G. Picotto

Antiproliferative action of menadione and 1,25(OH)2D3 on breast cancer cells.

Calcitriol or 1,25(OH)(2)D(3) is a negative growth regulator of MCF-7 breast cancer cells. The growth arrest is due to apoptosis activation, which involves mitochondrial disruption. This effect is blunted in vitamin D resistant cells (MCF-7(DRes) cells). Menadione (MEN), a glutathione (GSH)-depleting compound, may potentiate antitumoral effects of anticancer drugs. The aim of this study was to investigate whether MEN enhances cellular responsiveness of MCF-7 cells to 1,25(OH)(2)D(3). Cells were cultured and treated with different concentrations of 1,25(OH)(2)D(3)+/-MEN or vehicle for 96 h. GSH levels and the activity of antioxidant enzymes were determined by spectrophotometry and ROS production by flow cytometry. Both drugs decreased growth and enhanced ROS in MCF-7 cells, obtaining the maximal effects when 1,25(OH)(2)D(3) was combined with MEN (P<0.01 vs. Control and vs. each compound alone). MCF-7(DRes) cells were not responsive to 1,25(OH)(2)D(3), but the cell proliferation was slightly inhibited by the combined treatment. Calcitriol and MEN separately enhanced antioxidant enzyme activities, but when they were used in combination, the effect was more pronounced (P<0.05 vs. Control and vs. each compound alone). MEN, calcitriol and the combined treatment decreased GSH levels (P<0.05 vs. Control). The data indicate that MEN potentiates the effect of 1,25(OH)(2)D(3) on growth arrest in MCF-7 cells by oxidative stress and increases the activities of antioxidant enzymes, probably as a compensatory mechanism.

Intestinal Na+/Ca2+ exchanger protein and gene expression are regulated by 1,25(OH)2D3 in vitamin D-deficient chicks

The role of 1,25(OH)(2)D(3) on the intestinal NCX activity was studied in vitamin D-deficient chicks (-D) as well as the hormone effect on NCX1 protein and gene expression and the potential molecular mechanisms underlying the responses. Normal, -D and -D chicks treated with cholecalciferol or 1,25(OH)(2)D(3) were employed. In some experiments, -D chicks were injected with cycloheximide or with cycloheximide and 1,25(OH)(2)D(3) simultaneously. NCX activity was decreased by -D diet, returning to normal values after 50 IU daily of cholecalciferol/10 days or a dose of 1μg calcitriol/kg of b.w. for 15 h. Cycloheximide blocked NCX activity enhancement produced by 1,25(OH)(2)D(3). NCX1 protein and gene expression were diminished by -D diet and enhanced by 1,25(OH)(2)D(3). Vitamin D receptor expression was decreased by -D diet, effect that disappeared after 1,25(OH)(2)D(3) treatment. Rapid effects of 1,25(OH)(2)D(3) on intestinal NCX activity were also demonstrated. The abolition of the rapid effects through addition of Rp-cAMPS and staurosporine suggests that non genomic effects of 1,25(OH)(2)D(3) on NCX activity are mediated by activation of PKA and PKC pathways. In conclusion, 1,25(OH)(2)D(3) enhances the intestinal NCX activity in -D chicks through genomic and non genomic mechanisms.

Naringin prevents bone loss in a rat model of type 1 Diabetes mellitus

The aim of this work was to know whether naringin (NA) could prevent the bone complications in a model of streptozotocin (STZ) induced diabetes. Rats were divided in: 1) controls, 2) STZ-rats, 3) STZ-rats treated with 40xa0mg NA/kg, and 4) STZ-rats treated with 80xa0mg NA/kg. BMD and BMC were performed by DEXA. Bone histomorphometry and histology as well as TRAP staining were done in tibia. Osteocalcin (OCN) was determined in bone and serum. Glutathione content and SOD and catalase activities were assayed in bone marrow from femur. The data showed that NA80 increased the BMD and BMC from the long bones of STZ-rats. Both NA40 and NA80 normalized the trabecular number and the trabecular separations. An increase in the number of adipocytes and TRAP(+) cells in tibia from STZ-rats was blocked by NA. NA40 treatment increased the number of OCN(+) cells, but only the NA80 treatment allowed to reach the control values. NA normalized the SOD and catalase activities in bone marrow of femur from STZ-rats. In conclusion, NA avoids alterations in the physical properties and microstructure of bone from STZ-rats probably by stimulation of osteoblastogenesis, inhibition of the osteoclastogenesis and adipogenesis via blocking the oxidative stress.

