Treatment with Insulin Analog X10 and IGF-1 Increases Growth of Colon Cancer Allografts

<div><p>Obesity and type 2 diabetes are associated with an increased risk for development of certain forms of cancer, including colon cancer. The publication of highly controversial epidemiological studies in 2009 raised the possibility that use of the insulin analog glargine increases this risk further. However, it is not clear how mitogenic effects of insulin and insulin analogs measured <i>in vitro</i> correlate with tumor growth-promoting effects <i>in vivo</i>. The aim of this study was to examine possible growth-promoting effects of native human insulin, insulin X10 and IGF-1, which are considered positive controls <i>in vitro</i>, in a short-term animal model of an obesity- and diabetes-relevant cancer. We characterized insulin and IGF-1 receptor expression and the response to treatment with insulin, X10 and IGF-1 in the murine colon cancer cell line (MC38 cells) <i>in vitro</i> and <i>in vivo</i>. Furthermore, we examined pharmacokinetics and pharmacodynamics and monitored growth of MC38 cell allografts in mice with diet-induced obesity treated with human insulin, X10 and IGF-1. Treatment with X10 and IGF-1 significantly increased growth of MC38 cell allografts in mice with diet-induced obesity and we can therefore conclude that supra-pharmacological doses of the insulin analog X10, which is super-mitogenic <i>in vitro</i> and increased the incidence of mammary tumors in female rats in a 12-month toxicity study, also increase growth of tumor allografts in a short-term animal model.</p></div

Tumor growth <i>in vivo</i>.

<p>95% CI = 95% confidence interval.</p>†<p>fold change, i.e., mean value for a given treatment expressed relative to the mean value of the vehicle-treated group.</p>*<p>, ** and *** indicates <i>P</i><0.05, <0.001 and <0.0001 respectively.</p

Effect of HI, X10 and IGF-1 on proliferation <i>in vitro</i>.

<p>MCF-7 cells (A) and MC38 cells (B) were exposed to HI/X10/IGF for 24 h in medium with low serum content (MCF-7; 0.1% (v/v), MC38; 0.25% (v/v)). At the end of the treatment period relative cell numbers were assessed with an MTT assay. Panel A and B shows the mean of three independent experiments, each with four replicates per condition, error bars indicate SEM. HI, X10 and IGF-1 increased proliferation in MCF-7 and MC38 cells. In agreement with previously published data and receptor expression profile, the ranking of the test compounds according to mitogenic potency in MCF-7 cells was IGF-1>X10>HI. In the MC38 cells the ranking was X10≥IGF-1>HI. This suggests IR and IGF-1R are expressed at roughly comparable levels in MC38 cells.</p

Overview and aims of the animal experiments.

<p>Overview and aims of the animal experiments.</p

Expression of IR and IGF-1R in MC38 cell allografts and reference tissues.

<p>A: Representative Western blots for IRβ and IGF-1Rβ in liver, MC38 tumor, colon, muscle and MC38 cells cultured <i>in vitro</i>. For each sample 20 µg of total protein was loaded on the gel. B: Quantitation of Western blots for IRβ was done relative to the average band intensities of liver samples. The relative IR level in MC38 cell allografts was comparable to skeletal muscle and significantly lower than liver and colon. n = 3 or 4 per tissue, bars indicate mean of two experiments, error bars; SEM. *** indicate <i>P</i><0.0001. C: Quantitation of Western blots for IGF-1Rβ was done relative to the average band intensities of muscle samples. The relative IGF-1R level was significantly higher in MC38 cell allografts than muscle and liver and comparable to colon. n = 3 or 4 per tissue, bars indicate mean of two experiments, error bars; SEM. *** indicate <i>P</i><0.0001. D: Representative Western blots for P-mTOR, P-p70S6K, P-Akt and β-actin in samples of tumor allografts collected 1 h after sc injection of HI, X10 or IGF-1. Plasma concentrations of HI, X10 and IGF-1 in these animals at time of euthanasia and collection of tissue samples are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079710#pone-0079710-g003" target="_blank">Figure 3A</a>. Treatment with HI, X10 or IGF-1 resulted in activation of several kinases in the PI3K signalling pathway.</p

Effect of treatment with HI/X10/IGF-1 on tumor growth.

