Rajan Singh

Nitric oxide-induced cytostasis and cell cycle arrest of a human breast cancer cell line (MDA-MB-231): Potential role of cyclin D1

DETA-NONOate, a nitric oxide (NO) donor, induced cytostasis in the human breast cancer cells MDA-MB-231, and the cells were arrested in the G1 phase of the cell cycle. This cytostatic effect of the NO donor was associated with the down-regulation of cyclin D1 and hypophosphorylation of the retinoblastoma protein. No changes in the levels of cyclin E or the catalytic partners of these cyclins, CDK2, CDK4, or CDK6, were observed. This NO-induced cytostasis and decrease in cyclin D1 was reversible for up to 48 h of DETA-NONOate (1 mM) treatment. DETA-NONOate (1 mM) produced a steady-state concentration of 0.5 μM of NO over a 24-h period. Synchronized population of the cells exposed to DETA-NONOate remained arrested at the G1 phase of the cell cycle whereas untreated control cells progressed through the cell cycle after serum stimulation. The cells arrested at the G1 phase after exposure to the NO donor had low cyclin D1 levels compared with the control cells. The levels of cyclin E and CDK4, however, were similar to the control cells. The decline in cyclin D1 protein preceded the decrease of its mRNA. This decline of cyclin D1 was due to a decrease in its synthesis induced by the NO donor and not due to an increase in its degradation. We conclude that down-regulation of cyclin D1 protein by DETA-NONOate played an important role in the cytostasis and arrest of these tumor cells in the G1 phase of the cell cycle.

Estradiol Inhibits Leukocyte Adhesion and Transendothelial Migration in Rabbits In Vivo: Possible Mechanisms for Gender Differences in Atherosclerosis

The mechanism by which estrogens protect against atherosclerosis is not known. We evaluated in vivo whether there is a gender difference in monocyte adhesion and subendothelial migration in hypercholesterolemic rabbits and whether any gender differences observed are due to estradiol. Monocyte adhesion and subendothelial migration were assessed in a blinded fashion by analyzing a standardized segment of aorta using a scanning electron microscope. We also assessed whether estradiol modulates induction of vascular cell adhesion molecule-1 (VCAM-1) protein using Western blot and flow cytometric analyses. We observed that male rabbits develop more monocyte adhesion and subendothelial migration than do female rabbits during hypercholesterolemia. We also observed that oophorectomized rabbits given physiological estradiol supplementation demonstrate fewer adherent and subendothelial monocytes than do oophorectomized rabbits given placebo. VCAM-1 protein expression was increased in aortae obtained from hypercholesterolemic, oophorectomized animals supplemented with placebo, and this increase was attenuated by estradiol. Finally, in cultured rabbit aortic endothelial cells stimulated with lysophosphatidylcholine, we observed an increase in VCAM-1 protein that was inhibited in a concentration-dependent fashion by estradiol. We have demonstrated in vivo that there is a gender difference in monocyte adhesion to endothelial cells and transendothelial migration after hypercholesterolemia and that this gender difference is due in part to estradiol. Our results also suggest that estradiol inhibits monocyte adhesion by inhibiting expression of VCAM-1.

Estradiol Suppresses MCP-1 Expression In Vivo Implications for Atherosclerosis

The mechanisms by which 17beta-estradiol retards atherogenesis are not known. The adhesion of monocytes to endothelial cells followed by the migration of monocytes into the artery wall are key cellular events that occur throughout the entire atherogenic process and may be responsive to estradiol. Monocyte chemoattractant protein-1 (MCP-1), a chemokine that is expressed in atherosclerotic lesions, is thought to play a major role in stimulating the migration of blood monocytes into developing atherosclerotic lesions. We therefore assessed the effects of estradiol in vivo on MCP-1 protein and mRNA expression in the descending thoracic aorta of rabbits fed a cholesterol-enriched (0.5%) diet for 6 weeks and in animals fed normal chow. MCP-1 protein was quantified by Western blot analysis and monocyte chemotaxis bioassay, and reverse transcription-polymerase chain reaction was used to ascertain the level of MCP-1 mRNA expression. We observed that in both ovary-intact and ovariectomized (OVX) animals, MCP-1 protein and mRNA expression were significantly increased by 6 weeks in animals fed a high-cholesterol diet. The cholesterol-induced increase in MCP-1 protein and mRNA expression was significantly attenuated in OVX rabbits supplemented with estradiol pellets (1.5- and 10.0-mg 60-day-release pellets), which yielded a range of estradiol concentrations encompassing the physiological levels. MCP-1 protein and mRNA expression were increased in normocholesterolemic OVX rabbits compared with normocholesterolemic ovary-intact animals, and this increase was prevented in OVX animals supplemented with estradiol pellets. Our observations indicate that both basal and hypercholesterolemia-induced increases in MCP-1 protein are modulated by physiological concentrations of estradiol.

