Prostate carcinoma is characterized by the silencing of pi-class glutathione S-transferase gene (GSTP1), which encodes a detoxifying enzyme. The silencing of GSTP1, due to aberrant methylation at the CpG island in the promoter/5'-UTR, occurs in the vast majority of prostate tumors and precancerous lesions. It is a pathologic marker and probably an underlying cause of oxidative damage and inflammation at tumor initiation. Inhibition of the aberrant promoter methylation could therefore be an effective mean to prevent carcinogenesis. Several isothiocyanates, including phenethyl isothiocyanate (PEITC), found naturally in cruciferous vegetables, induced growth arrest and apoptosis in prostate cancer cells in culture and xenografts. The effects of PEITC to reactivate GSTP1 were investigated. Exposure of prostate cancer LNCaP cells to PEITC inhibited the activity and level of histone deacetylases (HDACs), and induced selective histone acetylation and methylation for chromatin unfolding. Concurrently PEITC demethylated the promoter and restored the unmethylated GSTP1 in both androgen-dependent and -independent LNCaP cancer cells to the level found in normal prostatic cells, as quantified by methylation-specific PCR and pyrosequencing. The dual action of PEITC on both the DNA and chromatin was more effective than 5'-Aza-2'-deoxycytidine, sodium butyrate, or trichostatin A (TSA), and may de-repress the methyl-binding domain (MBD) on gene transcription. The PEITC-mediated cross-talk between the DNA and chromatin in demethylating and reactivating GSTP1 genes, which is critically inactivated in prostate carcinogenesis, underlines a primary mechanism of cancer chemoprevention. Consequently, new approaches could be developed, with isothiocyanates to prevent and inhibit malignancies.

Abstract. Natural isothiocyanates, present in cruciferous vegetables and synthetic phenylhexyl isothiocyanate (PHI) are chemopreventive agents which act by blocking the initiation of carcinogen-induced tumors in rodents. We have demonstrated that isothiocyanates are also growth regulators, inhibiting cell cycle cdk activity and up-regulating inhibitor p21 WAF1 (p21) in cancer cells. The up-stream mechanism to modulate cell cycle progression remained to be elucidated. Here, we have demonstrated that exposure of HL-60 leukemia cells to PHI induced G1 arrest and apoptosis. The hypothesis that PHI inhibits cell growth via chromatin remodeling was investigated. PHI mediates the complex cross talk between chromatin and DNA, and it was demonstrated for the first time as an inhibitor of histone deacetylases (HDAC). Thus, the HDAC activity in PHI-exposed HL-60 cells was reduced. Additionally, PHI reduced the expression of HDAC and increased the level of acetyl transferase p300, in favor of accumulation of acetylated histones. Within hours, global acetylation of histones was enhanced. PHI further mediated selective alterations of histone methylation, with a pattern consistent to the marks of transcription competent chromatins. The relationship between acetylated histones and p21 was examined by chromatin immunoprecipitation (ChIP) assay. Chromatins from cells exposed to PHI contained more p21 DNA in the precipitates of hyperacetylated histones, indicating more accessibility of transcription machinery to the p21 promoter after chromatin unfolding. The cell cycle inhibitors were activated as a result. In contrast to the PHI-induced apoptosis in HL-60 cells, which was mediated by caspase-9 up-regulation and bcl-2 reduction, PHI did not induce significant apoptosis in the mononuclear cells from normal peripheral blood and bone marrow. The results revealed a potential selective effect of isothiocyanates to inhibit the growth of malignant cells.

