Variable effects of tyrosine kinase inhibitors on avian osteoclastic activity and reduction of bone loss in ovariectomized rats

Abstract

We compared the effects of the tyrosine kinase inhibitor genistein, a naturally occurring isoflavone, to those of tyrphostin A25, tyrphostin A47, and herbimycin on avian osteoclasts in vitro. Inactive analogs daidzein and tyrphostin A1 were used to control for nonspecific effects. None of the tyrosine kinase inhibitors inhibited bone attachment. However, bone resorption was inhibited by genistein and herbimycin with ID50s of 3 microM and 0.1 microM, respectively; tyrphostins and daidzein were inactive at concentrations below 30 microM, where nonspecific effects were noted. Genistein and herbimycin thus inhibit osteoclastic activity via a mechanism independent of cellular attachment, and at doses approximating those inhibiting tyrosine kinase autophosphorylation in vitro; the tyrphostins were inactive at meaningful doses. Because tyrosine kinase inhibitors vary widely in activity spectrum, effects of genistein on cellular metabolic processes were compared to herbimycin. Unlike previously reported osteoclast metabolic inhibitors which achieve a measure of selectivity by concentrating on bone, neither genistein nor herbimycin bound significantly to bone. Osteoclastic protein synthesis, measured as incorporation of 3H-leucine, was significantly inhibited at 10 microM genistein, a concentration greater than that inhibiting bone degradation, while herbimycin reduced protein synthesis at 10 nM. These data suggested that genistein may reduce osteoclastic activity at pharmacologically attainable levels, and that toxic potential was lower than that of herbimycin. To test this hypothesis in a mammalian system, bone mass was measured in 200 g ovariectomized rats treated with 44 mumol/day genistein, relative to untreated controls. During 30 d of treatment, weights of treated and control group animals were indistinguishable, indicating no toxicity, but femoral weight in the treated group was 12% greater than controls (P < 0.05). Our data indicate that the isoflavone inhibitor genistein suppresses osteoclastic activity in vitro and in vivo at concentrations consistent with its ID50s on tyrosine kinases, with a low potential for toxicity.

Health potential of soy isoflavones for menopausal women

“…6). The age and body weights of the rats in these three different studies were similar, and the duration of treatment ranged from 14 days 47 to 30 days 46,48 . All three of the studies incorporated the phyto-oestrogencontaining preparation into the diet; however, the doses used are difficult to compare.…”

“…Studies from three different laboratories using OVX rats have shown that treatment with the isoflavones present in soy, either unpurified 46 , partially purified 47 or purified 48 (as genistein) exerted significant improvement in bone mass and approximately the same per cent retention, which may approach the gain from treatment with replacement doses of oestradiol (Fig. 6).…”

“…Although the results from Blair et al 48 suggest that co-administration with soy protein is not required to show bone-conserving effects of soy isoflavones in the OVX rodent model, the effects of a range of doses have not been determined. Blair et al 48 used purified genistein at a single high dose.…”

“…Blair et al 48 used purified genistein at a single high dose. Only the study by Anderson et al 47 used more than one dose, and they reported that the lowest dose was the most effective, with less effect at the two higher doses, i.e.…”

“…The critical element is to provide isoflavones in the culture media at all times at a dose or doses sufficient to exert a threshold response. Our studies with isolated 46 , Anderson et al 47 and Blair et al 48 ) osteoblast-like MC3T3-E1 cells, treated with genistein between days 2 and 4 in culture, then incubated for a total of 12 days, suggest that genistein inhibits the synthesis and secretion of IL-6, an interleukin known to stimulate the activity of osteoclasts in bone (X. Chen et al, unpublished data, 1999). This inhibitory effect of genistein on IL-6 is similar to the inhibition of IL-6 following treatment of these cells with oestradiol (Fig.…”