ASPB10 insulin induction of increased mitogenic responses and phenotypic changes in human breast epithelial cells: Evidence for enhanced interactions with the insulin-like growth factor-I receptor

Abstract

The human insulin analogue ASPB10 has been reported to have increased affinity for the insulin receptor and to cause breast cancer in female rats. In the study reported here, we investigated whether ASPB10 has an increased mitogenic potency and induces a transformed phenotype in cultured human breast cells. In both MCF-10 cells (a non-malignant human breast line) and MCF-7 cells (a human breast cancer cell line), ASPB10 was approximately twofold more potent than insulin in competing for 125I-insulin binding but sevenfold to tenfold more potent than insulin in competing for 125I-insulin-like growth factor (IGF)-I binding. In addition, ASPB10 was twofold more potent than insulin in stimulating insulin receptor autophosphorylation but significantly more potent in stimulating IGF-I receptor autophosphorylation in both cell lines. Moreover, ASPB10 was approximately sevenfold more potent than insulin in stimulating the growth of MCF-10 and MCF-7 cells. This increased mitogenic effect of ASPB10 was significantly inhibited (but not abolished) when cells were cultured in the presence of alpha-IR3, a monoclonal antibody to the IGF-I receptor. ASPB10, but not insulin, caused phenotypic changes (focus formation) in MCF-10 cells. Neither agent caused colony formation in soft agar in MCF-10 cells, but ASPB10 was more potent than insulin in stimulating colony formation in MCF-7 cells. These observations indicate that in human breast cells, ASPB10 has enhanced mitogenic effects and induces phenotypic changes as a consequence of its activation of both insulin and IGF-I receptors.

Insulin X10 revisited: a super-mitogenic insulin analogue

“…In the majority of studies the binding affinity of insulin X10 to IR has been found to be increased 200-400% relative to that of human insulin. It was also noted by these early studies that the binding affinity of insulin X10 to IGF-1R was increased compared with that of human insulin [17,18] but with a fairly low affinity compared with IGF-1 itself, an effect that has subsequently been confirmed by a number of authors [19][20][21][22]. In addition to the increased receptor affinities, [12] insulin X10 was shown to have an increased in vitro ability to stimulate cell growth and DNA synthesis, and Bornfeldt and colleagues speculated that this was due to a substitution in the insulin X10 molecule that made this analogue more chemically similar to the IGF-1 molecule [18].…”

“…The binding of insulin X10 to hybrid receptors is unknown at present, but is currently under investigation. Some studies have found an increase in the maximal attainable response after stimulation with insulin X10 [18,21], whereas this has not been found by others [11,19,38]. However, it remains a possibility that in some cell types and for some responses, insulin X10 may be able to induce a higher response than human insulin.…”

“…However, unfortunately none of the knockdown studies included insulin X10 in the study of cellular growth, and these studies were carried out with cells expressing many IGF-1Rs, which may result in a bias towards IGF-1 mediated effects. Finally, blockade of IGF-1R-mediated growth effects using antibody directed against the receptor, almost completely abolished the effect of IGF-1 whereas the effect of insulin X10 was only partially blocked [21]. Thus, it is still unknown whether increased affinity for the IGF-1R or increased half-life of the receptor-ligand complex plays the more important role for the observed increased mitogenic potency, and consequently also whether either of these characteristics can explain the tumour findings in rats with insulin X10.…”

Effects of new insulin analogues HMR1964 (insulin glulisine) and HMR1423 on insulin receptors

“…5a). Quantification of five different experi- [10,14]. In contrast, the insulin analogue HMR1423 behaved like regular insulin, while HMR1964 was even less effective in stimulation of [ 3 H]thymidine incorporation (Fig.…”

Diabetes therapy by lentiviral hepatic insulin gene expression without transformation of liver

“…In this insulin analogue the histidine in position 10 of the B chain of the insulin molecule is replaced by aspartic acid [4]. The mutation of this insulin analogue has been shown to induce mitogenic effects [5][6][7], and according to the authors' [1] conclusion, could also be responsible for the observed liver transdifferentiation. Using the furin-cleavable insulin for the lentiviral transductions we detected a decrease in blood glucose concentration of STZ-diabetic rats from 22.3±3.4 to 15.6±2.1 mmol/l after 3 days and to 15.0±2.8 mmol/l after 30 days.…”