Borrelia burgdorferi binds strongly to the extracellular matrix and cells of the connective tissue, a binding apparently mediated by specific proteins and proteoglycans. We investigated the interactions between B. burgdorferi cells and intact type I collagen using hydrated lattices that reproduce features of in vivo collagen matrices. B. burgdorferi cells of several strains adhered avidly to these acellular matrices by a mechanism that was not mediated by decorin or other proteoglycans. Moreover, following adhesion to these matrices, B. burgdorferi grew and formed microcolonies. The collagen used in these studies was confirmed to lack decorin by immunoblot analysis; B. burgdorferi cells lacking the decorin adhesin bound readily to intact collagen matrices. B. burgdorferi also bound to collagen lattices that incorporated enzymes that degraded glycosaminoglycan chains in any residual proteoglycans. Binding of the bacteria to intact collagen was nonetheless specific, as bacteria did not bind agar and showed only minimal binding to bovine serum albumin, gelatin, pepsinized type I collagen, and intact collagen that had been misassembled under nonphysiological pH and ionic-strength conditions. Proteinase K treatment of B. burgdorferi cells decreased the binding, as did a lack of flagella, suggesting that surface-exposed proteins and motility may be involved in the ability of B. burgdorferi to interact with intact collagen matrices. The high efficiency of binding of B. burgdorferi strains to intact collagen matrices permits replacement of the commonly used isotopic binding assay with visual fluorescent microscopic assays and will facilitate future studies of these interactions.Borrelia burgdorferi, the etiologic agent of Lyme disease, is transmitted to animals and humans mainly by nymphae of the tick Ixodes scapularis, which during a blood meal deposit a small number of microorganisms into the skin (8,43,51). The inoculation of the bacteria by the tick bite results after a few days in a characteristic rash, erythema migrans, which may be accompanied by systemic symptoms, including malaise, fatigue, fever, headache, neck stiffness, arthralgias, or myalgias (30,51,58). Adhesion, colonization, and proliferation within the skin and other host organs and tissues by B. burgdorferi necessitates interaction between the spirochete and cells of the connective tissue, including macrophages, dendritic cells, fibroblasts (24,48), and the associated extracellular matrix (ECM) (24).B. burgdorferi expresses cell surface proteins that interact specifically with different components of the ECM of the host organism and of mammalian cells in culture (8,23,26,30). These B. burgdorferi surface proteins include the fibronectin receptor encoded by BBK32 (42); proteins that bind directly to glycosaminoglycans (GAGs), such as Bgp (39-41); and membrane lipoproteins, such as DbpA and DbpB, which bind to decorin (4,16,(20)(21)(22). The B. burgdorferi outer membrane protein p66 binds to the beta3 integrin chain of host ECM receptors (9-12). The var...

PEITC modulates the testosterone-influenced growth by repressing Sp1, thus down-regulating AR and proliferation. PEITC from cruciferous vegetables may represent a regulator for hormone-dependent growth of the prostate.

Col-6 is a compound found in certain edible plants. Col-6 has been synthesized, and structurely-characterized. This study investigated its effects on the growth of human promyelocytic leukemic cell line, HL-60. Exposure of HL-60 cells to Col-6 at high concentrations (≥40 μM) resulted in cytolysis in 90% cells within 72 hours. At lower concentrations (<20 μM) the cell density and viability were reduced as compared to untreated cells. A 30% reduction in cell growth rate was seen at 10 μM within 48 hours. A reduction of the proliferating cells in S and G2M phases along with an increase of G1 were detected, indicating a G1 arrest. Multiple assays were used to characterize apoptosis, including DNA strand breaks by TUNEL assay, and the presence of sub-G1 peak by flow cytometry. Col-6 induced apoptosis of HL-60 cells regularly at a concentration as low as 0.1 μM. The degree of apoptosis was proportional to the concentrations and the length of exposure. Mononuclear cells isolated from normal peripheral blood (PBMC) and bone marrow (BMMC) were similarly exposed to Col-6. At a concentration as high as 40 μM, Col-6 did not cause apoptosis of PBMC nor BMMC, suggesting that Col-6 does not have the apoptotic effects on normal non-dividing cells. Apoptosis was further correlated with the increase of caspase 9, and the cleavage of PARP (substrate of caspases), as defined by Western blot analyses. The expression of bcl-2 was markedly decreased in the treated cells. Conclusion: Col-6, a compound found in edible plants, induced apoptosis of HL-60 cells and down-regulation of bcl-2. Col-6 may be a potential novel anti-leukemia agent. Further in vivo study is indicated.