Complex context relationships between DNA methylation and accessibility, histone marks, and hTERT gene expression in acute promyelocytic leukemia cells: perspectives for all-trans retinoic acid in cancer therapy.

Telomerase (hTERT) reactivation and sustained expression is a key event in the process of cellular transformation. Therefore, the identification of the mechanisms regulating hTERT expression is of great interest for the development of new anticancer therapies. Although the epigenetic state of hTERT gene promoter is important, we still lack a clear understanding of the mechanisms by which epigenetic changes affect hTERT expression. Retinoids are well-known inducers of granulocytic maturation in acute promyelocytic leukemia (APL). We have previously shown that retinoids repressed hTERT expression in the absence of maturation leading to growth arrest and cell death. Exploring the mechanisms of this repression, we showed that transcription factor binding was dependent on the epigenetic status of hTERT promoter. In the present study, we used APL cells lines and publicly available datasets from APL patients to further investigate the integrated epigenetic events that promote hTERT promoter transition from its silent to its active state, and inversely. We showed, in APL patients, that the methylation of the distal domain of hTERT core promoter was altered and correlated with the outcome of the disease. Further studies combining complementary approaches carried out on APL cell lines highlighted the significance of a domain outside the minimal promoter, localized around 5 kb upstream from the transcription start site, in activating hTERT. This domain is characterized by DNA hypomethylation and H3K4Me3 deposition. Our findings suggest a cooperative interplay between hTERT promoter methylation, chromatin accessibility, and histone modifications that force the revisiting of previously proposed concepts regarding hTERT epigenetic regulation. They represent, therefore, a major advance in predicting sensitivity to retinoid-induced hTERT repression and, more generally, in the potential development of therapies targeting hTERT expression in cancers.

High-throughput screening with nucleosome substrate identifies small-molecule inhibitors of the human histone lysine methyltransferase NSD2.

The histone lysine methyltransferase nuclear receptor-binding SET domain protein 2 (NSD2, also known as WHSC1/MMSET) is an epigenetic modifier and is thought to play a driving role in oncogenesis. Both NSD2 overexpression and point mutations that increase its catalytic activity are associated with several human cancers. Although NSD2 is an attractive therapeutic target, no potent, selective, and bioactive small molecule inhibitors of NSD2 have been reported to date, possibly due to the challenges of developing high-throughput assays for NSD2. Here, to establish a platform for the discovery and development of selective NSD2 inhibitors, we optimized and implemented multiple assays. We performed quantitative high-throughput screening with full-length WT NSD2 and a nucleosome substrate against a diverse collection of bioactive small molecules comprising 16,251 compounds. We further interrogated 174 inhibitory compounds identified in the primary screen with orthogonal and counter assays and with activity assays based on the clinically relevant NSD2 variants E1099K and T1150A. We selected five confirmed inhibitors for follow-up, which included a radiolabeled validation assay, surface plasmon resonance studies, methyltransferase profiling, and histone methylation in cells. We found that all five NSD2 inhibitors bind the catalytic SET domain and one exhibited apparent activity in cells, validating the workflow and providing a template for identifying selective NSD2 inhibitors. In summary, we have established a robust discovery pipeline for identifying potent NSD2 inhibitors from small-molecule libraries.

Label-free protein quantification of sodium butyrate treated CHO cells by ESI-UHR-TOF-MS.

Effects of butyrate on CHO producer cells are contradictory, promoting productivity and at the same time repressing proliferation. Though in previous omics studies the background of butyrate impact on producer cells has been investigated, the knowledge about the mechanism is still very limited. As previous proteomic results on this field are mainly based on 2DE-gels, we conducted a label-free MS quantification, based on fast high resolution ESI-MS and a straight forward software solution, to gain insight in shifted cellular processes of CHO cells 25h after butyrate treatment. 118 proteins or subunits with significantly altered abundances were identified suggesting changes in carbohydrate, protein metabolic and cell cycle processes. Effects of butyrate on the nucleosome assembly as a known direct epigenetic influence on HDAC activity turned out to be unexpectedly fast and persistent, as confirmed by Western blots of histone-H4 acetylation. Contradictory to increased cell specific productivity, most elements of protein metabolism exhibited decreased levels after butyrate treatment. In comparison to published results some overlap of our label free MS data could be observed but also apparently diverging findings, showing the need for complementary omics techniques for a holistic view on cellular processes such as response to butyrate.

