in sarcolemma of murine heart, where it probably protects the cardiomyocyte by mediating efflux of endo- and xenobiotics. We used doxorubicin (DOX), a chemotherapeutic drug known to induce oxidative stress and thereby cardiac injury, as a model cardiotoxic compound and observed changes in the Mrp1 expression pattern in cardiac tissue of DOX-versus saline-treated mice. Confocal immunofluorescent and immunogold electron microscopy, together with subcellular fractionation followed by immunoblot analyses and transport measurements, localized functional Mrp1 to mitochondria after DOX. Expressions of Mrp1 in heart homogenate, sarcolemma, and submitochondrial particles (SMP) were increased 1.6-, 2-, and 3-fold, respectively, at 24 h after DOX. Mitochondrial Mrp1 expression was markedly increased 72 h after DOX, whereas transport of Mrp1 substrates in SMP was maximal at 24 h. ATP-dependent transport in SMP occurred into an osmotically sensitive space and was inhibited by the anti-MRP1 antibody QCRL3. Adduction of a 190-kDa protein with the reactive lipid peroxidation product 4-hydroxy-2-nonenal (HNE) was detected in SMP and was maximal at 72 h after DOX; immunoprecipitation confirmed Mrp1-HNE adduction. In vitro, HNE (10 muM) inhibited mitochondrial respiration and transport activity in SMP, suggesting that Mrp1 is adversely affected by oxidative stress. These data demonstrate that after DOX, functional Mrp1 is detected in mitochondria in addition to that in sarcolemma; however, adduction with HNE inhibits Mrp1 activity. Mrp1 may serve to protect the heart by mediating the efflux of toxic products of oxidative stress from mitochondria and cardiomyocytes.

Extracellular redox state regulates features associated with prostate cancer cell invasion.

We have examined the possible role of extracellular reduction-oxidation (redox) state in regulation of biological/biochemical features associated with prostate cancer cell invasion. DU145, PC-3, and RWPE1-derived human prostate cancer (WPE1-NB26) cell lines were used for the present in vitro analysis. Increasing levels of nitric oxide using S-nitroso-N-acetylpenicillamine resulted in a decrease in cell invasion ability, whereas increasing levels of extracellular superoxide radical (O(2)(*-)) using xanthine/xanthine oxidase resulted in an increase in cell invasion ability in these three cell lines. WPE1-NB26 cells exhibited an increased glutathione/glutathione disulfide ratio in the medium in comparison with RWPE1 cells (immortalized but nonmalignant prostate epithelial cells), suggesting an alteration of extracellular redox state of WPE1-NB26 cells. We hypothesized that O(2)(*-) production at or near the plasma membrane or in the adjacent extracellular matrix at least partially regulated prostate cancer cell invasion. Using adenovirus-mediated extracellular superoxide dismutase (EC-SOD) gene transduction to enzymatically decrease O(2)(*-) levels, we showed that in the presence of heparin, adenovirus EC-SOD gene transduction resulted in an increase in the expression of EC-SOD outside the cells with resultant inhibition of cell invasion ability. This inhibition correlated with reduced metalloproteinase [matrix metalloproteinase (MMP) 2/membrane type 1-MMP] activities and increased levels of extracellular nitrite. Our results suggest a prominent role of extracellular redox status in regulation of cell invasion, which may provide opportunities for therapeutic interventions.

Structural profiles of TP53 gene mutations predict clinical outcome in diffuse large B-cell lymphoma: an international collaborative study.