Effects of dihydrotestosterone on differentiation and proliferation of human mesenchymal stem cells and preadipocytes.

UNLABELLED The mechanisms by which androgens regulate fat mass are poorly understood. Although testosterone has been reported to increase lipolysis and inhibit lipid uptake, androgen effects on proliferation and differentiation of human mesenchymal stem cells (hMSCs) and preadipocytes have not been studied. Here, we investigated whether dihydrotestosterone (DHT) regulates proliferation, differentiation, or functional maturation of hMSCs and human preadipocytes from different fat depots. DHT (0-30 nM) dose-dependently inhibited lipid accumulation in adipocytes differentiated from hMSCs and downregulated expression of aP2, PPARgamma, leptin, and C/EBPalpha. Bicalutamide attenuated DHTs inhibitory effects on adipogenic differentiation of hMSCs. Adipocytes differentiated in presence of DHT accumulated smaller oil droplets suggesting reduced extent of maturation. DHT decreased the incorporation of labeled fatty acid into triglyceride, and downregulated acetyl CoA carboxylase and DGAT2 expression in adipocytes derived from hMSCs. DHT also inhibited lipid accumulation and downregulated aP2 and C/EBPalpha in human subcutaneous, mesenteric and omental preadipocytes. DHT stimulated forskolin-stimulated lipolysis in subcutaneous and mesenteric preadipocytes and inhibited incorporation of fatty acid into triglyceride in adipocytes differentiated from preadipocytes from all fat depots. CONCLUSIONS DHT inhibits adipogenic differentiation of hMSCs and human preadipocytes through an AR-mediated pathway, but it does not affect the proliferation of either hMSCs or preadipocytes. Androgen effects on fat mass represent the combined effect of decreased differentiation of fat cell precursors, increased lipolysis, and reduced lipid accumulation.

Regulation of Myogenic Differentiation by Androgens: Cross Talk between Androgen Receptor/ β-Catenin and Follistatin/Transforming Growth Factor-β Signaling Pathways

Androgens are important regulators of body composition and promote myogenic differentiation and inhibit adipogenesis of mesenchymal, multipotent cells. Here, we investigated the mechanisms by which androgens induce myogenic differentiation of mesenchymal multipotent cells. Incubation of mesenchymal multipotent C3H 10T1/2 cells with testosterone and dihydrotestosterone promoted nuclear translocation of androgen receptor (AR)/beta-catenin complex and physical interaction of AR, beta-catenin, and T-cell factor-4 (TCF-4). Inhibition of beta-catenin by small inhibitory RNAs significantly decreased testosterone-induced stimulation of myogenic differentiation. Overexpression of TCF-4, a molecule downstream of beta-catenin in Wnt signaling cascade, in C3H 10T1/2 cells significantly up-regulated expression of myoD and myosin heavy chain II proteins and of follistatin (Fst), which binds and antagonizes native ligands of the TGF-beta/Smad pathway. Gene array analysis of C3H 10T1/2 cells treated with testosterone revealed that testosterone up-regulated the expression of Fst and modified the expression of several signaling molecules involved in the TGF-beta/Smad pathway, including Smad7. Lowering of testosterone levels in mice by orchidectomy led to a significant decrease in Fst and Smad7 expression; conversely, testosterone supplementation in castrated mice up-regulated Fst and Smad7 mRNA expression in androgen-responsive levator ani muscle. Testosterone-induced up-regulation of MyoD and myosin heavy chain II proteins in C3H 10T1/2 cells was abolished in cells simultaneously treated with anti-Fst antibody, suggesting an essential role of Fst during testosterone regulation of myogenic differentiation. In conclusion, our data suggest the involvement of AR, beta-catenin, and TCF-4 pathway during androgen action to activate a number of Wnt target genes, including Fst, and cross communication with the Smad signaling pathway.

The Dynamic Structure of the Estrogen Receptor

The estrogen receptor (ER) mediates most of the biological effects of estrogens at the level of gene regulation by interacting through its site-specific DNA and with other coregulatory proteins. In recent years, new information regarding the dynamic structural nature of ER has emerged. The physiological effects of estrogen are manifested through ERs two isoforms, ERα and ERβ. These two isoforms (ERα and ERβ) display distinct regions of sequence homology. The three-dimensional structures of the DNA-binding domain (DBD) and ligand-binding domain (LBD) have been solved, whereas no three-dimensional natively folded structure for the ER N-terminal domain (NTD) is available to date. However, insights about the structural and functional correlations regarding the ER NTD have recently emerged. In this paper, we discuss the knowledge about the structural characteristics of the ER in general and how the structural features of the two isoforms differ, and its subsequent role in gene regulation.

