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Insulin receptor substrate 1 expression enhances the sensitivity of 32D cells to chemotherapy-induced cell death.

ABSTRACT: The adapters IRS1 and IRS2 link growth factor receptors to downstream signaling pathways that regulate proliferation and survival. Both suppress factor-withdrawal-induced apoptosis and have been implicated in cancer progression. However, recent studies suggest IRS1 and IRS2 mediate differential functions in cancer pathogenesis. IRS1 promoted breast cancer proliferation, while IRS2 promoted metastasis. The role of IRS1 and IRS2 in controlling cell responses to chemotherapy is unknown. To determine the role of IRS1 and IRS2 in the sensitivity of cells to chemotherapy, we treated 32D cells lacking or expressing IRS proteins with various concentrations of chemotherapeutic agents. We found that expression of IRS1, in contrast to IRS2, enhanced the sensitivity of 32D cells to chemotherapy-induced apoptosis. When IRS2 was expressed with IRS1, the cells no longer showed enhanced sensitivity. Expression of IRS1 did not alter the expression of pro- and anti-apoptotic proteins; however, 32D-IRS1 cells expressed higher levels of Annexin A2. In 32D-IRS1 cells, IRS1 and Annexin A2 were both located in cytoplasmic and membrane fractions. We also found that IRS1 coprecipitated with Annexin A2, while IRS2 did not. Decreasing Annexin A2 levels reduced 32D-IRS1 cell sensitivity to chemotherapy. These results suggest IRS1 enhances sensitivity to chemotherapy in part through Annexin A2.

SUBMITTER: Porter HA

PROVIDER: S-EPMC3395425 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Insulin receptor substrate 1 expression enhances the sensitivity of 32D cells to chemotherapy-induced cell death.

Porter Holly A HA Carey Gregory B GB Keegan Achsah D AD

Experimental cell research 20120528 14

The adapters IRS1 and IRS2 link growth factor receptors to downstream signaling pathways that regulate proliferation and survival. Both suppress factor-withdrawal-induced apoptosis and have been implicated in cancer progression. However, recent studies suggest IRS1 and IRS2 mediate differential functions in cancer pathogenesis. IRS1 promoted breast cancer proliferation, while IRS2 promoted metastasis. The role of IRS1 and IRS2 in controlling cell responses to chemotherapy is unknown. To determin ...[more]

PMID: 22652453

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Project description:To determine whether ?-adrenergic receptors require insulin receptor substrate (IRS)-1 activity to regulate apoptosis in retinal Müller cells.Müller cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) medium grown in normal (5 mm) or high glucose (25 mM) conditions. The medium was supplemented with 10% fetal bovine serum and antibiotics. Cells were allowed to reach 80%-90% confluence. After becoming appropriately confluent, cells were placed in medium with reduced serum (2%) for 18-24 h to eliminate any effects of fetal bovine serum. Cells were then transfected with 10 ug of IRS-1 small hairpin RNA (shRNA). Forty-eight hours following transfection, cells were lysed and harvested for protein analysis using western blotting. In additional experiments, some cells were treated with 10 uM salmeterol for 24 h following transfection with IRS-1 shRNA. To determine whether IRS-1 directly regulates apoptotic events in the insulin-signaling pathway in retinal Müller cells, a cell death assay kit was used. In tumor necrosis factor (TNF)? inhibitory studies, cells were treated with 5 ng/ml of TNF? alone for 30 min or 30 min pretreatment with TNF? followed by salmeterol for 4 h.Müller cells treated with 5 ng/ml TNF? in 25 mM glucose significantly increased phosphorylation of IRS-1(Ser307). Treatment with the selective beta-2-adrenergic receptor agonist, salmeterol, significantly decreased phosphorylation of IRS-1(Ser307). Following IRS-1 shRNA transfection+salmeterol treatment, Bcl-2-associated X protein (Bax) and cytochrome c levels were significantly decreased. Salmeterol+IRS-1 shRNA also decreased cell death and increased protein levels of B-cell lymphoma-extra large (Bcl-xL), an anti-apoptotic factor.In these studies, we show for the first time that salmeterol, a beta-2-adrenergic receptor agonist, can reduce retinal Müller cell death through IRS-1 actions. These findings also suggest the importance of IRS-1 in beta-adrenergic receptor signaling in the prevention of cell death in retinal Müller cells.

Project description:BACKGROUND: Insulin receptor substrate (IRS)-1 is associated with tumorigenesis; its levels are elevated in several human cancers. IRS-1 protein binds to several oncogene proteins. Oxidative stress and reactive oxygen species (ROS) are involved in the initiation and progression of cancers. Cancer cells produce greater levels of ROS than normal cells do because of increased metabolic stresses. However, excessive production of ROS kills cancer cells. Autophagy usually serves as a survival mechanism in response to stress conditions, but excessive induction of autophagy results in cell death. In addition to inducing necrosis and apoptosis, ROS induces autophagic cell death. ROS inactivates IRS-1 mediated signaling and reduces intracellular IRS-1 concentrations. Thus, there is a complex relationship between IRS-1, ROS, autophagy, and cancer. It is not fully understood how cancer cells grow rapidly and survive in the presence of high ROS levels. METHODS AND RESULTS: In this study, we established mouse NIH/3T3 cells that overexpressed IRS-1, so mimicking cancers with increased IRS-1 expression levels; we found that the IRS-1 overexpressing cells grow more rapidly than control cells do. Treatment of cells with glucose oxidase (GO) provided a continuous source of ROS; low dosages of GO promoted cell growth, while high doses induced cell death. Evidence for GO induced autophagy includes increased levels of isoform B-II microtubule-associated protein 1 light chain 3 (LC3), aggregation of green fluorescence protein-tagged LC3, and increased numbers of autophagic vacuoles in cells. Overexpression of IRS-1 resulted in inhibition of basal autophagy, and reduced oxidative stress-induced autophagy and cell death. ROS decreased the mammalian target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase signaling, while overexpression of IRS-1 attenuated this inhibition. Knockdown of autophagy-related gene 5 inhibited basal autophagy and diminished oxidative stress-induced autophagy and cell death. CONCLUSION: Our results suggest that overexpression of IRS-1 promotes cells growth, inhibits basal autophagy, reduces oxidative stress-induced autophagy, and diminishes oxidative stress-mediated autophagy-dependent cell death. ROS-mediated autophagy may occur via inhibition of IRS-1/phosphatidylinositol 3-kinase/mTOR signaling. Our data afford a plausible explanation for IRS-1 involvement in tumor initiation and progression.

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Insulin receptor substrate 1 expression enhances the sensitivity of 32D cells to chemotherapy-induced cell death.

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Insulin receptor substrate 1 expression enhances the sensitivity of 32D cells to chemotherapy-induced cell death.

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