The purpose of this study is to correlate the presence of TP53 gene mutations with the clinical outcome of a cohort of patients with diffuse large B-cell lymphoma (DLBCL) assembled from 12 medical centers. TP53 mutations were identified in 102 of 477 patients, and the overall survival (OS) of patients with TP53 mutations was significantly worse than those with wild-type TP53 (P < .001). However, subsets of TP53 mutations were found to have different effects on OS. Mutations in the TP53 DNA-binding domains were the strongest predictors of poor OS (P < .001). Mutations in the Loop-Sheet-Helix and Loop-L3 were associated with significantly decreased OS (P = .002), but OS was not significantly affected by mutations in Loop-L2. A subset of missense mutations (His158, His175, Ser245, Gln248, His273, Arg280, and Arg282) in the DNA-binding domains had the worst prognosis. Multivariate analysis confirmed that the International Prognostic Index and mutations in the DNA-binding domains were independent predictors of OS. TP53 mutations also stratified patients with germinal center B cell-like DLBCL, but not nongerminal center B cell-like DLBCL, into molecularly distinct subsets with different survivals. This study shows the prognostic importance of mutations in the TP53 DNA-binding domains in patients with DLBCL.

Differential tumor biology effects of double-initiation in a mouse skin chemical carcinogenesis model comparing wild type versus protein kinase Cepsilon overexpression mice.

Our previous studies showed that protein kinase Cepsilon (PKCepsilon) verexpression in mouse skin resulted in metastatic squamous cell carcinoma (SCC) elicited by single 7,12-dimethylbenz(a)anthracene (DMBA)-initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promotion in the absence of preceding papilloma formation as is typically observed in wild type mice. The present study demonstrates that double-DMBA initiation modulates tumor incidence, multiplicity, and latency period in both wild type and PKCepsilon overexpression transgenic (PKCepsilon-Tg) mice. After 17 weeks (wks) of tumor promotion, a reduction in papilloma multiplicity was observed in double- versus single-DMBA initiated wild type mice. Papilloma multiplicity was inversely correlated with cell death indices of interfollicular keratinocytes, indicating decreased papilloma formation was caused by increased cell death and suggesting the origin of papillomas is in interfollicular epidermis. Double-initiated PKCepsilon-Tg mice had accelerated carcinoma formation and cancer incidence in comparison to single-initiated PKCepsilon-Tg mice. Morphologic analysis of mouse skin following double initiation and tumor promotion showed a similar if not identical series of events to those previously observed following single initiation and tumor promotion: putative preneoplastic cells were observed arising from hyperplastic hair follicles (HFs) with subsequent cancer cell infiltration into the dermis. Single-initiated PKCepsilon-Tg mice exhibited increased mitosis in epidermal cells of HFs during tumor promotion.

Protein kinase Cepsilon interacts with signal transducers and activators of transcription 3 (Stat3), phosphorylates Stat3Ser727, and regulates its constitutive activation in prostate cancer.

Prostate cancer is the most common type of cancer in men and ranks second only to lung cancer in cancer-related deaths. The management of locally advanced prostate cancer is difficult because the cancer often becomes hormone insensitive and unresponsive to current chemotherapeutic agents. Knowledge about the regulatory molecules involved in the transformation to androgen-independent prostate cancer is essential for the rational design of agents to prevent and treat prostate cancer. Protein kinase Cepsilon (PKCepsilon), a member of the novel PKC subfamily, is linked to the development of androgen-independent prostate cancer. PKCepsilon expression levels, as determined by immunohistochemistry of human prostate cancer tissue microarrays, correlated with the aggressiveness of prostate cancer. The mechanism by which PKCepsilon mediates progression to prostate cancer remains elusive. We present here for the first time that signal transducers and activators of transcription 3 (Stat3), which is constitutively activated in a wide variety of human cancers, including prostate cancer, interacts with PKCepsilon. The interaction of PKCepsilon with Stat3 was observed in human prostate cancer, human prostate cancer cell lines (LNCaP, DU145, PC3, and CW22rv1), and prostate cancer that developed in transgenic adenocarcinoma of mouse prostate mice. In reciprocal immunoprecipitation/blotting experiments, prostatic Stat3 coimmunoprecipitated with PKCepsilon. Localization of PKCepsilon with Stat3 was confirmed by double immunofluorescence staining. The interaction of PKCepsilon with Stat3 was PKCepsilon isoform specific. Inhibition of PKCepsilon protein expression in DU145 cells using specific PKCepsilon small interfering RNA (a) inhibited Stat3Ser727 phosphorylation, (b) decreased both Stat3 DNA-binding and transcriptional activity, and (c) decreased DU145 cell invasion. These results indicate that PKCepsilon activation is essential for constitutive activation of Stat3 and prostate cancer progression.

