Russell D. Klein

Low‐dose genistein induces cyclin‐dependent kinase inhibitors and G1 cell‐cycle arrest in human prostate cancer cells

Genistein, a naturally occurring isoflavone found chiefly in soy products, reportedly has antiprostate cancer effects, but the mechanisms underlying these effects are unknown. We studied the antiproliferative and apoptosis‐inducing effects of genistein in the androgen‐sensitive human prostate cancer cell line LNCaP. Viable cell number was assessed by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay; cell‐cycle progression and apoptosis were evaluated by flow cytometry; apoptosis was also assessed by a histone enzyme‐linked immunosorbent assay; and the expression of several cell‐cycle– and apoptosis‐related genes and their gene products was determined by northern blot analysis, western blot analysis, and/or assays based on polymerase chain reaction. Physiologic concentrations of genistein (≤ 20 μM) decreased LNCaP viable cell number in a dose‐dependent manner, induced a G1 cell‐cycle block, decreased prostate‐specific antigen mRNA expression, and increased p27KIP1 and p21WAF1 (mRNA and protein) but had no effect on apoptosis or the mRNA expression of the apoptosis‐ and cell‐cycle–related markers bcl‐2, bax, Rb, and proliferating cell nuclear antigen. Higher concentrations of genistein (> 20 μM) did induce apoptosis. We conclude that genistein (at physiologic concentrations) exerts potent antiproliferative effects on LNCaP cells by inducing a G1 cell‐cycle block. The antiproliferative effects of genistein may be mediated by increased levels of p27KIP1 and p21WAF1, which are negative cell‐cycle regulators that act as cyclin‐dependent kinase inhibitors and that have been recently linked with prostate carcinogenesis. These findings may provide insights into the mechanisms underlying the apparent antiprostate cancer effects of soy consumption observed in epidemiologic studies. Mol. Carcinog. 29:92–102, 2000.

Formation and antiproliferative effect of prostaglandin E3 from eicosapentaenoic acid in human lung cancer cells

We investigated the formation and pharmacology of prostaglandin E3 (PGE3) derived from fish oil eicosapentaenoic acid (EPA) in human lung cancer A549 cells. Exposure of A549 cells to EPA resulted in the rapid formation and export of PGE3. The extracellular ratio of PGE3 to PGE2 increased from 0.08 in control cells to 0.8 in cells exposed to EPA within 48 h. Incubation of EPA with cloned ovine or human recombinant cyclooxygenase 2 (COX-2) resulted in 13- and 18-fold greater formation of PGE3, respectively, than that produced by COX-1. Exposure of A549 cells to 1 μM PGE3 inhibited cell proliferation by 37.1% (P < 0.05). Exposure of normal human bronchial epithelial (NHBE) cells to PGE3, however, had no effect. When A549 cells were exposed to EPA (25 μM) or a combination of EPA and celecoxib (a selective COX-2 inhibitor), the inhibitory effect of EPA on the growth of A549 cells was reversed by the presence of celecoxib (at both 5 and 10 μM). This effect appears to be associated with a 50% reduction of PGE3 formation in cells treated with a combination of EPA and celecoxib compared with cells exposed to EPA alone. These data indicate that exposure of lung cancer cells to EPA results in a decrease in the COX-2-mediated formation of PGE2, an increase in the level of PGE3, and PGE3-mediated inhibition of tumor cell proliferation.

Transitional Cell Hyperplasia and Carcinomas in Urinary Bladders of Transgenic Mice with Keratin 5 Promoter-Driven Cyclooxygenase-2 Overexpression

The inducible form of cyclooxygenase (COX), COX-2, is up-regulated in many epithelial cancers and its prostaglandin products increase proliferation, enhance angiogenesis, and inhibit apoptosis in several tissues. Pharmacologic inhibition and genetic deletion studies showed a marked reduction of tumor development in colon and skin. COX-2 has also been strongly implicated in urinary bladder cancer primarily by studies with nonselective COX- and COX-2-selective inhibitors. We now show that forced expression of COX-2, under the control of a keratin 5 promoter, is sufficient to cause transitional cell hyperplasia (TCH) in 17% and 75% of the heterozygous and homozygous transgenic lines, respectively, in an age-dependent manner. TCH was strongly associated with inflammation, primarily nodules of B lymphocytes; some T cells and macrophage infiltration were also observed. Additionally, transitional cell carcinoma was observed in approximately 10% of the K5.COX-2 transgenic mice; no TCH or transitional cell carcinoma was observed in wild-type bladders. Immunohistochemistry for vascular proliferation and vascular endothelial growth factor showed significant increases above that in wild-type urinary bladders. Our results suggest that overexpression of COX-2 is sufficient to cause hyperplasia and carcinomas in the urinary bladder. Therefore, inhibition of COX-2 should continue to be pursued as a potential chemopreventive and therapeutic strategy.

OSU-HDAC42, a Histone Deacetylase Inhibitor, Blocks Prostate Tumor Progression in the Transgenic Adenocarcinoma of the Mouse Prostate Model

Histone deacetylase (HDAC) inhibitors suppress tumor cell growth via a broad spectrum of mechanisms, which should prove advantageous in the context of cancer prevention. Here, we examined the effect of dietary administration of OSU-HDAC42, a novel HDAC inhibitor, on prostate tumor progression in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Based on a series of pilot studies, an AIN-76A diet was formulated containing 208 ppm OSU-HDAC42, which was estimated to deliver approximately 25 mg/kg of drug per day to each mouse and found to cause a suppression of PC-3 xenograft tumor growth equivalent to that achieved by gavage administration of a similar dose. At 6 weeks of age, TRAMP mice received this drug-containing or control diet for 4 or 18 weeks and were evaluated for prostatic intraepithelial neoplasia (PIN) and carcinoma development, respectively. OSU-HDAC42 not only decreased the severity of PIN and completely prevented its progression to poorly differentiated carcinoma (74% incidence in controls versus none in drug-treated mice), but also shifted tumorigenesis to a more differentiated phenotype, suppressing absolute and relative urogenital tract weights by 86% and 85%, respectively, at 24 weeks of age. This tumor suppression was associated with the modulation of intraprostatic biomarkers, including those indicative of HDAC inhibition, increased apoptosis and differentiation, and decreased proliferation. With the exception of completely reversible hematologic alterations and testicular degeneration, no significant changes in body weight or other indicators of general health were observed in drug-treated mice. These results suggest that OSU-HDAC42 has value in prostate cancer prevention. [Cancer Res 2008;68(10):3999-4009].