Cell Cycle Arrest and Apoptosis Induction via Modulation of Mitochondrial Integrity by Bcl-2 Family Members and Caspase Dependence in Dracaena cinnabari-Treated H400 Human Oral Squamous Cell Carcinoma.

Dracaena cinnabari Balf.f. is a red resin endemic to Socotra Island, Yemen. Although there have been several reports on its therapeutic properties, information on its cytotoxicity and anticancer effects is very limited. This study utilized a bioassay-guided fractionation approach to determine the cytotoxic and apoptosis-inducing effects of D. cinnabari on human oral squamous cell carcinoma (OSCC). The cytotoxic effects of D. cinnabari crude extract were observed in a panel of OSCC cell lines and were most pronounced in H400. Only fractions DCc and DCd were active on H400 cells; subfractions DCc15 and DCd16 exhibited the greatest cytotoxicity against H400 cells and D. cinnabari inhibited cells proliferation in a time-dependent manner. This was achieved primarily via apoptosis where externalization of phospholipid phosphatidylserine was observed using DAPI/Annexin V fluorescence double staining mechanism studied through mitochondrial membrane potential assay cytochrome c enzyme-linked immunosorbent and caspases activities revealed depolarization of mitochondrial membrane potential (MMP) and significant activation of caspases 9 and 3/7, concomitant with S phase arrest. Apoptotic proteins array suggested that MMP was regulated by Bcl-2 proteins family as results demonstrated an upregulation of Bax, Bad, and Bid as well as downregulation of Bcl-2. Hence, D. cinnabari has the potential to be developed as an anticancer agent.

Total triterpenoids from Ganoderma Lucidum suppresses prostate cancer cell growth by inducing growth arrest and apoptosis.

In this study, one immortalized human normal prostatic epithelial cell line (BPH) and four human prostate cancer cell lines (LNCaP, 22Rv1, PC-3, and DU-145) were treated with Ganoderma Lucidum triterpenoids (GLT) at different doses and for different time periods. Cell viability, apoptosis, and cell cycle were analyzed using flow cytometry and chemical assays. Gene expression and binding to DNA were assessed using real-time PCR and Western blotting. It was found that GLT dose-dependently inhibited prostate cancer cell growth through induction of apoptosis and cell cycle arrest at G1 phase. GLT-induced apoptosis was due to activation of Caspases-9 and -3 and turning on the downstream apoptotic events. GLT-induced cell cycle arrest (mainly G1 arrest) was due to up-regulation of p21 expression at the early time and down-regulation of cyclin-dependent kinase 4 (CDK4) and E2F1 expression at the late time. These findings demonstrate that GLT suppresses prostate cancer cell growth by inducing growth arrest and apoptosis, which might suggest that GLT or Ganoderma Lucidum could be used as a potential therapeutic drug for prostate cancer.

Enhanced antitumor efficacy with combined administration of astragalus and pterostilbene for melanoma.

Astragalus, a commonly used traditional Chinese medicine, has exhibited antitumor actions in patients. In this study, in vitro and in vivo antitumor effects of astragalus and synergistic antitumor efficacy in combination with pterostilbene were investigated. Melanoma cells were treated with pterostilbene (Pt), graduated doses of astragalus injection (AI), or these in combination. Cell viability was measured using a MTT assay. Released nucleosomes and caspase activity were measured using enzyme-linked immunosorbent assay. Growth inhibition in vitro and in vivo was also assessed. Analysis of variance and t tests were used for statistical analysis. Significant reduction (p<0.05) in cellular proliferation were observed with AI and AI-Pt in a time- and concentration-dependent manner. Apoptosis and caspase-3/7 activity were significantly increased by AI and AI-Pt treatment (p<0.05). In vivo, AI inhibited melanoma tumor growth, with inhibition rates ranging from 36.5 to 62.3%, by inducing apoptosis via up-regulation Bax expression and the Bax/Bcl-2 ratio and down-regulating Bcl-2 expression. AI significantly inhibits the growth of melanoma in vitro and in vivo by inducing apoptosis. These data suggest that combined treatment of astragalus with pterostilbene enhances antitumor efficacy.