Characterization of redox state of two human prostate carcinoma cell lines with different degrees of aggressiveness.

We characterized the redox profiles in two different human prostate carcinoma cell lines (LNCaP vs PC3) that are known to exhibit varying degrees of invasiveness/metastatic ability. We confirmed that PC3 cells were more invasive than LNCaP cells through an in vitro analysis. The present study documented higher 8-hydroxy-2'-deoxyguanosine levels in PC3 cells than in LNCaP cells. The levels of lipid peroxidation were higher in LNCaP cells than in PC3 cells. The reduced glutathione (GSH)/glutathione disulfide (GSSG) ratio increased to a greater extent during cell growth in PC3 cells than in LNCaP cells, whereas both reduced GSH and GSSG levels were higher in the medium of PC3 cells than in that of LNCaP cells. The levels of reactive oxygen (ROS) and reactive nitrogen species (RNS), both intracellularly and in the medium, were higher for LNCaP cells than for PC3 cells during cell growth. In addition, our results demonstrated higher ROS/RNS levels in LNCaP cells than in PC3 cells in S and G(2)/M phases of the cell cycle during logarithmic growth. Each cell type showed distinct cytotoxic responses to low-molecular-weight redox-modulating compounds. Our results document that human prostate cancer cell lines of varying degrees of aggressive behavior have distinct redox properties, findings that could lead to novel therapeutic interventions.

Mitochondrial and nuclear p53 localization in cardiomyocytes: redox modulation by doxorubicin (Adriamycin)?

Reactive oxygen (ROS) and nitrogen species (RNS) generation have been proposed to be an important mechanism of doxorubicin (Adriamycin; ADR)-induced cardiotoxicity and cardiomyocyte apoptosis, processes that may be mediated by p53 protein. We note that ADR treatment resulted in increased levels of p53 protein in cardiomyocyte mitochondria and nuclei. Modulation of the cardiomyocyte redox state in genetically engineered mice by modulation of enzymes involved in metabolism of ROS/RNS, manganese superoxide dismutase (MnSOD), or inducible nitric oxide synthase (iNOS), or a combination of these, regulated levels of mitochondrial/nuclear p53 in cardiomyocytes after ADR administration. These observations led to the hypothesis that mitochondrial/nuclear p53 localization and function in the cardiomyocyte response to ADR may be regulated through redox-dependent mechanism(s).

Phenylbutyrate, a histone deacetylase inhibitor, protects against Adriamycin-induced cardiac injury.

Cardiac injury is a major complication for oxidative-stress-generating anticancer agents exemplified by Adriamycin (ADR). Recently, several histone deacetylase inhibitors (HDACIs) including phenylbutyrate (PBA) have shown promise in the treatment of cancer with little known toxicity to normal tissues. PBA has been shown to protect against oxidative stress in normal tissues. Here, we examined whether PBA might protect heart against ADR toxicity in a mouse model. The mice were i.p. injected with ADR (20 mg/kg). PBA (400 mg/kg/day) was i.p. injected 1 day before and daily after the ADR injection for 2 days. We found that PBA significantly decreased the ADR-associated elevation of serum lactate dehydrogenase and creatine kinase activities and diminished ADR-induced ultrastructural damages of cardiac tissue by more than 70%. Importantly, PBA completely rescued ADR-caused reduction of cardiac functions exemplified by ejection fraction and fraction shortening, and increased cardiac manganese superoxide dismutase (MnSOD) protein and activity. Our results reveal a previously unrecognized role of HDACIs in protecting against ADR-induced cardiac injury and suggest that PBA may exert its cardioprotective effect, in part, by the increase of MnSOD. Thus, combining HDACIs with ADR could add a new mechanism to fight cancer while simultaneously decrease ADR-induced cardiotoxicity.