Synthesis of new indole-based bisphosphonates and evaluation of their chelating ability in PE/CA-PJ15 cells.

Bisphosphonates are the most important class of antiresorptive agents used against osteoclast-mediated bone loss, and, more recently, in oncology. These compounds have high affinity for calcium ions (Ca(2+)) and therefore target bone mineral, where they appear to be internalized selectively by bone-resorbing osteoclasts and inhibit osteoclast function. They are extensively used in healthcare, however they are affected by severe side effects; pharmacological properties of bisphosphonates depend on their molecular structure, which is frequently the cause of poor intestinal adsorption and low distribution. In this work we synthesized six novel bisphosphonate compounds having a variably substituted indole moiety to evaluate their extra- and intracellular calcium chelating ability in PE/CA-PJ15 cells. Preliminary in silico and in vitro ADME studies were also performed and the results suggested that the indole moiety plays an important role in cell permeability and metabolism properties.

The impact of nitric oxide on calcium homeostasis in PE/CA-PJ15 cells.

OBJECTIVE: Nitric oxide (NO) production and Ca(2+) homeostasis are key determinants for the control of many cell functions. NO is known to be a mediator of Ca(2+) homeostasis in a highly complex and cell-specific manner and although Ca(2+) homeostasis has been explored in human oral cancer cells, the exact mechanisms are not completely understood. In this study we investigated the impact of exogenous NO on [Ca(2+)]c homeostasis in PE/CA-PJ15 cells. DESIGN: Cells were treated with S-nitrosocysteine as NO-donor and the determinations of cytosolic Ca(2+) concentrations were performed using FURA-2 AM. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) and oligomycin were used to challenge mitochondrial functionality, whereas thapsigargin (TG) and La(3+) were employed to perturb intracellular calcium levels. RESULTS: NO derived from S-nitrosocysteine (CySNO) induced a dose-dependent reduction of cytosolic calcium [Ca(2+)]c whereas oxy-haemoglobin (oxyHb) completely counteracted this effect. Subsequently, we assessed possible relationships between NO and cellular structures responsible for Ca(2+) homeostasis. We found that uncoupling of mitochondrial respiration with carbonyl-cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) and oligomycin strongly reduced the effect of NO on [Ca(2+)]c. Moreover, we found that during this mitochondrial energetic deficit, the effect of NO on [Ca(2+)]c was also reduced in the presence of La(3+) or thapsigargin. CONCLUSIONS: NO induces a concentration-dependent [Ca(2+)]c reduction in PE/CA-PJ15 human oral cancer cells and potentiates mitochondrial Ca(2+) buffering in the presence of TG or La(3+). Further, we show that exogenous NO deregulates Ca(2+) homeostasis in PE/CA-PJ15 cells with fully energized mitochondria.

Nitric oxide depletion alters hematopoietic stem cell commitment toward immunogenic dendritic cells.

BACKGROUND: NO* is a key molecule involved in the regulation of cell survival, proliferation and differentiation in many cell types. In this study we investigated the contribution of NO* during the differentiation of human peripheral blood hemopoietic stem cells (CD34+HSCs) toward immunogenic dendritic cells (i-DCs). METHODS: We depleted autocrine NO* production, using NG-monomethyl-L-arginine monoacetate (L-NMMA) and paracrine NO', using oxy-hemoglobin (HbO2) as a NO* scavenger during in vitro differentiation of CD34+HSCs to i-DCs. We monitored the NO* level, cell proliferation, phenotype and differentiation potential. RESULTS: We found that the depletion of paracrine or autocrine NO* correlated with (I) an active proliferation state at the end of differentiation, when control cells were not proliferating; (II) a significant reduction in the expression levels of differentiative markers (CD1a and HLA-DR) with a parallel high expression of the CD34 marker (III) with a retrieved clonogenic ability compared to control cells. CONCLUSIONS: On the whole, our data indicate that the depletion of NO* during the commitment stage blocks CD34+HSC differentiation into i-DCs and maintains an undifferentiated, highly proliferating cell population, indicating/revealing a novel role for NO* in the commitment of CD34+HSCs into i-DCs. GENERAL SIGNIFICANCE: The essential finding of the present study is that NO*, produced in HSCs by NOS enzymes, may act as autocrine and paracrine effectors regulating the in vitro differentiation process of CD34+-HSCs toward i-DCs.