Cytotoxic effects of high concentrations of sodium ascorbate on human myeloid cell lines.

The effect of high doses of intravenous (sodium) ascorbate (ASC) in the treatment of cancer has been controversial although there is growing evidence that ASC in high (pharmacologic) concentrations induces dose-dependent pro-apoptotic death of tumor cells, in vitro. Very few data are available on the role of ASC in the treatment of acute myeloid leukemia (AML). Ascorbate behaves as an antioxidant at low (physiologic), and as pro-oxidant at pharmacologic, concentrations, and this may account for the differences reported in different experimental settings, when human myeloid cell lines, such as HL60, were treated with ASC. Considering the myeloid origin of HL60 cells, and previous literature reports showing that some cell lines belonging to the myeloid lineage could be sensitive to the pro-apoptotic effects of high concentrations of ASC, we investigated in more details the effects of high doses (0.5 to 7 mM) of ASC in vitro, on a variety of human myeloid cell lines including the following: HL60, U937, NB4, NB4-R4 (retinoic acid [RA]-resistant), NB4/AsR (ATO-resistant) acute promyelocytic leukemia (APL)-derived cell lines, and K562 as well as on normal CD34+ progenitors derived from human cord blood. Our results indicate that all analyzed cell lines including all-trans retinoic acid (ATRA)- and arsenic trioxide (ATO)-resistant ones are highly sensitive to the cytotoxic, pro-oxidant effects of high doses of ASC, with an average 50 % lethal concentration (LC50) of 3 mM, depending on cell type, ASC concentration, and time of exposure. Conversely, high doses of ASC neither did exert significant cytotoxic effects nor impaired the differentiation potential in cord blood (CB) CD34+ normal cells. Since plasma ASC concentrations within the millimolar (mM) range can be easily and safely reached by intravenous administration, we conclude that phase I/II clinical trials using high doses of ASC should be designed for patients with advanced/refractory AML and APL.

Alternative Pathways of Cancer Cell Death by Rottlerin: Apoptosis versus Autophagy.

Since the ability of cancer cells to evade apoptosis often limits the efficacy of radiotherapy and chemotherapy, autophagy is emerging as an alternative target to promote cell death. Therefore, we wondered whether Rottlerin, a natural polyphenolic compound with antiproliferative effects in several cell types, can induce cell death in MCF-7 breast cancer cells. The MCF-7 cell line is a good model of chemo/radio resistance, being both apoptosis and autophagy resistant, due to deletion of caspase 3 gene, high expression of the antiapoptotic protein Bcl-2, and low expression of the autophagic Beclin-1 protein. The contribution of autophagy and apoptosis to the cytotoxic effects of Rottlerin was examined by light, fluorescence, and electron microscopic examination and by western blotting analysis of apoptotic and autophagic markers. By comparing caspases-3-deficient (MCF-7(3def)) and caspases-3-transfected MCF-7 cells (MCF-7(3trans)), we found that Rottlerin induced a noncanonical, Bcl-2-, Beclin 1-, Akt-, and ERK-independent autophagic death in the former- and the caspases-mediated apoptosis in the latter, in not starved conditions and in the absence of any other treatment. These findings suggest that Rottlerin could be cytotoxic for different cancer cell types, both apoptosis competent and apoptosis resistant.

Rottlerin exhibits antiangiogenic effects in vitro.

Rottlerin, a natural product purified from Mallotus philippinensis, has a number of target molecules and biological effects. We recently found that Rottlerin caused growth arrest in MCF-7 breast cancer cells and human immortalized keratinocytes, through inhibition of NFκB and downregulation of cyclin D-1. To evaluate whether this effect could be generalized to primary cells, human microvascular endothelial cells were treated with Rottlerin. In this study, we demonstrated that Rottlerin prevents basal and TNFα-stimulated NFκB nuclear migration and DNA binding also in human microvascular endothelial cell, where NFκB inhibition was accompanied by the downregulation of NFκB target gene products, such as cyclin D-1 and endothelin-1, which are essential molecules for endothelial cell proliferation and survival. Rottlerin, indeed, inhibited human microvascular endothelial cells proliferation and tube formation on Matrigel. Rottlerin also increases cytoplasmic free calcium and nitric oxide levels and downregulates endothelin converting enzyme-1 expression, thus contributing to the drop in endothelin-1 and growth arrest. These results suggest that Rottlerin may prove useful in the development of therapeutic agents against angiogenesis.

Athletic humans and horses: comparative analysis of interleukin-6 (IL-6) and IL-6 receptor (IL-6R) expression in peripheral blood mononuclear cells in trained and untrained subjects at rest.