Solution scattering and FRET studies on nucleosomes reveal DNA unwrapping effects of H3 and H4 tail removal.

Using a combination of small-angle X-ray scattering (SAXS) and fluorescence resonance energy transfer (FRET) measurements we have determined the role of the H3 and H4 histone tails, independently, in stabilizing the nucleosome DNA terminal ends from unwrapping from the nucleosome core. We have performed solution scattering experiments on recombinant wild-type, H3 and H4 tail-removed mutants and fit all scattering data with predictions from PDB models and compared these experiments to complementary DNA-end FRET experiments. Based on these combined SAXS and FRET studies, we find that while all nucleosomes exhibited DNA unwrapping, the extent of this unwrapping is increased for nucleosomes with the H3 tails removed but, surprisingly, decreased in nucleosomes with the H4 tails removed. Studies of salt concentration effects show a minimum amount of DNA unwrapping for all complexes around 50-100mM of monovalent ions. These data exhibit opposite roles for the positively-charged nucleosome tails, with the ability to decrease access (in the case of the H3 histone) or increase access (in the case of the H4 histone) to the DNA surrounding the nucleosome. In the range of salt concentrations studied (0-200mM KCl), the data point to the H4 tail-removed mutant at physiological (50-100mM) monovalent salt concentration as the mononucleosome with the least amount of DNA unwrapping.

SSH gene expression profile of Eisenia andrei exposed in situ to a naturally contaminated soil from an abandoned uranium mine.

The effects of the exposure of earthworms (Eisenia andrei) to contaminated soil from an abandoned uranium mine, were assessed through gene expression profile evaluation by Suppression Subtractive Hybridization (SSH). Organisms were exposed in situ for 56 days, in containers placed both in a contaminated and in a non-contaminated site (reference). Organisms were sampled after 14 and 56 days of exposure. Results showed that the main physiological functions affected by the exposure to metals and radionuclides were: metabolism, oxireductase activity, redox homeostasis and response to chemical stimulus and stress. The relative expression of NADH dehydrogenase subunit 1 and elongation factor 1 alpha was also affected, since the genes encoding these enzymes were significantly up and down-regulated, after 14 and 56 days of exposure, respectively. Also, an EST with homology for SET oncogene was found to be up-regulated. To the best of our knowledge, this is the first time that this gene was identified in earthworms and thus, further studies are required, to clarify its involvement in the toxicity of metals and radionuclides. Considering the results herein presented, gene expression profiling proved to be a very useful tool to detect earthworms underlying responses to metals and radionuclides exposure, pointing out for the detection and development of potential new biomarkers.

Identification and characterization of a bactericidal and proapoptotic peptide from Cycas revoluta seeds with DNA binding properties.

Nowadays, novel pharmacies have been screened from plants. Among them are the peptides, which show multiple biotechnological activities. In this report, a small peptide (Ala-Trp-Lys-Leu-Phe-Asp-Asp-Gly-Val) with a molecular mass of 1,050 Da was purified from Cycas revoluta seeds by using reversed-phase liquid chromatography. This peptide shows clear deleterious effects against human epidermoid cancer (Hep2) and colon carcinoma cells (HCT15). It caused inhibition of cancer cell proliferation and further disruption of nucleosome structures, inducing apoptosis by direct DNA binding. A remarkable antibacterial activity was also observed in this same peptide. Nevertheless, no significant lysis of normal RBC cells was observed in the presence of peptide. Additionally, an acetylation at the N-termini portion is able to reduce both activities. Bioinformatics tools were also utilized for construction of a three-dimensional model showing a single amphipathic helix. Since in vitro binding studies show that the target of this peptide seems to be DNA, theoretical docking studies were also performed to better understand the interaction between peptide and nucleic acids and also to shed some light on the acetyl group role. Firstly, binding studies showed that affinity contacts basically occur due to electrostatic attraction. The complex peptide-ssDNA was clearly oriented by residues Ala(1), Lys(3), and Asp(6), which form several hydrogen bonds that are able to stabilize the complex. When acetyl was added, hydrogen bonds are broken, reducing the peptide affinity. In summary, it seems that information here provided could be used to design a novel derivative of this peptide which a clear therapeutic potential.