SAGE profiling of UV-induced mouse skin squamous cell carcinomas, comparison with acute UV irradiation effects

Ultraviolet (UV) irradiation is the primary environmental insult responsible for the development of most common skin cancers. To better understand the multiple molecular events that contribute to the development of UV‐induced skin cancer, in a first study, serial analysis of gene expression (SAGE) was used to compare the global gene expression profiles of normal SKH‐1 mice epidermis with that of UV‐induced squamous cell carcinomas (SCCs) from SKH‐1 mice. More than 200 genes were found to be differentially expressed in SCCs compared to normal skin (P < 0.0005 level of significance). As expected, genes related to epidermal proliferation and differentiation were deregulated in SCCs relative to normal skin. However, various novel genes, not previously associated with skin carcinogenesis, were also identified as deregulated in SCCs. Northern blot analyses on various selected genes validated the SAGE findings: caspase‐14 (reduced 8.5‐fold in SCCs); cathepsins D and S (reduced 3‐fold and increased 11.3‐fold, respectively, in SCCs); decorin, glutathione S‐transferase omega‐1, hypoxia‐inducible factor 1α, insulin‐like growth factor binding protein‐7, and matrix metalloproteinase‐13 (increased 18‐, 12‐, 12‐, 18.3‐, and 11‐folds, respectively, in SCCs). Chemokine (C‐C motif), ligand 27 (CCL27), which was found downregulated 12.7‐fold in SCCs by SAGE, was also observed to be strongly downregulated 6–24 h after a single and multiple UV treatments. In a second independent study we compared the expression profile of UV‐irradiated versus sham‐treated SKH‐1 epidermis. Interestingly, numerous genes determined to be deregulated 8 h after a single UV dose were also deregulated in SCCs. For instance, genes whose expression was upregulated both after acute UV‐treated skin and SCCs included keratins 6 and 16, small proline‐rich proteins, and S100 calcium binding protein A9. Studies like those described here do not only provide insights into genes and pathways involved in skin carcinogenesis but also allow us to identify early UV irradiation deregulated surrogate biomarkers of potential use in chemoprevention studies.

Aberrant expression of fibroblast growth factor receptor-1 in prostate epithelial cells allows induction of promatrilysin expression by fibroblast growth factors

Matrix metalloproteinases (MMPs) degrade extracellular matrix proteins, and there is evidence that they play a role in tumor cell growth, invasion and metastasis. Matrilysin (MMP‐7) is over‐expressed in prostate cancer cells and increases prostate cancer cell invasion. Prostate stromal fibroblasts secrete a factor(s), including fibroblast growth factor‐1 (FGF‐1), which induces promatrilysin expression in the prostate carcinoma cell line LNCaP but not in normal prostate epithelial cells (PrECs). Since FGF‐1 is present in the prostate, an altered sensitivity to FGF‐1 might explain the up‐regulation of matrilysin expression in prostate cancer cells compared to normal prostate epithelium. FGF receptor‐1 (FGFR‐1) is not normally expressed by normal prostate epithelial cells; however, aberrant expression of this receptor has been reported in prostate cancer cells, including the LNCaP cell line. We hypothesized that aberrant expression of FGFR‐1 in PrECs would render them sensitive to induction of promatrilysin expression by recombinant FGF‐1. To test this hypothesis, we transiently transfected PrECs with an FGFR‐1 expression vector, which resulted in over‐expression of FGFR‐1 protein in approximately 40% of cells. FGF‐1 increased promatrilysin expression in FGFR‐1–transfected PrECs 4‐fold over mock‐transfected cells, and this induction was inhibited by a specific FGFR‐1 inhibitor, SU5402, and by co‐expression of a dominant negative FGFR‐1 protein. Our results demonstrate that aberrant FGFR‐1 expression, an epigenetic phenomenon that has been associated with prostate cancer progression, allows induction of promatrilysin expression by FGF‐1 in PrECs.

Lipoxygenases as Targets for Cancer Prevention

Although the cyclooxygenase (COX) metabolites of arachidonic acid (AA; 20:4; eicosatetraenoic acid) metabolism have received more attention than the metabolites of the lipoxygenases (LOX), there is growing evidence that inhibition of LOX offers an effective means of cancer (and other disease) prevention. As a family, LOX are dioxygenase enzymes that incorporate molecular oxygen into some polyunsaturated fatty acids, particularly AA and linoleic acid (LA; 18:2; octadecadienoic acid). LOX are widely distributed in plants, fungi, and mammals, but are absent in most bacteria and yeasts (1–3). The recognition that AA can be metabolized to COX and LOX products that are ligands for specific receptors has suggested their potential importance in regulating cell growth and/or differentiation. However, there is uncertainty regarding which metabolites are the most important or how they contribute to transformation, tumor growth, and metastases. It is the goal of this chapter to provide information on the known tissue distribution of the various LOX family members, their products, and the effect of inhibiting LOX in specific organ models of cancer.