Buthionine Sulfoximine and 1,25-Dihydroxyvitamin D Induce Apoptosis in Breast Cancer Cells via Induction of Reactive Oxygen Species

Calcitriol or 1,25(OH)2D3 is a negative growth regulator of breast cancer cells. The aim of this study was to determine whether L-buthionine-S,R-sulfoximine, a glutathione-depleting drug, modifies the antiproliferative effects of 1,25(OH)2D3 on MCF-7 cells. For comparison, we included studies in MCF-7 cells selected for vitamin D resistance and in human mammary epithelial cells transformed with SV40 and ras. Our data indicate that L-buthionine-S,R-sulfoximine enhances the growth inhibition of 1,25(OH)2D3 in all transformed breast cell lines. This effect is mediated by ROS leading to apoptosis. In conclusion, BSO alters redox state and sensitizes breast cancer cells to 1,25(OH)2D3-mediated apoptosis.

Combined calcitriol and menadione reduces experimental murine triple negative breast tumor

BACKGROUNDnCalcitriol (D) or 1,25(OH)2D3 inhibits the growth of several tumor cells including breast cancer cells, by activating cell death pathways. Menadione (MEN), a glutathione-depleting compound, may be used to potentiate the antiproliferative actions of D on cancer cells. We have previously shown in vitro that MEN improved D-induced growth arrest on breast cancer cell lines, inducing oxidative stress and DNA damage via ROS generation. Treatment with MEN+D resulted more effective than D or MEN alone.nnnOBJECTIVEnTo study the in vivo effect of calcitriol, MEN or their combination on the development of murine transplantable triple negative breast tumor M-406 in its syngeneic host.nnnMETHODSnTumor M-406 was inoculated s.c., and when tumors reached the desired size, animals were randomly assigned to one of four groups receiving daily i.p. injections of either sterile saline solution (controls, C), MEN, D, or both (MEN+D). Body weight and tumor volume were recorded three times a week. Serum calcium was determined before and at the end of the treatment, at which time tumor samples were obtained for histological examination.nnnRESULTSnNone of the drugs, alone or in combination, affected mice body weight in the period studied. The combined treatment reduced tumor growth rate (C vs. MEN+D, P<0.05) and the corresponding histological sections exhibited small remaining areas of viable tumor only in the periphery. A concomitant DNA fragmentation was observed in all treated groups and MEN potentiated the calcitriol effect on tumor growth.nnnCONCLUSIONSnAs previously observed in vitro, treatment with MEN and D delayed tumor growth in vivo more efficiently than the individual drugs, with evident signals of apoptosis induction. Our results propose an alternative protocol to treat triple negative breast cancer, using GSH depleting drugs together with calcitriol, which would allow lower doses of the steroid to maintain the antitumor effect while diminishing its adverse pharmacological effects.

Association between bone mineral density and VDR and IGF-1 gene polymorphisms in patients with congenital adrenal hyperplasia treated with glucocorticoids

