%0 Generic
%A Shukla, Ashish
%D 2009
%F heidok:8991
%K Insulin Analogues
%R 10.11588/heidok.00008991
%T INSULIN ANALOGUES: ANALYSIS OF PROLIFERATIVE POTENCY AND CHARACTERIZATION OF RECEPTORS AND SIGNALLING PATHWAYS ACTIVATED IN HUMAN MAMMARY EPITHELIAL CELLS
%U https://archiv.ub.uni-heidelberg.de/volltextserver/8991/
%X Insulin analogues have been developed with the aim to provide better glycaemic control to diabetic patients. They are generated by modifying the insulin backbone which, however, may alter relevant biochemical characteristics such as the affinity to insulin receptor and type I insulin-like growth factor receptor (IGF-IR), and the insulin receptor dissociation rate. As a result insulin analogues may exhibit stronger mitogenic potency than regular insulin. Normal mammary epithelial cells show high expression of insulin receptor and IGF-IR and mammary cancer cells frequently even show overexpression of both receptors, thus suggesting mammary epithelial cells to be a sensitive target for insulin analogue - mediated proliferation. Indeed, treatment of female Sprague-Dawley rats with the insulin analogue B10Asp resulted in strong increase in the incidence of mammary tumours. Of all the insulin analogues available nowadays for therapeutical use, a standard two-year carcinogenicity study has been performed only for Insulin Glargine (Lantus®). The study showed similar incidence of mammary tumours in rats treated with Insulin Glargine or normal insulin. However, this study reported a very high mortality rate in all experimental groups thus raising questions on the conclusions drawn. In vitro studies on the effect of insulin analogues on mammary cell lines are scarce and lack comprehensiveness. In addition, the biochemical mechanism of the proliferative effect of the insulin analogues has not been clarified. This thesis aimed to study and compare in detail the proliferative potency of insulin analogues available for therapeutical use in insulin responsive mammary epithelial cell lines and to clarify the molecular and biochemical mechanism behind the proliferative potency. The role of insulin receptor, IGF-IR and related signalling pathways was analysed for the insulin analogue showing the strongest proliferative effect in comparison to regular insulin. Among a panel of seven mammary epithelial cell lines, MCF7 (a tumour cell line) and MCF10A (a benign cell line) showed the strongest insulin response. Proliferation assays on MCF10A cells demonstrated equipotency of four insulin analogues (Insulin Aspart, Insulin Lispro, Insulin Glargine and Insulin Detemir) to regular insulins (human and bovine insulin). However, proliferation assays performed in MCF7 cells revealed that Insulin Glargine induced significantly stronger proliferation than regular insulin and the other three insulin analogues. This finding was corroborated by BrdU incorporation studies in MCF7 cells. Activation of the two insulin-related signalling pathways - PI3K and MAPK pathway - was determined by studying the phosphorylation status of key signalling molecules (Akt and GSK3α/β for PI3K and Erk1/2 for MAPK pathway). In MCF10A cells, all insulin analogues were equipotent to regular insulin in inducing phosphorylation of GSK3α/β and Erk1/2. Interestingly, Insulin Glargine induced significantly higher phosphorylation of Akt in comparison to regular insulin in MCF10A cells. On the contrary, in MCF7 cells, Insulin Glargine induced strong phosphorylation of the three signalling molecules studied. The signalling potency of Insulin Glargine was significantly stronger than that of regular insulin and all other insulin analogues. Use of specific inhibitors showed that MAPK is the major proliferation pathway activated by Insulin Glargin in MCF7 cells. In order to determine the contribution of insulin receptor and IGF-IR to the strong mitogenic potency of Insulin Glargine, the RNAi technique was utilized to specifically target insulin receptor and IGF-IR. Study of signalling pathways and proliferation under knockdown conditions clearly demonstrated the activation of IGF-IR by Insulin Glargine whereas the other compounds activated the insulin receptor. Thus, the increased proliferative ability of Insulin Glargine in comparison to regular insulin is the result of IGF-IR activation. IGF-IR immunoprecipitated from cells treated with Insulin Glargine or regular insulin showed much higher tyrosine phosphorylation levels in the Insulin Glargine - treated cells, which substantiates the findings from the knockdown experiments. Moreover, analysis of expression levels of cyclin D1, an IGF-I responsive gene, by quantitative RT-PCR showed higher expression levels in MCF7 cells treated with Insulin Glargine than in cells treated with regular insulin, again corroborating the strong activation of IGF-IR by Insulin Glargine. In order to clarify the potential activation of the established cross-talk between insulin receptor/IGF-IR and estrogen receptor-α (ERα) by insulin analogues, we determined the activation of ERα by analysing the phosphorylation status of Ser118 at ERα as well as ERE-dependent luciferase gene expression. In comparison to regular insulin, Insulin Glargine induced significantly stronger phosphorylation of ERα at Ser118 and slightly higher luciferase activity. However, since ERα was only weakly activated by Insulin Glargine, modulation of ERα activity is unlikely to play a strong role in rendering high proliferative ability to Insulin Glargine. Finally, the possible tumour-promoting potential of Insulin Glargine was studied by wound healing, transwell and matrigel assays. The assays demonstrated similar migration-inducing potential of Insulin Glargine and regular human insulin in MCF7 cells. In summary, this study shows that different from regular insulin and other insulin analogues studied, which activate the insulin receptor and the PI3K pathway, Insulin Glargine activates the IGF-IR and the MAPK pathway too, and is a strong mitogen in breast cancer cells showing high IGF-IR expression. Insulin Glargine may therefore be of risk for patients with breast cancer or as yet undetected (pre-) cancerous lesions.