Michael N. Alexis

Coactivation of GR and NFKB alters the repertoire of their binding sites and target genes

Glucocorticoid receptor (GR) exerts anti-inflammatory action in part by antagonizing proinflammatory transcription factors such as the nuclear factor kappa-b (NFKB). Here, we assess the crosstalk of activated GR and RELA (p65, major NFKB component) by global identification of their binding sites and target genes. We show that coactivation of GR and p65 alters the repertoire of regulated genes and results in their association with novel sites in a mutually dependent manner. These novel sites predominantly cluster with p65 target genes that are antagonized by activated GR and vice versa. Our data show that coactivation of GR and NFKB alters signaling pathways that are regulated by each factor separately and provide insight into the networks underlying the GR and NFKB crosstalk.

Pilot phase III immunotherapy study in early-stage breast cancer patients using oxidized mannan-MUC1 [ISRCTN71711835].

IntroductionMucin 1 (MUC1) is a high molecular weight glycoprotein overexpressed on adenocarcinoma cells and is a target for immunotherapy protocols. To date, clinical trials against MUC1 have included advanced cancer patients. Herein, we report a trial using early stage breast cancer patients and injection of oxidized mannan-MUC1.MethodIn a randomized, double-blind study, 31 patients with stage II breast cancer and with no evidence of disease received subcutaneous injections of either placebo or oxidized mannan-MUC1, to immunize against MUC1 and prevent cancer reoccurrence/metastases. Twenty-eight patients received the full course of injections of either oxidized mannan-MUC1 or placebo. Survival and immunological assays were assessed.ResultsAfter more than 5.5 years had elapsed since the last patient began treatment (8.5 years from the start of treatment of the first patient), the recurrence rate in patients receiving the placebo was 27% (4/15; the expected rate of recurrence in stage II breast cancer); those receiving immunotherapy had no recurrences (0/16), and this finding was statistically significant (P = 0.0292). Of the patients receiving oxidized mannan-MUC1, nine out of 13 had measurable antibodies to MUC1 and four out of 10 had MUC1-specific T cell responses; none of the placebo-treated patients exhibited an immune response to MUC1.ConclusionThe results suggest that, in early breast cancer, MUC1 immunotherapy is beneficial, and that a larger phase III study should be undertaken.

5α,8α-Epidioxysterols from the gorgonian Eunicella cavolini and the ascidian Trididemnum inarmatum: Isolation and evaluation of their antiproliferative activity

Three new (1, 4, 9) and nine previously reported (2, 3, 5-8, 10-12) 5alpha,8alpha-epidioxysterols were isolated from the organic extracts of the gorgonian Eunicella cavolini and the ascidian Trididemnum inarmatum. The structures and relative configurations of 1-12 were established on the basis of detailed NMR spectroscopic analyses and comparison with the literature. The growth inhibitory effects of 1-12 were evaluated against MCF-7 human breast cancer cells. Compound 1, bearing a cyclopropyl moiety in the side chain, exhibited the highest antiproliferative activity.

Microarray analysis of the differential transformation mediated by Kirsten and Harvey Ras oncogenes in a human colorectal adenocarcinoma cell line

