Differential effects of genistein on apoptosis induced by fluoride and pertussis toxin in human and rat pancreatic islets and RINm5F cells

Abstract

Clonal pancreatic -cell lines have been used widely for the study of the factors involved in the regulation of apoptosis but it has not been firmly established that the response of normal islets mirrors that found in transformed -cells. In the present work, the role of pertussis toxin (Ptx)-sensitive G-proteins in the control of -cell apoptosis was studied in isolated rat and human islets of Langerhans and compared with the clonal -cell line, RINm5F. Annexin-V and deoxycarboxyfluoroscein diacetate staining was used to identify viable, apoptotic and necrotic cells directly, under fluorescence illumination. Treatment of human and rat islet cells with the G-protein activator fluoride (NaF; 5 mM) caused a marked increase in apoptosis that was further potentiated in islets pretreated with Ptx. The tyrosine kinase inhibitor genistein (100 µM) also increased islet cell apoptosis and the combination of 100 µM genistein and 5 mM NaF did not lead to any diminution of the apoptotic response. This latter effect was quite different from that seen in RINm5F cells where the combination of 100 µM genistein and 5 mM NaF resulted in much less apoptosis than was observed with either agent alone. In islets treated with a lower concentration of genistein (25 µM; that did not, itself, increase cell death), the drug attenuated NaF-induced apoptosis and also blocked the enhancement mediated by Ptx. These results revealed that human (and rat) islets are equipped with a Ptx-sensitive pathway that may be regulated by tyrosine phosphorylation and is anti-apoptotic. However, they also define conditions under which marked differences in response between RINm5F cells and normal islets were observed and they suggest that care should be taken when extrapolating data obtained with clonal cell lines to the situation in normal islet cells.

Conditional expression of hepatocyte nuclear factor-1β, the maturity-onset diabetes of the young-5 gene product, influences the viability and functional competence of pancreatic β-cells

“…We were unable to detect GADD45a at the protein level in uninduced INS-1 cells, although exposure of INS-1 cells to 5 mM NaF (which induces b-cell apoptosis (Elliott et al 2002)) resulted in the appearance of immunoreactive GADD45a. This suggests that, as in other cell types, GADD45a may play a role in regulating the apoptotic response of b-cells to certain stimuli.…”

“…GADD45a protein was not detectable in control INS-1 cells, but its expression was clearly induced by NaF, a reagent that has previously been shown to cause apoptosis in islets and b-cell lines (Hollander et al 1999, Sheikh et al 2000, Elliott et al 2002, Hildesheim et al 2002. However, despite the evidence of increased GADD45a mRNA expression in response to WT HNF1b in microarray studies, no GADD45a protein was detectable after either 24 or 48 h of induction of the transcription factor (results not shown).…”

“…The second label, 6-carboxyfluorescein diacetate (6-CFDA), is cell permeable and is used as a measure of cell viability as it is hydrolysed by esterases present in living cells to produce the green fluorescent compound 6-carboxyfluorescein (6-CF). Dual staining with these two labels can distinguish between live, necrotic and apoptotic cells (Elliott et al 2002). Following exposure to test reagents, INS-1 cells were labelled with the double-staining solution (AnnCy3 and 6-CFDA) according to the manufacturer's instructions and viewed by fluorescence microscopy.…”

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.…”

“…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].…”