Duchesnea phenolic fraction inhibits in vitro and in vivo growth of cervical cancer through induction of apoptosis and cell cycle arrest.

Duchesnea indica (Andr.) Focke has been commonly used to treat cancer in Asian countries for centuries, and recently has been shown to possess anticancer properties in vitro and in vivo. But the underlying mechanism of the anticancer action is unclear, especially in in vivo studies. In this study, we investigated the anticancer effect and associated mechanisms of Duchesnea phenolic fraction (DPF) on cervical cancer in vitro and in vivo. Our results showed that DPF significantly inhibited cervical cancer cell proliferation in dose- and time-dependent manners. DPF induced apoptosis as determined by AO/EB staining, DNA fragmentation and flow cytometry. Some apoptosis correlated proteins were altered following DPF treatment. Bax was up-regulated while Bcl-2 was down-regulated, and the expression ratio of Bax/Bcl-2 was increased. These resulted in the translocation of Bax to mitochondria, the release of cytochrome c from the mitochondria to the cytosol and caspase-3 activation. Concurrently, DPF provoked S phase arrest along with significant down-regulation of S phase-associated proteins, such as cyclin A, cyclin E, cyclin D1 and cdk2. Transplanted U14 cervical cancer mouse model was used to evaluate the antitumor effect of DPF in vivo. Compared with control, DPF treatment markedly prolonged survival of tumor-bearing mice and dose-dependently reduced the tumor weight. DPF could induce apoptosis in tumor tissues as evidenced by increased TUNEL-positive cells, activation of caspase-3, up-regulation of Bax and down-regulation of Bcl-2. In addition, DPF significantly decreased the expression of cell proliferation markers PCNA and ki67 in tumors. All together, these data sustain our contention that DPF has anticancer properties and merits further investigation as a potential therapeutic agent.

Adaptation and impairment of DNA repair function in pollen of Betula verrucosa and seeds of Oenothera biennis from differently radionuclide-contaminated sites of Chernobyl.

BACKGROUND AND AIMS: The plants that have remained in the contaminated areas around Chernobyl since 1986 encapsulate the effects of radiation. Such plants are chronically exposed to radionuclides that they have accumulated internally as well as to alpha-, beta- and gamma-emitting radionuclides from external sources and from the soil. This radiation leads to genetic damage that can be countered by DNA repair systems. The objective of this study is to follow DNA repair and adaptation in haploid cells (birch pollen) and diploid cells (seed embryos of the evening primrose) from plants that have been growing in situ in different radionuclide fall-out sites in monitored regions surrounding the Chernobyl explosion of 1986. METHODS: Radionuclide levels in soil were detected using gamma-spectroscopy and radiochemistry. DNA repair assays included measurement of unscheduled DNA synthesis, electrophoretic determination of single-strand DNA breaks and image analysis of rDNA repeats after repair intervals. Nucleosome levels were established using an ELISA kit. KEY RESULTS: Birch pollen collected in 1987 failed to perform unscheduled DNA synthesis, but pollen at gamma/beta-emitter sites has now recovered this ability. At a site with high levels of combined alpha- and gamma/beta-emitters, pollen still exhibits hidden damage, as shown by reduced unscheduled DNA synthesis and failure to repair lesions in rDNA repeats properly. Evening primrose seed embryos generated on plants at the same gamma/beta-emitter sites now show an improved DNA repair capacity and ability to germinate under abiotic stresses (salinity and accelerated ageing). Again those from combined alpha- and gamma/beta-contaminated site do not show this improvement. CONCLUSIONS: Chronic irradiation at gamma/beta-emitter sites has provided opportunities for plant cells (both pollen and embryo cells) to adapt to ionizing irradiation and other environmental stresses. This may be explained by facilitation of DNA repair function.

Influence of galangin on HL-60 cell proliferation and survival.