Colorectal cancer arises after a series of mutational events in the colon epithelia and is often used as a model of the multistep progression of tumorigenesis. Mutations in Ki‐Ras have been detected in some 50% of cases and are thought to occur at an early stage. Almost never do mutations arise in the loci of other Ras isoforms (Ha‐ and N‐), leading to the assumption that Ki‐Ras plays a unique role in tumorigenesis. In order to examine the distinctive function that Ki‐Ras plays in cancer development in the colon, we introduced constitutively active mutant Ki‐ and Ha‐Ras genes into an intermediate‐stage colon adenoma cell line (Caco‐2). We found that mutant active Ha‐RasV12 was more efficient at transforming these colon epithelial cells as assessed by anchorage‐independent growth, tumor formation in SCID mice and the development of mesenchymal morphology compared to transformation by Ki‐RasV12. We conducted microarray analysis in an attempt to reveal the genes whose aberrant expression is a direct result of overexpression of either Ki‐RasV12 or Ha‐RasV12. We used Clontechs Atlas cancer cDNA (588 genes) and RZPDs Onco Set 1 (1,544 genes) arrays. We identified fewer genes that were commonly regulated than were differentially expressed between Ki‐ and Ha‐RasV12 isoforms. Specifically, we found that Ki‐RasV12 regulated genes involved in cytokine signaling, cell adhesion and colon development, whereas Ha‐RasV12 mainly regulated genes involved in controlling cell morphology, correlating to an epithelial‐mesenchymal transition only observed in these cells. Our results demonstrate how 2 Ras isoforms regulate disparate biologic processes, revealing a number of genes whose deregulated expression may influence colon carcinogenesis (supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020‐7136/suppmat/index.html).

Estrogen receptor α and β in uterine fibroids: a basis for altered estrogen responsiveness

OBJECTIVE To investigate the relative expression and the DNA-binding status of estrogen receptors alpha and beta in fibroids and normal myometrial tissue to explore the molecular basis of altered estrogen responsiveness of leiomyomas. DESIGN Biopsy samples from uterine fibroids and adjacent normal myometrial tissue at the follicular phase of the menstrual cycle. SETTING Aretaieio University Hospital and the National Hellenic Research Foundation, Athens, Greece. PATIENT(S) Thirty-five patients who underwent hysterectomy or myomectomy because of myoma symptoms. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Deoxyribonucleic acid-binding status of estrogen receptors alpha and beta. RESULT(S) The level of messenger RNA expression of estrogen receptor alpha and beta and the level of estrogen receptor as a whole are increased on average to a similar extent in leiomyomas compared with normal myometrium. Occasionally, however, estrogen receptor alpha is disproportionately increased in leiomyomas, and this appears to increase the amount of estrogen receptor alpha that binds to the estrogen-responsive element of estrogen target genes as homodimer rather than as heterodimer with estrogen receptor beta. CONCLUSION(S) The estrogen receptor alpha-to-estrogen receptor beta expression ratio rather than the individual expression levels determines the fraction of DNA-binding homodimers of estrogen receptor alpha and possibly the growth potential of myomas.

Design and synthesis of novel neuroprotective 1,2-dithiolane/chroman hybrids

Novel 1,2-dithiolane/chroman hybrids bearing heterocyclic rings such as 1,2,4- and 1,3,4-oxadiazole, 1,2,3-triazole and tetrazole were designed and synthesized. The neuroprotective activity of the new analogues was tested against oxidative stress-induced cell death of glutamate-challenged HT22 hippocampal neurons. Our results show that bioisosteric replacement of amide group in 2-position of the chroman moiety, by 1,3,4-oxadiazole did not affect activity. However, analogue 5 bearing the 1,2,4-oxadiazole moiety showed improved neuroprotective activity. The presence of nitrogen heterocycles strongly influences the neuroprotective activity of 5-substituted chroman derivatives, depending on the nature of heterocycle. Replacement of the amide group of the first generation analogues by 1,2,4-oxadiazole or 1,2,3-triazole resulted in significant improvement of the activity against glutamate induced oxidative stress.

Quantitative Fluorescence Cytometric Measurement of Estrogen and Progesterone Receptors: Correlation with the Hormone Binding Assay

