Effects of tyrosine kinase inhibitors on cell death induced by sodium fluoride and pertussis toxin in the pancreatic β‐cell line, RINm5F

Abstract

1 Sodium¯uoride causes apoptosis of pancreatic b-cells and this response is enhanced by pretreatment with pertussis toxin. In the present study, tyrosine kinase inhibitors were used to investigate the mechanisms of action of NaF and pertussis toxin in the b-cell line, RINm5F. 2 Exposure of RINm5F cells to low concentrations of genistein or tyrphostin A25 resulted in signi®cant inhibition of cell death induced by 5 mM NaF. Higher concentrations (425 mM) were cytotoxic in the absence of NaF but, paradoxically, the combination of genistein and NaF induced less cell death than when each agent was used alone. 3 The increase in cell death induced by 100 mM genistein was markedly inhibited by cipro¯oxacin, a drug which binds to topoisomerase II. Etoposide (which inhibits topoisomerase II but has no e ect on tyrosine kinase activity) also caused an increase in RINm5F cell death. Neither etoposide nor cipro¯oxacin altered the response to 5 mM NaF. 4 Pertussis toxin markedly enhanced the extent of RINm5F cell death induced by NaF and this e ect was completely prevented by 25 mM genistein. The inhibition caused by genistein was not a ected by cipro¯oxacin but was reproduced by a structurally dissimilar tyrosine kinase inhibitor, herbimycin A. 5 The results demonstrate that RINm5F b-cells express a pertussis toxin sensitive pathway that is antiapoptotic. The activity of this pathway is most evident in cells exposed to pro-apoptotic stimuli where the e ects of pertussis toxin can be blocked by inhibitors of tyrosine kinase enzymes. A genisteinsensitive tyrosine kinase does not appear to be involved in RINm5F cell survival under basal conditions.

Molecular Mechanisms of Cytotoxicity and Apoptosis Induced by Inorganic Fluoride

“…A positive role of tyrosine phosphorylation in the manifestations of toxic fluoride effects was established in A549 cells, where NaF-induced apoptosis was partially reduced by tyrosine kinase inhibitor genistein [26]. The proapoptotic effects of NaF in both RINm5F pancreatic cell line and human and rat pancreatic islet cells were also associated with the activation of genisteinsensitive tyrosine kinases [36,58]. In the fluoride-treated osteoblastic cells, pertussis toxin-insensitive G proteins were suggested to activate several cytoplasmic protein tyrosine kinases from Src family, Pyk2 (proline-rich tyrosine kinase 2) and Fak (focal adhesion kinase) [61,62], what modulates the adhesion properties of these cells and, consequently, differentiation, migration, and apoptosis.…”

“…Thus, treatment of the epithelial lung cells A549 with inhibitor of PI3 kinase wortmannin augmented their responses to NaF, indicating that activation of PI3 kinase is a survival factor [26]. In contrast, in the pancreatic RINm5F cells wortmannin failed to decrease the cell viability and did not attenuated the NaF-induced cell death [36]. 12/13 proteins are specific guanine-nucleotide exchange factors RhoGEFs, which stimulate small Ras G-proteins of the Rho subfamily catalyzing the exchange of GDP for GTP [79].…”

“…As early as in 1985, Blackmore et al [57] suggested that inhibition of some G protein (possibly Gα i ) by fluoroaluminate creates "some degree of regulatory chaos" in the rat hepatocytes. Lately, the role of Gα i protein in execution of toxic fluoride effects was demonstrated in the clonal RINm5F pancreatic β-cells and rat Langerhans islets, where apoptosis induced by 5 mM NaF was markedly enhanced by pretreatment with pertussis toxin (Ptx) modifying the α-subunit of Gα i/o family [35,36,58]. The rat proximal tubules also respond to fluoride through Gα i protein [59].…”

“…Many of these cellular events ultimately lead to cell death. NaFinduced apoptosis was demonstrated in the cells from different organs and tissues including lungs [25,26], kidneys [27,28], liver [29][30][31], brain [32][33][34], pancreas [35,36], thymus [37], endometrium [38,39], bone marrow [40], hair follicles [41], erythrocytes [42][43][44], as well as leukemic cells [45][46][47].…”

Apoptotic Signaling in Mouse Odontogenesis

“…Sodium fluoride (NaF) is used as a prophylactic for caries; however, extreme acute or chronic exposure may result in enamel and skeletal fluorosis and other failures (Robinson et al, 2004). Toxic effects of NaF consist of inhibition of protein synthesis, alteration in cellular metabolism, induction of inflammatory cytokines, and apoptosis (Elliot et al, 2001). In ameloblasts treated with NaF an increased level of casapase-8 was observed, whereas in odontoblasts, an increased level of Bax was detected.…”