Testosterone inhibits transforming growth factor-β signaling during myogenic differentiation and proliferation of mouse satellite cells: potential role of follistatin in mediating testosterone action.

Testosterone (T) administration is associated with increased satellite cell number and skeletal muscle hypertrophy, although there is considerable heterogeneity in the response of different skeletal muscle groups to T in vivo. We investigated the effects of T on the growth and differentiation of satellite cells isolated from levator ani (LA) and gastrocnemius (gastroc) muscles. T up regulated follistatin (Fst) expression, but down regulated the mRNA and protein expression of a number of genes in the transforming growth factor-beta (TGF-β)-signaling pathway. Inhibition of Fst expression by small interfering RNA (siRNA) inhibited myogenic differentiation and blocked the pro-myogenic effects of T. Treatment of satellite cells with T or Fst up regulated the expression of Pax7 and PCNA, and increased their proliferation. T and Fst blocked TGF-β induced inhibition of growth and myogenic differentiation and down regulated TGF-β-dependent transcriptome in both LA and gastroc cells. We conclude that T stimulation of satellite cell proliferation and myogenic differentiation are associated with up regulation of Fst and inhibition of TGF-β-signaling.

Myostatin promotes a fibrotic phenotypic switch in multipotent C3H 10T1/2 cells without affecting their differentiation into myofibroblasts

Tissue fibrosis, the excessive deposition of collagen/extracellular matrix combined with the reduction of the cell compartment, defines fibroproliferative diseases, a major cause of death and a public health burden. Key cellular processes in fibrosis include the generation of myofibroblasts from progenitor cells, and the activation or switch of already differentiated cells to a fibrotic synthetic phenotype. Myostatin, a negative regulator of skeletal muscle mass, is postulated to be involved in muscle fibrosis. We have examined whether myostatin affects the differentiation of a multipotent mesenchymal mouse cell line into myofibroblasts, and/or modulates the fibrotic phenotype and Smad expression of the cell population. In addition, we investigated the role of follistatin in this process. Incubation of cells with recombinant myostatin protein did not affect the proportion of myofibroblasts in the culture, but significantly upregulated the expression of fibrotic markers such as collagen and the key profibrotic factors transforming growth factor-beta1 (TGF-beta1) and plasminogen activator inhibitor (PAI-1), as well as Smad3 and 4, and the pSmad2/3. An antifibrotic process evidenced by the upregulation of follistatin, Smad7, and matrix metalloproteinase 8 accompanied these changes. Follistatin inhibited TGF-beta1 induction by myostatin. Transfection with a cDNA expressing myostatin upregulated PAI-1, whereas an shRNA against myostatin blocked this effect. In conclusion, myostatin induced a fibrotic phenotype without significantly affecting differentiation into myofibroblasts. The concurrent endogenous antifibrotic reaction confirms the view that phenotypic switches in multipotent and differentiated cells may affect the progress or reversion of fibrosis, and that myostatin pharmacological inactivation may be a novel therapeutic target against fibrosis.

Caspase-8-mediated BID Cleavage and Release of Mitochondrial Cytochrome c duringN ω-Hydroxy-l-arginine-induced Apoptosis in MDA-MB-468 Cells ANTAGONISTIC EFFECTS OF l-ORNITHINE

We have previously reported thatN ω-hydroxy-l-arginine (NOHA), a stable intermediate product formed during the conversion ofl-arginine to nitric oxide, induced apoptosis in MDA-MB-468 cells, and this action was antagonized in the presence ofl-ornithine. We also reported that apoptosis induced by NOHA in this cell line could not be explained on the basis of a reduction of intracellular polyamines. In the current study, we investigated other potential mechanism(s) by which NOHA may have induced apoptosis in this cell line. We observed that NOHA initially activated caspase-8 and induced cleavage of BH3 interacting domain. This was followed by release of cytochrome c and subsequently, activation of downstream caspases-9 and -3 to cleave poly(ADP-ribose) polymerase. We also observed that NOHA induced a rapid and persistent hyperpolarization of the mitochondrial membrane potential rather than depolarization indicating that the release of cytochrome c by NOHA was by a mechanism independent of the mitochondrial transition pore. Exogenous l-ornithine did not inhibit NOHA-induced caspase-8 activation and cleavage of BH3 interacting domain but acted at the mitochondrial level and inhibited the NOHA-induced cytochrome c release and apoptosis.

Nitric oxide, Nω-hydroxy-l-arginine and breast cancer

Nitric oxide has varied effects on human breast cancer cells. At low concentration (micromolar range) it increases proliferation by increasing synthesis of some cells cycle protein and in higher concentration (nanomolar range) it leads to cytostasis or apoptosis by decreasing the translation of some cell cycle proteins.