We describe, here, a rapid flow cytometry technique for the detection and quantification of estrogen (ER) and progesterone (PgR) receptors in several human cell lines and in clinical samples obtained from breast cancer tumors. ER and PgR quantitation can be very useful in patients with breast cancer as their role in diagnosis and prognosis is well established. However ligand binding assays and immunohistochemical assays are difficult to measure heterogeneity in individual cells. On the other hand, flow cytometry is a convenient tool for quantification in individual cells. Flow cytometric results with breast cancer cell lines and clinical samples were compared to those obtained by quantitative biochemical ER and PgR performed by the standard dextran-coated charcoal biochemical assay. The latter assay is affected by the level of endogenous steroids. This is also the case in the routine measurement of ER/PgR in patients tumor cells whereby estradiol molecules in patients serum produced negative or low values in the biochemical assay. The mAbs used in our flow cytometric method bind to their specific ER or PgR independently of whether they are preoccupied by their ligands and they produce reliable results. With the use of beads calibrated in MESF (Molecules of Equivalent Soluble Fluorochrome) units, the ER and PgR can be measured on a per cell basis. The flow cytometric method showed a strong correlation with biochemical receptor assessments of either ERα (ERαDCC, r = 0.918, p = 0.073) or PgR (PgRDCC, r = 0.75, p = 0.001). This study demonstrates that ERα and PgR can be detected by flow cytometry on a per cell basis in intact cells, and can be quantitated reliably in terms of MESF without the limitations of competition with serums estradiol molecules.

Synthesis of tropolone derivatives and evaluation of their in vitro neuroprotective activity.

beta-Thujaplicin (hinokitiol or 2-hydroxy-4-isopropyl-2,4,6-cycloheptatrien-1-one), a natural tropolone, shows numerous activities while its synthetic analogues were found to exhibit anticancer and anti-ischemic activity. However, the ability of tropolone derivatives to protect neuronal cells from oxidative stress-induced cell damage has not been studied so far. As an ongoing effort toward highly effective antioxidants with potential neuroprotective activity, we have synthesized 7-substituted derivatives of beta-thujaplicin and its methoxy analogue. The substituents were heterocycles (piperazine, morpholine) or heteroaromatics (triazoles, pyridine). Only the piperazine derivatives of beta-thujaplicin were able to protect HT22 hippocampal neurons from oxidative stress-induced cell death.

Isoxazole substituted chromans against oxidative stress-induced neuronal damage.

We have previously reported that catechol-bearing regioisomers of 5-isoxazolyl-6-hydroxy-chroman display higher in vitro neuroprotective activity, compared to hybrids with other nitrogen heterocycles, but their activity is hampered by cytotoxicity at higher concentrations. In an effort to discover non-cytotoxic isoxazole substituted chromans of high neuroprotective activity, 20 new 3- and 5-substituted (chroman-5-yl)-isoxazoles and (chroman-2-yl)-isoxazoles were synthesized using the copper(I)-catalysed cycloaddition reaction between in situ generated nitrile oxides and terminal acetylenes. An additional aim was to further explore the effect of the isoxazole ring substituents on the neuroprotective activity. The activity of these compounds against oxidative stress-induced death (oxytosis) of neuronal HT22 cells was evaluated and interesting SARs for this group of analogues were derived. The vast majority of new chroman analogues displayed high in vitro neuroprotective activity displaying EC(50) values below 1 μM and lacked cytotoxicity. The position of substituents on the isoxazole ring influences the activity of the regioisomers, with the 3-aryl-5-(chroman-5-yl)-isoxazoles, 17 and 18 and bis-chroman 20 displaying higher neuroprotective activity (EC(50)∼0.3 μM) compared to other (chroman-5-yl) and (chroman-2-yl)-isoxazoles.

Ebenfurans IV-VIII from Onobrychis ebenoides: Evidence that C-Prenylation is the Key Determinant of the Cytotoxicity of 3-Formyl-2-arylbenzofurans

Phytochemical investigation of a methanol extract of Onobrychis ebenoides yielded five new 3-formyl-2-arylbenzofurans, namely, ebenfurans IV-VIII (1-5), together with the known compounds ebenfurans I, II (6), and III (7). Only 1 and 7 exhibited growth inhibitory activity against MCF-7 and Ishikawa cells, suggesting that the prenyl moiety at position C-5 is the key determinant of the cytotoxic activity of this group of compounds.