BACKGROUND: Horses and humans share a natural proclivity for athletic performance. In this respect, horses can be considered a reference species in studies designed to optimize physical training and disease prevention. In both species, interleukin-6 (IL-6) plays a major role in regulating the inflammatory process induced during exercise as part of an integrated metabolic regulatory network. The aim of this study was to compare IL-6 and IL-6 receptor (IL-6R) mRNA expression in peripheral blood mononuclear cells (PBMCs) in trained and untrained humans and horses. RESULTS: Nine highly trained male swimmers (training volume: 21.6 ± 1.7 h/wk in 10-12 sessions) were compared with two age-matched control groups represented by eight lightly trained runners (training volume: 6.4 ± 2.6 h/wk in 3-5 sessions) and nine untrained subjects. In addition, eight trained horses (training volume: 8.0 ± 2.1 h/wk in 3-4 sessions) were compared with eight age-matched sedentary mares. In humans, IL-6 mRNA levels in PBMCs determined by quantitative reverse transcription-polymerase chain reaction were significantly higher in highly trained subjects, whereas IL-6R expression did not differ among groups. In horses, transcripts of both IL-6 and IL-6R were significantly up-regulated in the trained group. CONCLUSIONS: Up-regulation of IL-6R expression in PBMCs in horses could reflect a mechanism that maintains an adequate anti-inflammatory environment at rest through ubiquitous production of anti-inflammatory cytokines throughout the body. These findings suggest that the system that controls the inflammatory response in horses is better adapted to respond to exercise than that in humans.

Differential expression of follistatin and FLRG in human breast proliferative disorders.

BACKGROUND: Activins are growth factors acting on cell growth and differentiation. Activins are expressed in high grade breast tumors and they display an antiproliferative effect inducing G0/G1 cell cycle arrest in breast cancer cell lines. Follistatin and follistatin- related gene (FLRG) bind and neutralize activins. In order to establish if these activin binding proteins are involved in breast tumor progression, the present study evaluated follistatin and FLRG pattern of mRNA and protein expression in normal human breast tissue and in different breast proliferative diseases. METHODS: Paraffin embedded specimens of normal breast (NB - n = 8); florid hyperplasia without atypia (FH - n = 17); fibroadenoma (FIB - n = 17); ductal carcinoma in situ (DCIS - n = 10) and infiltrating ductal carcinoma (IDC - n = 15) were processed for follistatin and FLRG immunohistochemistry and in situ hybridization. The area and intensity of chromogen epithelial and stromal staining were analyzed semi-quantitatively. RESULTS: Follistatin and FLRG were expressed both in normal tissue and in all the breast diseases investigated. Follistatin staining was detected in the epithelial cytoplasm and nucleus in normal, benign and malignant breast tissue, with a stronger staining intensity in the peri-alveolar stromal cells of FIB at both mRNA and protein levels. Conversely, FLRG area and intensity of mRNA and protein staining were higher both in the cytoplasm and in the nucleus of IDC epithelial cells when compared to NB, while no significant changes in the stromal intensity were observed in all the proliferative diseases analyzed. CONCLUSION: The present findings suggest a role for follistatin in breast benign disease, particularly in FIB, where its expression was increased in stromal cells. The up regulation of FLRG in IDC suggests a role for this protein in the progression of breast malignancy. As activin displays an anti-proliferative effect in human mammary cells, the present findings indicate that an increased FST and FLRG expression in breast proliferative diseases might counteract the anti-proliferative effects of activin in human breast cancer.

Rottlerin: a multifaced regulator of keratinocyte cell cycle.

In this study we showed that Rottlerin (also called Kamala or Mallotoxin), a natural product purified from Mallotus phillippinensis, is a potent suppressor of human keratinocytes (HaCaT cell line) proliferation. Following Rottlerin treatment, Thymidine incorporation into DNA and re-epithelialisation in a scratch wound model was decreased. At the molecular level, Rottlerin hampered the NFkB activation process, causing loss of cyclin D1 and promoting, in a PKCdelta-dependent pathway, ERK activation, which, in turn induced the cell cycle inhibitor p21 Cip1/Kip1. The NFkB-dependent drop in cyclin D1, along with the PKCdelta/ERK-dependent induction of p21 Cip1/Kip1, is responsible for growth arrest. These results open the way to further investigation on the Rottlerin therapeutic potential against keratinocyte hyper-proliferative disorders.

Changes in salivary cortisol and corticosteroid receptor-alpha mRNA expression following a 3-week multidisciplinary treatment program in patients with fibromyalgia.