<p>A: Data for tumor volume day 14 in experiment A–E. Treatment with IGF-1 significantly increased tumor volume compared to all other treatments and treatment with X10 increased tumor growth compared to vehicle. B: Area under the tumor growth curves day 0–14 in experiment A–E. These data were in excellent agreement with the data describing tumor volume at day 14; treatment with IGF-1 increased tumor growth compared to all other treatments and treatment with X10 increased tumor growth compared to the vehicle-treated group. Open circles: observations from individual animals, horizontal bars: group mean, error bars: SEM. * and *** indicates <i>P</i><0.05 and 0.0001, respectively.</p

Mean values ± SEM of selected metabolic parameters in DIO-mice and lean, age-matched controls.

†<p>Area under blood glucose curves during a glucose tolerance test was calculated as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079710#pone.0079710-Andrikopoulos1" target="_blank">[52]</a>.</p>††<p>Whole-body insulin sensitivity indices during a glucose tolerance test was calculated as described in a previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079710#pone.0079710-Matsuda1" target="_blank">[43]</a>.</p>*<p>, ** and *** indicate <i>P</i><0.05, <0.001 and <0.0001, respectively, when the difference between DIO-mice and lean mice was analyzed (student t-test).</p

Agonistic (mitogenic) effect of S961 in MCF-7 cells.

<p>Cells were stimulated with increasing concentrations of S961 or IGF-I. The graph is representative for three experiments. The increased response for S961 at 1 nM compared to the response at the three lowest concentrations is statistically significant (P<0.001, two-tailed t-test). Data points represent means of triplicate determinations. Error bars show one standard deviation.</p

Current model of IR activation and proposed binding mechanism for S961. A

<p>. Current model of IR activation. The four blue circles represent the receptor binding sites (sites 1 and 2) seen from a top view. Insulin is depicted as a yellow circle. For a detailed explanation of binding sites 1 and 2, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051972#pone.0051972-Whitesell1" target="_blank">[24]</a>. <b>B</b>. Proposed binding mechanism for S961. The four blue circles represent the receptor binding sites (sites 1 and 2) seen from a top view. For a detailed explanation of binding sites 1 and 2, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051972#pone.0051972-Whitesell1" target="_blank">[24]</a>. The S961 peptide (Site 1–2 peptide) is shown as two connected yellow circles. At concentrations of 1–10 nM, S961 crosslinks the receptor, leading to agonist activity. At concentrations of above 10 nM, the higher flexibility of S961 in comparison to the insulin molecule allows simultaneous crosslinking of both pairs of binding sites, leading to an inactive conformation and antagonism. The corresponding activation and inactivation sigmoids are also shown. <b>C</b>. Orientation of peptide binding sites. If site 1 is located N-terminally and site 2 C-terminally, a longer distance between the binding sites in S961 in comparison to S661 can be achieved.</p

S961 has antagonist as well as agonist activity on IR-mediated mitogenic effect in L6-hIR cells. A,

<p>"10 nM S961" and "100 nM S961" curves: Cells were pretreated for 2h with 10 nM or 100 nM S961, and stimulated with increasing concentrations of insulin (as indicated on the x-axis) in the continued presence of S961. "HI" curve, insulin stimulation only (without S961). "DMSO" curve, insulin stimulation with equal volume DMSO added instead of S961. <b>B,</b> "10 nM S961" and "100 nM S961" curves: Cells were pretreated for 2h with 10 nM or 100 nM S961, and stimulated with increasing concentrations of IGF-I (as indicated on the x-axis) in the continued presence of S961. "IGF-I" curve, IGF-I stimulation only (without S961). "DMSO" curve, IGF-I stimulation with equal volume DMSO added instead of S961. <b>C,</b> "0.01 nM HI", "0.025 nM HI" and "0.05 nM HI" curves: Cells were pretreated for 2 h with increasing concentrations of S961 (as indicated on the x-axis), and stimulated with 0.01 nM, 0.025 nM or 0.05 nM HI in the continued presence of S961. "S961 alone" curve, insulin was omitted. <b>D</b>, "1 nM HI", "10 nM HI" and "100 nM HI" curves: Cells were pretreated for 2 h with increasing concentrations of S961 (as indicated on the x-axis), and stimulated with 1 nM, 10 nM or 100 nM HI in the continued presence of S961. "S961 alone" curve, insulin was omitted. <b>A</b> and <b>B,</b> Graphs are representative for three independent experiments, each experiment comprising triplicate determinations of each ligand concentration. <b>C,</b> The graph is performed in triplicates once. <b>D,</b> The graph is representative for two independent experiments each performed in triplicates. Error bars indicate one standard deviation.</p