s from the XXIX Annual Meeting of the Argentine Association of Osteology and Mineral Metabolism, 23–25th August 2012, Buenos Aires, Argentina Androgen regulations of cellular events involve in bone perfusion A.C. Campelo, P.H. Cutini, M.B. Rauschemberger, V.L. Massheimer Catedra de Bioquimica Clinica II, Departamento de Biologia, Bioquimica y Farmacia, Universidad Nacional del Sur, Argentina, CONICET We studied the effect of testosterone (T) on nitric oxide (NO) production, endothelial cell (EC) migration and VEGF synthesis; key factors for bone perfusion and vascularization. We previously reported that in rat aortic strips 1–20 min treatment with T (0.01–100 nM) significantly increased NO production in a non-gender specific manner. We found that the stimulatory action of T on NO synthesis decreased with age (7.84±0.83 vs 3.82±0.60 nmol NO/mg prot, young vs adult, pb0.01). Evidence of the participation of Ca influx from extracellular medium was obtained. Incubation of EC in Ca2+ free medium or in the presence of a calcium channel blocker (nifedipine) partially reduced the effect of T (79.0%, 26.5% and 28.3% above control; T, T+EGTA and T+nifedipine, pb0.01). The mechanism of action of the steroid was investigated. The effect of T did not depend on its aromatization to estradiol since presence of the aromatase inhibitor anastrozole did not affect the stimulatory action of T on NO synthesis. In contrast, preincubation of EC with finasteride (fin), an inhibitor that blocks T conversion to DHT, partially suppressed T stimulus (4.20±0.39 vs 7.34±1.01, 4.32±0.53 vs 6.00±0.91 nmol NO/mg prot; C vs T; C+fin vs T+fin, pb0.05) suggesting that both T and DHT mediate the androgen action. We observed that PI3K signal pathway is involved in the androgen-dependent NO synthesis since the inhibitor Wortmanin, completely suppressed the steroid action. In migration assays we determined that 72 h of T treatment stimulated EC migration (63±7.8 vs 115±15.8 migrated cells/field C vs T, pb0.025). Indeed the androgen enhanced the expression mRNA for VEGF. These results indicate that T regulates cellular and molecular events associated with bone perfusion. doi:10.1016/j.bone.2012.12.019 Association between bone mineral density and VDR and IGF-1 gene polymorphisms in patients with congenital adrenal hyperplasia treated with glucocorticoids G. Diaz de Barboza, A. Perez, S. Martin, M. Miras, G. Picotto, L. Munoz, A. Carpentieri, G. Sobrero, M. Ochetti, L. Silvano, M. Signorino, C. Ruperez, P. Bertolotto, C. Pellizas, M. Montesinos, M.R. Ulla, N. Tolosa de Talamoni Bioquimica y Biologia Molecular FCM UNC, Argentina Servicio de Endocrinologia Hospital de Ninos, Argentina FAMAF-UNC, Argentina FCQ UNC, Argentina CEOM, Argentina We investigated clinical and biochemical indicators of the outcome of glucocorticoid therapy in patients with congenital adrenal hyperplasia (CAH). The frequencies of different gene polymorphisms and their associations with bone metabolism and IGF system parameters were analyzed. Sixty six patients with CAH treated with glucocorticoids (GC) and/or mineralocorticoids (MC) were classified in two groups according to the presence of an adequate (Acl) or inadequate (Icl) clinical and biochemical control. Bone mineral density (BMD), osteocalcin and β-crosslaps were determined. Vitamin D receptor gene polymorphisms (Bsm I and Fok I sites) and IGF1 microsatellites were evaluated by using PCR-RFLP methods. The distribution by sex and clinical forms was similar between Acl and Icl groups. Frequencies of Fok I genotypes were no different between controls and CAH patients. Patients carrying FF genotypes showed the lowest z-score of spine BMD. The analysis of IGF-1 gene polymorphisms revealed that the frequency of allele 192 in the Icl group was lower vs controls. CAH patients with genotype 0/0 (absence of allele 192) presented higher values of β-crosslaps and lower BMDs. Altogether, our results suggest that CHA patients lacking allele 192 have an altered bone turnover and inadequate response to therapy. In addition, patients with genotype FF have the worse bone mass. Based on our data, the determination of these polymorphisms may be useful as predictors of low bone mass in patients with CAH. doi:10.1016/j.bone.2012.12.020 Bone mass and vitamin D levels in patients with rheumatoid arthritis M.L. Brance, L. Brun, J. Chiarpenello, A. Sanchez, M. Abdala, B. Oliveri Rheumatology Department, Centenario Hospital, Rosario, Argentina Bone Biology Laboratory, Argentina Rosario Endocrinology Center, Argentina Medical Osteopathy Division, Hospital de Clinicas, BsAs, Argentina Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease. Immobility due to pain, disease activity, muscle atrophy and the use of glucocorticoids, contributes to bone loss and osteoporosis. The aim of this work was to study bone mass and vitamin D (VitD) levels in patients with RA. We included 31 female patients with RA and 28 women as control group. Bone densitometry (BMD) in the lumbar spine was performed in patients younger than 60 years and in the femoral neck in patients aged more than 60 years. VitD levels were determined by chemiluminescence (Oct–Nov 2011). Results (mean±SD): Age (years): C:53.4± 11.8; RA:52.0±10.8. BMI (kg/m2): C:26.4±4.9; RA:26.1±5.1. VitD (ng/ml): C:27.2± 12.2; RA:20.0±4.9*. Calcium (mg/dl): C:9.6±0.7; RA:9.4±0.5. P (mg/dl): C:3.9±0.8; RA:3.4±0.8. PTH (pg/ml): C:40.0±16.9; RA:40.9±19.5. FAL (IU/L): C:118.7±71.8; RA:195.2±81.9*. T-score femoral neck: C:−1.730±0.338 (n=10); RA:−2.654± 0.314 (n=9)*. T-score L2–L4: C:−1.367±0.389 (n=18); RA:−1.482±0.221 (n=22). * pb0.05, Mann Whitney test. C: 28.6% had normal BMD, 46.4% osteopenia and 25% osteoporosis; RA: 21.9% had normal BMD, 46.9% osteopenia and 31.2% osteoporosis (p=0.47 Chi-square test). VitD (ng/ml): b10 ng/ml: C:4.1%, RA:2.8%; 10–20 ng/ml: C:16.7%, RA:57.1%; 20–30 ng/ml: C:62.5%, RA:34.3%; N30 ng/ml: C:16.7%, RA:5.7% (p=0.02 Chi test). VitD levels and RA activity (DAS-28): inactive (n=5): 21.3±3.9, moderate (n=23): 19.7±5.2, active (n=3): 17.3±0.9. We conclude that RA patients have lower levels of VitD and long-term impairment of bone mass, as evidenced by the difference found in the T-score at the femoral neck, with no difference at the lumbar spine. doi:10.1016/j.bone.2012.12.021 8756-3282/

Código 10Naringin: A possible bone protector for experimental type I diabetes mellitus

– see front matter. Bone 53 (2012) 599–604