The aim of the present study was to investigate the effects of a 3-week residential multidisciplinary non-pharmacological treatment program (including individually prescribed aerobic exercise and cognitive-behavioral therapy) on fibromyalgia symptoms and hypothalamic-pituitary-adrenal (HPA) axis function. Salivary and venous blood samples were collected from 12 female patients with fibromyalgia (age: 25-58) the day before and the day after the treatment period: saliva, eight times (every two hours from 0800 to 2200 h); venous blood, at 0800 h. Peripheral blood mononuclear cells (PBMC) were separated and analyzed for glucocorticoid receptor-alpha (GR-alpha) mRNA expression by semi-quantitative RT-PCR, while the salivary cortisol concentration was determined by RIA. At the same time, pain and aerobic capacity were evaluated. Aerobic capacity improved at the end of the treatment program. The slope of the regression of salivary cortisol values on sampling time was steeper in all patients after treatment, indicating that the cortisol decline was more rapid. Concomitantly, the area under the cortisol curve "with respect to increase" (AUC(i)) was higher and there was a significant increase in GR-alpha mRNA expression in PBMC. The number of positive tender points, present pain, pain area and CES-D score were significantly reduced after the treatment, while the pressure pain threshold increased at most of the tender points. Our findings suggest that one of the active mechanisms underlying the effects of our treatment is an improvement of HPA axis function, consisting in increased resiliency and sensitivity of the stress system probably related to stimulation of GR-alpha synthesis by the components of the treatment.

Role of PTHrp and PTHrp-engaged pathways in MCF-7 cells migration/invasion.

In this paper the effect of N-terminal parathyroid hormone-related protein (PTHrp) and PTHrp-engaged pathways on MCF-7 breast cancer cell migration/invasivity and matrix metalloproteinases (MMPs) production were investigated. We found that: a) migration is not affected by PTHrp and Forskolin (FK)-activated PKA, while Phorbol Myristate Acetate (PMA)-activated PKC strongly stimulates MCF-7 cells motility. b) MMPs production was unaffected by PTHrp, but FK reduced membrane-type (MT)-1 MMP expression. Conversely, PMA induced a marked increase of MT1-MMP and MMP-9. c) Chemical activation of PKC is not sufficient, by itself, to confer invasive ability to MCF-7 cells, unless they were provided with additional factors, supplied by fibroblasts. d) Matrix invasion likely occurs through an activation cascade, involving at least three components: pro-MMP-9 and MT-1 MMP (supplied by PMA-stimulated MCF-7 cells) and pro MMP-2 (supplied by fibroblasts). e) The selective chemical inhibition of the adenylylciclase (AC)/PKA and phospholipase C (PLC)/PKC pathways confirmed that MCF-7 cells invasivity is not affected by exogenous PTHrp, which can only modulate their growth. However, the PTHrp responsibility in breast cancer invasion cannot be completely excluded. Indeed, fibroblasts are known to respond to PTHrp (which is a normal product of MCF-7 as well as other breast cancer cells) with enhanced release of MMP-2. On the basis of the documented requirement of fibroblast-derived MMP-2 for MCF-7 cell invasivity, a novel humoral fibroblast-breast cancer cell interaction, mediated by PTHrp, can be recognised.

Estrogen and mu-opioid receptor antagonists counteract the 17 beta-estradiol-induced licking increase and interferon-gamma reduction occurring during the formalin test in male rats.

Women have a higher incidence of chronic pain syndromes than men and are generally more sensitive to experimental pain. Numerous studies have shown that the female gonadal hormones, estrogens, can profoundly affect the nervous and immune systems, including mechanisms involved in pain and nociception. In the present study, we used antagonists of estrogen receptors (ER) or mu-opioid receptors (mu OR) to evaluate the effects of estrogens on formalin-induced behavioural and immune responses in male rats. After two days of priming with 17 beta-estradiol or saline (i.c.v.), animals were subjected to the formalin test; 15 min prior to formalin (50 microl, 5%) or sham injection in the hind paw, animals were treated with an ER antagonist (ICI 182,780, ICI) or a mu OR antagonist (beta-funaltrexamine, FNA) or saline. The spontaneous behaviours, pain-related behaviours and interferon-gamma (IFN-gamma) production by peripheral blood mononuclear cells were studied in all groups. We found that central administration of estradiol increased the amount of licking of the formalin-injected paw in the second phase of the formalin test. Whereas ICI and FNA had no effect on pain behaviour in saline-pre-treated animals, both antagonists reversed the estradiol-induced increase in licking. The immune system was differently affected by formalin and estradiol treatment. Indeed, formalin injection per se decreased IFN-gamma production; estradiol had no effect on sham-injected animals but strongly reduce the decrease of IFN-gamma production in formalin-injected animals. The results demonstrate that centrally acting estrogens affect ER- and mu OR-mediated pain processing and influence immune function.

Prepro-endothelin-1 mRNA and its mature peptide in human appendix.

