14 research outputs found
Insulin Receptor Isoform A and Insulin-like Growth Factor II as Additional Treatment Targets in Human Osteosarcoma
Abstract
Despite the frequent presence of an insulin-like growth factor I receptor (IGFIR)-mediated autocrine loop in osteosarcoma (OS), interfering with this target was only moderately effective in preclinical studies. Here, we considered other members of the IGF system that might be involved in the molecular pathology of OS. We found that, among 45 patients with OS, IGF-I and IGFBP-3 serum levels were significantly lower, and IGF-II serum levels significantly higher, than healthy controls. Increased IGF-II values were associated with a decreased disease-free survival. After tumor removal, both IGF-I and IGF-II levels returned to normal values. In 23 of 45 patients, we obtained tissue specimens and found that all expressed high mRNA level of IGF-II and >IGF-I. Also, isoform A of the insulin receptor (IR-A) was expressed at high level in addition to IGFIR and IR-A/IGFIR hybrids receptors (HRA). These receptors were also expressed in OS cell lines, and simultaneous impairment of IGFIR, IR, and Hybrid-Rs by monoclonal antibodies, siRNA, or the tyrosine kinase inhibitor BMS-536924, which blocks both IGFIR and IR, was more effective than selective anti-IGFIR strategies. Also, anti–IGF-II-siRNA treatment in low-serum conditions significantly inhibited MG-63 OS cells that have an autocrine circuit for IGF-II. In summary, IGF-II rather than IGF-I is the predominant growth factor produced by OS cells, and three different receptors (IR-A, HRA, and IGFIR) act complementarily for an IGF-II–mediated constitutive autocrine loop, in addition to the previously shown IGFIR/IGF-I circuit. Cotargeting IGFIR and IR-A is more effective than targeting IGF-IR alone in inhibiting OS growth. [Cancer Res 2009;69(6):2443–52
Inhibition of insulin-like growth factor-1 receptor signaling enhances growth-inhibitory and proapoptotic effects of gefitinib (Iressa) in human breast cancer cells
INTRODUCTION: Gefitinib (Iressa, ZD 1839, AstraZeneca) blocks the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) and inhibits proliferation of several human cancer cell types including breast cancer. Phase II clinical trials with gefitinib monotherapy showed an objective response of 9 to 19% in non-small-cell lung cancer patients and less than 10% for breast cancer, and phase III results have indicated no benefit of gefitinib in combination with chemotherapy over chemotherapy alone. In order to improve the antineoplastic activity of gefitinib, we investigated the effects of blocking the signalling of the insulin-like growth factor 1 receptor (IGF-1R), a tyrosine kinase with a crucial role in malignancy that is coexpressed with EGFR in most human primary breast carcinomas. METHODS: AG1024 (an inhibitor of IGF-1R) was used with gefitinib for treatment of MDA468, MDA231, SK-BR-3, and MCF-7 breast cancer lines, which express similar levels of IGF-1R but varying levels of EGFR. Proliferation assays, apoptosis induction studies, and Western blot analyses were conducted with cells treated with AG1024 and gefitinib as single agents and in combination. RESULTS: Gefitinib and AG1024 reduced proliferation in all lines when used as single agents, and when used in combination revealed an additive-to-synergistic effect on cell growth inhibition. Flow cytometry measurements of cells stained with annexin V-propidium iodide and cells stained for caspase-3 activation indicated that adding an IGF-1R-targeting strategy to gefitinib results in higher levels of apoptosis than are achieved with gefitinib alone. Gefitinib either reduced or completely inhibited p42/p44 Erk kinase phosphorylation, depending on the cell line, while Akt phosphorylation was reduced by a combination of the two agents. Overexpression of IGF-1R in SK-BR-3 cells was sufficient to cause a marked enhancement in gefitinib resistance. CONCLUSION: These results indicate that IGF-1R signaling reduces the antiproliferative effects of gefitinib in several breast cancer cell lines, and that the addition of an anti-IGF-1R strategy to gefitinib treatment may be more effective than a single-agent approach
PRODUCT ION
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Plagiorchis elegans in the molluscan intermediate host : infection, susceptibility, growth, reproduction, mortality and cercarial production