The effect of galangin, a flavonol component of India root spice and the 'herbal' medicine propolis, on HL-60 human leukaemia cell survival is characterised. Galangin (1-100 microM) exerted an antiproliferative effect that, with dose and exposure longevity, was progressively associated with an elevated hypodiploid DNA content and expression of the active form of caspase-3, principally prior to membrane damage. At >or=50 microM, plasmamembrane phosphatidylserine exposure was observed. There was no evidence for intracellular oxidative stress as an orchestrator of cytotoxicity and significant phagocyte-like differentiation was not detected. We discuss whether such cytotoxicity will be therapeutically exploitable or contribute to cancer prevention within a pharmacological or dietary context.

Induction of apoptosis by pterocarpans from Platymiscium floribundum in HL-60 human leukemia cells.

(+)-2,3,9-Trimethoxy-pterocarpan (1) (+)-3,9-dimethoxy-pterocarpan [(+)-homopterocarpin] (2), (+)-3-hydroxy-9-methoxy-pterocarpan [(+)-medicarpin] (3) and (+)-3,4-dihydroxy-9-methoxy-pterocarpan [(+)-vesticarpan] (4) are cytotoxic pterocarpans isolated from the native Brazilian plant Platymiscium floribundum. The purpose of the present study was to examine whether induction of apoptosis and/or inhibition of DNA synthesis is involved in the cytotoxicity of these pterocarpans in human leukemia cells. The effect on cell viability determined using the trypan exclusion assay revealed that all compounds tested reduced the number of viable cells, while only in the presence of 3 and 4, there was an increase of nonviable cells. The analysis of membrane integrity and morphological modifications by flow cytometry in the presence of these two compounds indicated that treated cells undergo necrosis, while 1 and 2 trigger apoptosis. DNA synthesis seemed to be affected since BrdU incorporation was inhibited in a dose-dependent manner in the presence of all tested compounds. Pterocarpan treatment also induced an increase in the amount of subdiploid DNA, indicating internucleosomal DNA breakdown, mitochondrial depolarization and caspase-3 activation, which indicate apoptosis induction.

In vivo DNase I-mediated footprinting analysis along the human bradykinin B1 receptor (BDKRB1) gene promoter: evidence for cell-specific regulation.

By applying in vivo dimethyl sulphate and UV light type C-footprinting analysis, we previously showed that specific DNA sequences in the -1349/+42 core promoter region of the inducible human BDKRB1 (bradykinin B1 receptor) gene correlated with its transcriptional activity. In the present study we used the highly sensitive DNase I in vivo footprinting approach to delineate more precisely the functional domains of the BDKRB1 gene promoter in human SMCs (smooth muscle cells). Human lymphocytes that do not express a functional BDKRB1 were also studied as a reference using dimethyl sulphate, UV light type C and DNase I treatments. An obvious difference was found in the DNase I-footprinting patterns between cellular systems that express a functional BDKRB1 (SMCs) in comparison with human lymphocytes, where randomly distributed nucleosome-like footprinting patterns were found in the bulk of the core promoter region studied. Gel-shift assays and expression studies pointed to the implication of the YY1 and a TBP/TFIIB (TATA-box-binding protein/transcription factor IIB) transcription factor in the regulation of BDKRB1 gene expression in SMCs and possible YY1 involvement in the mechanisms of nuclear factor kappaB-mediated regulation of the receptor expression. No significant changes in the promoter foot-printing pattern were found after treatment with interleukin-1beta or serum (known BDKRB1 gene inducers), indicating that definite regulatory motifs could exist outside the BDKRB1 gene core promoter region studied.

Cytotoxic activities of Coriolus versicolor (Yunzhi) extract on human leukemia and lymphoma cells by induction of apoptosis.