Because the precise immunopathological events occurring in appendicitis are not completely understood, possible local production of endothelin-1 (ET-1) in human appendix was investigated. We used immunohistochemistry and in situ hybridization to detect the presence, distribution, and phenotype of ET-1-positive cells and prepro-ET-1 (pp-ET-1) mRNA-expressing cells. ET-1-positive stromal cells and pp-ET-1 mRNA-expressing cells were detected with different distributions and relative frequencies in normal control appendix, histologically normal appendix, and inflamed appendix. Six of 20 histologically normal appendixes from patients with a clinical diagnosis of acute appendicitis had many ET-1-positive stromal cells and high pp-ET-1 mRNA expression, similar to inflamed appendix. Forty percent of the pp-ET-1 mRNA-expressing cells were neutrophils, and the other positive cells were mast cells and macrophages. We suggest that local production of ET-1 by neutrophils and other inflammatory cells could be a molecular sign of focal inflammation in histologically normal appendixes and that ET-1 could be implicated, with other cytokines, in the pathogenesis of appendicitis by inducing appendiceal ischemia through vasoconstriction.

IFN-gamma production in rabbits: behavioral and endocrine correlates.

Freely interacting male rabbits were studied to establish the effect of exogenous testosterone on interferon-gamma (IFN-gamma) production in peripheral blood mononuclear cells (PBMCs) and to evaluate if this effect is related to season, social rank, plasma corticosterone and glucocorticoid receptors (GcR) in PBMCs. Dominance behavior increases after testosterone propionate (TP) administration only in rank 1 animals, while submission behavior increases after TP only in rank 4 animals, indicating a reinforcing effect of TP on the behavior. Corticosterone and IFN-gamma production are higher and GcR binding capacity is lower in spring than in autumn, suggesting that seasonal fluctuations in the immune system may be related to the pattern of secretion of immunomodulatory hormones. In autumn, corticosterone decreases after TP treatment and increases after social interaction, while GcR binding capacity decreases after TP treatment and social interaction. IFN-gamma production decreases in spring and increases in autumn after TP treatment plus social interaction, indicating that the modulating action of testosterone is related to the current immune status. The relationship between dominance, testosterone and the immune system in spring is suggested by the finding that GcR binding capacity after TP treatment is directly related to social rank, as confirmed by the positive correlation with dominance behavior frequency. The dominance index is positively correlated with GcR binding capacity and negatively with IFN-gamma production before TP treatment, indicating that high receptor activity in immunocompetent cells and low immunoreactivity could be prerequisites for dominance behavior. The immunosuppressive effect of corticosterone and the mechanism of down-regulation on GcR are confirmed by the negative correlations with IFN-gamma production and GcR binding capacity.

Gender-related differences in erythrocyte glutathione peroxidase activity in healthy subjects.

OBJECTIVE: The antioxidant property of oestrogens may partly explain the gender differences in atherosclerotic heart disease and a reduction in the incidence of coronary heart disease, as well as mortality from cardiovascular disease in women undergoing postmenopausal oestrogen therapy. In the present study, we tested the hypothesis that the antioxidant glutathione peroxidase (GSH-Px) erythrocyte activity is gender related and is correlated with oestradiol serum levels. PATIENTS: One hundred and fifty healthy women (90 premenopausal; 60 postmenopausal) and 150 age-matched healthy men were recruited during routine health screening. DESIGN AND MEASUREMENTS: Serum was analysed for oestradiol (E2), progesterone and testosterone concentrations, and erythrocytes for GSH-Px enzyme activity. Thirty of the 60 postmenopausal women were treated with transdermal therapy for 30 days with 8-mg E2 patches (nominal daily dose 100 micro g), while the remaining 30 acted as controls. RESULTS: Erythrocyte GSH-Px activity was significantly higher in premenopausal than in postmenopausal women (P = 0.0014), and higher in premenopausal women than in age-matched men (P = 0.025). By contrast, no significant differences were observed between postmenopausal women and the age-matched male population. In postmenopausal women, E2 replacement therapy induced a significant increase in erythrocyte GSH-Px activity (P < 0.001). GSH-Px was positively correlated with serum E2 levels in both premenopausal (r = 0.26, P = 0.011) and postmenopausal women treated with oestrogen replacement therapy (r = 0.42, P = 0.022). No significant correlations in the female population were found between GSH-Px erythrocyte activity and the other sex steroid hormones examined (r2 < or = 0.10, P > or = 0.45). No statistically significant correlations were found between GSH-Px and E2, progesterone and testosterone in the male population (r2 < or = 0.15, P > or = 0.30). CONCLUSIONS: Our findings support the hypothesis that oestrogens are responsible for the sex-related differences in glutathione peroxidase activity. The hypothesis generated from the present study can be tested directly in experimental models involving specific bone marrow cells by analysis of specific glutathione peroxidase transcriptional changes.