Ajit K. Verma

A novel transformation suppressor, Pdcd4, inhibits AP-1 transactivation but not NF-κB or ODC transactivation

Pdcd4 is a novel transformation suppressor that is highly expressed in promotion-resistant (P−) mouse epidermal JB6 cells but not in susceptible (P+) cells. Overexpression of pdcd4 cDNA in stably transfected P+ cells rendered cells resistant to tumor promoter-induced transformation, indicating that elevated expression of Pdcd4 protein is sufficient to suppress neoplastic transformation. To determine whether Pdcd4 suppresses neoplastic transformation through inhibiting known transformation required events, we examined the possibility that pdcd4 inhibited the activation of AP-1 or NF-κB dependent transcription or of ornithine decarboxylase (ODC) activity. Activation of AP-1-dependent transcriptional activity was inhibited by pdcd4 expression in a concentration dependent manner. In contrast, Pdcd4 slightly increased NF-κB-dependent transcription and did not alter ODC enzymatic activity. Previous studies suggested that activation of AP-1 was required for P+ cell transformation as well as for tumor promotion in vivo. These results indicate that Pdcd4 functions as a transformation suppressor, possibly through inhibiting AP-1 activation in combination with other factors such as enhancing NF-κB activation. Pdcd4 may thus constitute a useful molecular target for cancer prevention.

Plumbagin, a Medicinal Plant–Derived Naphthoquinone, Is a Novel Inhibitor of the Growth and Invasion of Hormone-Refractory Prostate Cancer

Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men. Hormone-refractory invasive PCa is the end stage and accounts for the majority of PCa patient deaths. We present here that plumbagin (PL), a quinoid constituent isolated from the root of the medicinal plant Plumbago zeylanica L., may be a potential novel agent in the control of hormone-refractory PCa. Specific observations are the findings that PL inhibited PCa cell invasion and selectively induced apoptosis in PCa cells but not in immortalized nontumorigenic prostate epithelial RWPE-1 cells. In addition, i.p. administration of PL (2 mg/kg body weight), beginning 3 days after ectopic implantation of hormone-refractory DU145 PCa cells, delayed tumor growth by 3 weeks and reduced both tumor weight and volume by 90%. Discontinuation of PL treatment in PL-treated mice for as long as 4 weeks did not result in progression of tumor growth. PL, at concentrations as low as 5 micromol/L, inhibited in both cultured PCa cells and DU145 xenografts (a) the expression of protein kinase Cepsilon (PKCepsilon), phosphatidylinositol 3-kinase, phosphorylated AKT, phosphorylated Janus-activated kinase-2, and phosphorylated signal transducer and activator of transcription 3 (Stat3); (b) the DNA-binding activity of transcription factors activator protein-1, nuclear factor-kappaB, and Stat3; and (c) Bcl-xL, cdc25A, and cyclooxygenase-2 expression. The results indicate for the first time, using both in vitro and in vivo preclinical models, that PL inhibits the growth and invasion of PCa. PL inhibits multiple molecular targets including PKCepsilon, a predictive biomarker of PCa aggressiveness. PL may be a novel agent for therapy of hormone-refractory PCa.

Prostaglandins and skin tumor promotion: Inhibition of tumor promoter-induced ornithine decarboxylase activity in epidermis by inhibitors of prostaglandin synthesis☆

Abstract Application of 12-O-tetradecanoylphorbol-13-acetate to mouse skin led to a dramatic induction of epidermal ornithine decarboxylase (EC 4.1.1.17; L-ornithine carboxy-lyase) activity. The degree of induction was remarkably depressed by prior treatment of skin with indomethacin, acetylsalicylic acid or flufenamic acid, inhibitors of prostaglandin synthesis. In contrast, dexamethasone, a steroidal anti-inflammatory drug, was ineffective. The inhibition of tumor promoter-induced ornithine decarboxylase activity by the non-steroidal anti-inflammatory drugs was completely counteracted by treatment with prostaglandin E1 and E2 but not with prostaglandin F1α or F2α.

Protein Kinase Cε 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.

Polyamine biosynthesis and skin tumor promotion: Inhibition of 12-0-tetradecanoylphorbol-13-acetate-promoted mouse skin tumor formation by the irreversible inhibitor of ornithine decarboxylase α-difluoromethylornithine

Abstract Application of 12-0-tetradecanoylphorbol-13-acetate (TPA) to mouse skin leads to the induction of ornithine decarboxylase (EC 4.1.1.17) and the accumulation of putrescine. The relevance of these TPA-induced changes to the mechanism of tumor promotion was determined using α-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase. α-Difluoromethylornithine applied to the skin of mice or administered in drinking water in conjunction with applications of TPA to 7,12-dimethylbenz[a]anthracene-initiated mouse skin inhibited the formation of mouse skin papillomas by 50 and 90% respectively; TPA-induced ornithine decarboxylase activity and the accumulation of putrescine were almost completely inhibited.

Protein kinase Cvarepsilon mediates Stat3Ser727 phosphorylation, Stat3-regulated gene expression, and cell invasion in various human cancer cell lines through integration with MAPK cascade (RAF-1, MEK1/2, and ERK1/2).