Coriolus versicolor (CV), also known as Yunzhi, is one of the commonly used Chinese medicinal herbs. Although recent studies have demonstrated its antitumour activities on cancer cells in vitro and in vivo, the exact mechanism is not fully elucidated. Hence, the objective of this study was to examine the in vitro cytotoxic activities of a standardized aqueous ethanol extract prepared from Coriolus versicolor on a B-cell lymphoma (Raji) and two human promyelocytic leukemia (HL-60, NB-4) cell lines using a MTT cytotoxicity assay, and to test whether the mechanism involves induction of apoptosis. Cell death ELISA was employed to quantify the nucleosome production resulting from nuclear DNA fragmentation during apoptosis. The present results demonstrated that CV extract at 50 to 800 microg/ml dose-dependently suppressed the proliferation of Raji, NB-4, and HL-60 cells by more than 90% (p < 0.01), with ascending order of IC50 values: HL-60 (147.3 +/- 15.2 microg/ml), Raji (253.8 +/- 60.7 microg/ml) and NB-4 (269.3 +/- 12.4 microg/ml). The extract however did not exert any significant cytotoxic effect on normal liver cell line WRL (IC50 > 800 microg/ml) when compared with a chemotherapeutic anticancer drug, mitomycin C (MMC), confirming the tumour-selective cytotoxicity. Nucleosome productions in HL-60, NB-4 and Raji cells were significantly increased by 3.6-, 3.6- and 5.6-fold respectively upon the treatment of CV extract, while no significant nucleosome production was detected in extract-treated WRL cells. The CV extract was found to selectively and dose-dependently inhibit the proliferation of lymphoma and leukemic cells possibly via an apoptosis-dependent pathway.

Pb2+-induced toxicity is associated with p53-independent apoptosis and enhanced by glutamate in GT1-7 neurons.

Recent studies indicate that the glutamatergic neurotransmitter system is involved in neurotoxicity caused by inorganic lead (Pb2+). We studied the role of apoptosis in the effects induced by Pb2+ (0.01-100 microM) and glutamate (0.1 and 1 mM) in mouse hypothalamic GT1-7 neurons. Although glutamate alone had no effect on cell viability, it enhanced neuronal cell death induced by Pb2+ (1-100 microM) within 72 h. Glutamate alone neither induced caspase-3-like protease activity nor promoted internucleosomal DNA fragmentation, both biochemical hallmarks of apoptosis. However, concurrent exposure to Pb2+ (10 or 100 microM) and glutamate (1 mM) resulted in more prominent cleavage of the fluorogenic caspase-3 substrate (Ac-DEVD-AMC) than caused by the same Pb2+ concentrations alone at 24-72 h. The highest caspase-3-like protease activities were measured at 48 h. Internucleosomal DNA fragmentation caused by Pb2+ (10 or 100 microM) alone or together with glutamate (1 mM) was evident at 96 h, less clear at 72 h and absent at 48 h. Immunoblotting did not reveal any changes in p53 protein levels in cells exposed to Pb2+, glutamate or their combination at any studied time point (3-72 h). Our results suggest that Pb2+-induced neurotoxicity may partially be mediated through p53-independent apoptosis and enhanced by glutamate.

Apoptotic cell death induced by baccatin III, a precursor of paclitaxel, may occur without G(2)/M arrest.

PURPOSE: Paclitaxel has been demonstrated to possess significant cell-killing activity in a variety of tumor cells by induction of apoptosis, but the mechanism by which paclitaxel leads to cell death and its relationship with mitotic arrest is not entirely clear. In this study, baccatin III, a synthetic precursor of paclitaxel, was used to analyze whether paclitaxel-induced apoptosis can be a separate event from microtubule bundling and G(2)/M arrest. METHODS: Several different methods including DNA fragmentation, flow cytometric analyses, TdT-mediated dUTP nick end labeling (TUNEL) and time-lapse video microscopy were used to analyze apoptotic cell death induced by baccatin III and its possible correlation with cell cycle distribution. RESULTS: Our results demonstrated that baccatin III could also cause apoptotic cell death in both BCap37 (a human breast cancer cell line) and KB cells (derived from human epidermoid carcinoma), but had less effect on microtubule bundling and G(2)/M arrest. Furthermore, we demonstrated that most apoptotic events induced by baccatin III were not coupled with G(2)/M arrest. Instead, these apoptotic events occurred predominantly in the cells in other phases of the cell cycle. CONCLUSION: Baccatin III, which contains the core taxane ring, is the fundamental piece of paclitaxel structure. The finding of baccatin III-induced apoptosis independent of cell cycle arrest, on the one hand, implies that the core taxane ring may play a critical role in inducing cell death and, on the other hand, suggests that paclitaxel might induce apoptosis from other phases of the cell cycle by a similar mechanism.