The host/parasite association between the pulmonate snail Stagnicola elodes and the digenean Plagiorchis elegans was examined with a view to identifying some of the biotic and abiotic factors that govern parasite egg development, infectivity and survival, the dynamics of cercarial production, as well as host susceptibility to infection, growth, reproduction and mortality. Plagiorchis elegans eggs passed with the feces of the hamster (Mesocricetus auratus) experimental definitive host were unembryonated. Between 0 and 30 C, the rate of embryonation continued to increase with temperature, whereas infectivity and survival were optimal between 10 to 20 C. Light adversely affected embryonation. Parasite acquisition increased with the intensity of exposure and sexual maturity. Infection invariably enhanced pre-patent host growth in a dose-dependent manner. Cercarial production increased with time and reached a plateau. Snails infected prior to sexual maturity released more cercariae than mature individuals. Heavily infected snails tended to die prematurely regardless of age at infection and parasite dose, thereby reducing their total production of cercariae to levels below those of more lightly infected individuals. Even light infections castrated the snail host. Snails infected when immature never reproduced, whereas reproductive snails ceased egg production within days of infection. Similar effects were observed in the incompatible snail host Biomphalaria glabrata, although no cercariae were ever produced. Both enhanced growth and parasitic castration are attributable to the mother sporocyst
Metformin Inhibits Mammalian Target of Rapamycin–Dependent Translation Initiation in Breast Cancer Cells
Alterations in Cellular Energy Metabolism Associated with the Antiproliferative Effects of the ATM Inhibitor KU-55933 and with Metformin
<div><p>KU-55933 is a specific inhibitor of the kinase activity of the protein encoded by <em>Ataxia telangiectasia mutated</em> (ATM), an important tumor suppressor gene with key roles in DNA repair. Unexpectedly for an inhibitor of a tumor suppressor gene, KU-55933 reduces proliferation. In view of prior preliminary evidence suggesting defective mitochondrial function in cells of patients with Ataxia Telangiectasia (AT), we examined energy metabolism of cells treated with KU-55933. The compound increased AMPK activation, glucose uptake and lactate production while reducing mitochondrial membrane potential and coupled respiration. The stimulation of glycolysis by KU-55933 did not fully compensate for the reduction in mitochondrial functions, leading to decreased cellular ATP levels and energy stress. These actions are similar to those previously described for the biguanide metformin, a partial inhibitor of respiratory complex I. Both compounds decreased mitochondrial coupled respiration and reduced cellular concentrations of fumarate, malate, citrate, and alpha-ketogluterate. Succinate levels were increased by KU-55933 levels and decreased by metformin, indicating that the effects of ATM inhibition and metformin are not identical. These observations suggest a role for ATM in mitochondrial function and show that both KU-55933 and metformin perturb the TCA cycle as well as oxidative phosphorylation.</p> </div
Subcellular localization of ATM.
<p>Total MCF-7 cell lysate and MCF-7 cells fractionated into cytoplasmic, nuclear and mitochondrial extracts were immunoblotted with ATM antibody, α-Tubulin (cytoplasmic marker), Ki67 (nuclear marker) and VDAC (mitochondrial marker). The results indicate ATM immunoreactivity in mitochondrial extracts that are negative for cytoplasmic and nuclear markers.</p
Effects of KU-55933 and metformin on TCA metabolites.
<p>(<b>A</b>) TCA metabolites were measured by NMR. <i>P</i> values for changes in TCA metabolites are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049513#pone.0049513.s003" target="_blank">Table S1</a> (<b>B</b>) Interpretation of metabolic changes observed. <i>[a]</i> ATM is hypothesized to have a role in oxidative phosphorylation, effecting respiratory complex II. Therefore, the ATM inhibitor KU-55933 leads not only to reduced ATP production, but also to accumulation of succinate. <i>[b]</i> KU-55933 also may reduce oxidative phosphorylation by a mechanism involving SCO2, as discussed in the text. <i>[c]</i> Metformin acts to inhibit oxidative phosphorylation, but prior evidence together with our findings of decreased NAD<sup>+</sup> suggest a site of action involving respiratory complex I. <i>[d]</i> Both KU-55933 and metformin exposure lead to increased glucose uptake and lactate production, consistent with a compensatory increase in glycolysis following decreased oxidative phosphorylation. <i>[e]</i> Our observations provide evidence for reduced concentrations of TCA cycle intermediates with exposure to either KU-55933 or metformin, but we postulate different reasons for this: metformin may reduce TCA cycle activity because of a reduction in supply of complex I-generated NAD<sup>+</sup>, while KU-55933 may act to inhibit conversion of succinate to fumarate. ATM, Ataxia Telangiectasia Mutated protein; SCO2, Synthesis of Cytochrome C Oxidase 2; AMPK, AMP-activated protein kinase; TSC1/TSC2, Tuberous Sclerosis 1/Tuberous Sclerosis 2; mTOR, Mammalian Target of Rapamycin complex 1; rpS6, ribosomal protein S6.</p
Inhibition of ATM by KU-55933 decreases SCO2 expression in MCF-7 cells.
<p>MCF-7 cells were exposed to KU-55933 (10 µM) for the indicated time. After harvesting, cells were lysed and prepared for immunoblot analyses using antibodies against SCO2, phospho-ATM (Ser<sup>1981</sup>), ATM, phosphorylated p53 (Ser<sup>15</sup>), p53, phospho-S6 (Ser<sup>235/236</sup>), S6rp, phospho-AMPK (Thr<sup>172</sup>) and AMPK. ß-actin is shown as a loading control. The results are representative of three individual experiments.</p