Protein kinase C epsilon (PKCɛ), a novel calcium-independent PKC isoform, has been shown to be a transforming oncogene. PKCɛ-mediated oncogenic activity is linked to its ability to promote cell survival. However, the mechanisms by which PKCɛ signals cell survival remain elusive. We found that signal transducers and activators of transcription 3 (Stat3), which is constitutively activated in a wide variety of human cancers, is a protein partner of PKCɛ. Stat3 has two conserved amino-acid (Tyr705 and Ser727) residues, which are phosphorylated during Stat3 activation. PKCɛ interacts with Stat3α isoform, which has Ser727, and not with Stat3β isoform, which lacks Ser727. PKCɛ–Stat3 interaction and Stat3Ser727 phosphorylation was initially observed during induction of squamous cell carcinomas and in prostate cancer. Now we present that (1) PKCɛ physically interacts with Stat3α isoform in various human cancer cells: skin melanomas (MeWo and WM266-4), gliomas (T98G and MO59K), bladder (RT-4 and UM-UC-3), colon (Caco-2), lung (H1650), pancreatic (PANC-1), and breast (MCF-7 and MDA:MB-231); (2) inhibition of PKCɛ expression using specific siRNA inhibits Stat3Ser727 phosphorylation, Stat3-DNA binding, Stat3-regulated gene expression as well as cell invasion; and (3) PKCɛ mediates Stat3Ser727 phosphorylation through integration with the MAPK cascade (RAF-1, MEK1/2, and ERK1/2). The results indicate that PKCɛ-mediated Stat3Ser727 phosphorylation is essential for constitutive activation of Stat3 and cell invasion in various human cancers.

Relation of the induction of epidermal ornithine decarboxylase and hyperplasia to the different skin tumor–promotion susceptibilities of protein kinase Cα, ‐δ and ‐ϵ transgenic mice

To define the in vivo role of individual PKC isoforms in mouse skin carcinogenesis, we previously characterized FVB/n transgenic mice that over‐expressed epitope‐tagged PKCδ (T7‐PKCδ) or PKCϵ (T7‐PKCϵ) isoforms under the regulation of the human K14 promoter. In continuation of our prior PKC isoform specificity studies, we now report the generation of FVB/n transgenic mice with K14‐regulated, epitope‐tagged PKCα (T7‐PKCα). T7‐PKCα transgenic mice (line 115) express 8‐fold more PKCα protein than wild‐type mice. Using high‐resolution immunogold cytochemistry, we determined that transgenic over‐expression of T7‐PKCα did not alter the subcellular localization of PKCα but that the density of PKCα staining increased. PKCα localized primarily to the cytoskeleton (tonofilaments, tight junctions) and cell membranes, with modest but definite nuclear labeling also identified. Also, PKCα over‐expression did not alter the immunoreactive protein levels of other PKC isoforms (δ, ϵ, η, ζ, μ) in the epidermis. Skin tumor‐promotion susceptibility was compared among all 3 lines of T7‐PKC transgenic mice (α, δ and ϵ). While T7‐PKCα had no effect on skin tumor promotion by TPA, T7‐PKCδ reduced papilloma burden by 76% compared to wild‐type controls. T7‐PKCϵ further reduced papilloma burden to 93% compared to wild‐type controls but still resulted in the development of squamous‐cell carcinoma. To find potential mechanisms of PKC‐associated differences in tumor promotion, the induction of known downstream effectors of tumor promotion, ornithine decarboxylase (ODC) activity and epidermal hyperplasia, was determined. Despite long‐term papilloma inhibition in both PKCδ and PKCϵ transgenic mice, the induction of ODC by TPA was not attenuated in PKC δ and ϵ mouse lines. Both PKC transgenic and wild‐type mice exhibited sustained hyperplasia after repeated TPA treatments. However, TPA‐induced epidermal hyperplasia in T7‐PKCϵ mice was significantly increased (52%) compared with T7‐PKCα, T7‐PKCδ and wild‐type mice. TPA‐induced ODC activity and the resultant accumulation of polyamines may play different roles (e.g., induction of apoptosis vs. proliferation) in the pathways leading to the induction of cancer in PKCα, PKCδ and PKCϵ transgenic mice.

Uncertainty analysis based on probability bounds (p-box) approach in probabilistic safety assessment.

A wide range of uncertainties will be introduced inevitably during the process of performing a safety assessment of engineering systems. The impact of all these uncertainties must be addressed if the analysis is to serve as a tool in the decision-making process. Uncertainties present in the components (input parameters of model or basic events) of model output are propagated to quantify its impact in the final results. There are several methods available in the literature, namely, method of moments, discrete probability analysis, Monte Carlo simulation, fuzzy arithmetic, and Dempster-Shafer theory. All the methods are different in terms of characterizing at the component level and also in propagating to the system level. All these methods have different desirable and undesirable features, making them more or less useful in different situations. In the probabilistic framework, which is most widely used, probability distribution is used to characterize uncertainty. However, in situations in which one cannot specify (1) parameter values for input distributions, (2) precise probability distributions (shape), and (3) dependencies between input parameters, these methods have limitations and are found to be not effective. In order to address some of these limitations, the article presents uncertainty analysis in the context of level-1 probabilistic safety assessment (PSA) based on a probability bounds (PB) approach. PB analysis combines probability theory and interval arithmetic to produce probability boxes (p-boxes), structures that allow the comprehensive propagation through calculation in a rigorous way. A practical case study is also carried out with the developed code based on the PB approach and compared with the two-phase Monte Carlo simulation results.