Differential response of human melanoma and Ehrlich ascites cells in vitro to the ribosome-inactivating protein luffin.

The cytotoxicity and inhibitory effect on proliferation of the type 1 ribosome-inactivating protein luffin purified from the seeds of Luffa aegyptiaca were investigated both in human metastatic melanoma cells and in murine Ehrlich ascites tumour cells. Results indicate that luffin from the seeds of Luffa aegyptiaca is cytotoxic to the cell lines tested, with approximately 10 times greater potency in Ehrlich cells. Luffin was found to induce an increase in cytosolic oligonucleosome-bound DNA in both melanoma and Ehrlich ascites tumour cells, the level of DNA fragmentation in the former cell line being higher than in the latter. Experiments with melanoma cells indicate that an increase in cytosolic nucleosomes could be supportive of apoptosis as the type of cell death induced by luffin.

Inhibition of arachidonate 5-lipoxygenase triggers massive apoptosis in human prostate cancer cells.

Diets high in fat are associated with an increased risk of prostate cancer, although the molecular mechanism is still unknown. We have previously reported that arachidonic acid, an omega-6 fatty acid common in the Western diet, stimulates proliferation of prostate cancer cells through production of the 5-lipoxygenase metabolite, 5-HETE (5-hydroxyeicosatetraenoic acid). We now show that 5-HETE is also a potent survival factor for human prostate cancer cells. These cells constitutively produce 5-HETE in serum-free medium with no added stimulus. Exogenous arachidonate markedly increases the production of 5-HETE. Inhibition of 5-lipoxygenase by MK886 completely blocks 5-HETE production and induces massive apoptosis in both hormone-responsive (LNCaP) and -nonresponsive (PC3) human prostate cancer cells. This cell death is very rapid: cells treated with MK886 showed mitochondrial permeability transition between 30 and 60 min, externalization of phosphatidylserine within 2 hr, and degradation of DNA to nucleosomal subunits beginning within 2-4 hr posttreatment. Cell death was effectively blocked by the thiol antioxidant, N-acetyl-L-cysteine, but not by androgen, a powerful survival factor for prostate cancer cells. Apoptosis was specific for 5-lipoxygenase-programmed cell death was not observed with inhibitors of 12-lipoxygenase, cyclooxygenase, or cytochrome P450 pathways of arachidonic acid metabolism. Exogenous 5-HETE protects these cells from apoptosis induced by 5-lipoxygenase inhibitors, confirming a critical role of 5-lipoxygenase activity in the survival of these cells. These findings provide a possible molecular mechanism by which dietary fat may influence the progression of prostate cancer.

The influence of selenium and neuroleptics on the rat brain nuclear regulatory mechanism in carcinogenesis.

The potential clinical benefit of the antitumor effect of selenium (Se) has recently been confirmed in tumor-bearing animals and human tumor cells in culture. In clinical medicine, the reduced incidence of cancer among schizophrenic patients was attributed to neuroleptic medication. However, there has been little information on the effect of Se, carcinogen, and neuroleptic on the brain cells. This investigation was carried out on the brains of male Wistar rats treated with inorganic Se, 9,10-dimethyl-1,2-benzanthracene, and chlorpromazine. Chromatin was prepared and purified from isolated brain cell nuclei. Various protein species (histones and nonhistone proteins), RNA, and DNA were extracted by different extraction procedures. A higher relative content of nonhistone proteins was found in the group of animals treated with Se alone, carcinogen alone, and Se plus carcinogen administered simultaneously when compared with other experimental and control groups. The ratio of nonhistone proteins and histones of < 1.0 in the group of animals treated with neuroleptic + carcinogen or with neuroleptic indicates a lower content of nonhistone proteins when compared to histones. We obtained a more pronounced susceptibility to degradation by DNase I in the group of animals treated with neuroleptic + carcinogen or with neuroleptic compared to chromatin in the animals treated with carcinogen alone. We conclude that neuroleptic increases protein synthesis, as well as chromatin susceptibility to enzymatic degradation, thus achieving an opposite effect of carcinogen.