A randomized, double-blind, placebo-controlled phase 3 skin cancer prevention study of {alpha}-difluoromethylornithine in subjects with previous history of skin cancer.

Preclinical studies have shown that the inhibition of ornithine decarboxylase (ODC) by α-difluoromethylornithine (DFMO) and resultant decreases in tissue concentrations of polyamines (putrescine and spermidine) prevents neoplastic developments in many tissue types. Clinical studies of oral DFMO at 500 mg/m2/day revealed it to be safe and tolerable and resulted in significant inhibition of phorbol ester–induced skin ODC activity. Two hundred and ninety-one participants (mean age, 61 years; 60% male) with a history of prior nonmelanoma skin cancer (NMSC; mean, 4.5 skin cancers) were randomized to oral DFMO (500 mg/m2/day) or placebo for 4 to 5 years. There was a trend toward a history of more prior skin cancers in subjects randomized to placebo, but all other characteristics including sunscreen and nonsteroidal anti-inflammatory drug use were evenly distributed. Evaluation of 1,200 person-years of follow-up revealed a new NMSC rate of 0.5 events/person/year. The primary end point, new NMSCs, was not significantly different between subjects taking DFMO and placebo (260 versus 363 cancers, P = 0.069, two-sample t test). Evaluation of basal cell (BCC) and squamous cell cancers separately revealed very little difference in squamous cell cancer between treatment groups but a significant difference in new BCC (DFMO, 163 cancers; placebo, 243 cancers; expressed as event rate of 0.28 BCC/person/year versus 0.40 BCC/person/year, P = 0.03). Compliance with DFMO was >90% and it seemed to be well tolerated with evidence of mild ototoxicity as measured by serial audiometric examination when compared with placebo subjects. The analysis of normal skin biopsies revealed a significant (P < 0.05) decrease in 12-0-tetradecanoylphorbol-13-acetate–induced ODC activity (month 24, 36, and 48) and putrescine concentration (month 24 and 36 only) in DFMO subjects. Subjects with a history of skin cancer taking daily DFMO had an insignificant reduction (P = 0.069) in new NMSC that was predominantly due to a marked reduction in new BCC. Based on these data, the potential of DFMO, alone or in combination, to prevent skin cancers should be explored further. Cancer Prev Res; 3(1); 35–47

Protein Kinase C ε Is an Endogenous Photosensitizer That Enhances Ultraviolet Radiation-Induced Cutaneous Damage and Development of Squamous Cell Carcinomas1

Chronic exposure to UV radiation (UVR), especially in the UVA (315–400 nm) and UVB (280–315 nm) spectrum of sunlight, is the major risk factor for the development of nonmelanoma skin cancer. UVR is a complete carcinogen, which both initiates and promotes carcinogenesis. We found that protein kinase C ε (PKCε), a member of the phospholipid-dependent threonine/serine kinase family, is an endogenous photosensitizer, the overexpression of which in the epidermis increases the susceptibility of mice to UVR-induced cutaneous damage and development of squamous cell carcinoma. The PKCε transgenic mouse (FVB/N) lines 224 and 215 overexpressed 8- and 18-fold PKCε protein, respectively, over endogenous levels in basal epidermal cells. UVR exposure (1 kJ/m2 three times weekly) induced irreparable skin damage in high PKCε-overexpressing mouse line 215. However, the PKCε transgenic mouse line 224, when exposed to UVR (2 kJ/m2 three times weekly), exhibited minimum cutaneous damage but increased squamous cell carcinoma multiplicity by 3-fold and decreased tumor latency by 12 weeks. UVR exposure of PKCε transgenic mice compared with wild-type littermates (1) elevated the levels of neither cyclobutane pyrimidine dimer nor pyrimidine (6-4) pyrimidone dimer, (2) reduced the appearance of sunburn cells, (3) induced extensive hyperplasia and increased the levels of mouse skin tumor promoter marker ornithine decarboxylase, and (4) elevated the levels of tumor necrosis factor α (TNFα) and other growth stimulatory cytokines, granulocyte colony–stimulating factor, and granulocyte macrophage colony–stimulating factor. The role of TNFα in UVR-induced cutaneous damage was evaluated using PKCε transgenic mice deficient in TNFα. UVR treatment three times weekly for 13 weeks at 2 kJ/m2 induced severe cutaneous damage in PKCε transgenic mice (line 215), which was partially prevented in PKCε-transgenic TNFα-knockout mice. Taken together, the results indicate that PKCε signals UVR-induced TNFα release that is linked, at least in part, to the photosensitivity of PKCε transgenic mice.