IGF-1R siRNA Research Articles

Biodegradable Sucrose Ester-Based Cationic Lipids as Novel Vectors for Efficient and Safe Delivery of IGF-1R siRNA

The key to the success of siRNA therapy depends on the use of siRNA delivery agents that effectively penetrate tumor cells and simultaneously protect siRNA from RNase attacks. Sucrose esters (SEs) are found to have tremendous potential applications in siRNA delivery because of their biocompatibility and low toxicity. However, the esterification of sucrose yields complex mixtures that greatly limit their progress in this field. We synthesized a series of novel sucrose monoester-based cationic lipids (SECLs) by using 1′,4,6′-trichlorogalactosucrose (TGS) to achieve monoesterification of sucrose. SECL-based liposomes formed from these lipids and colipids [dioleoylphosphatidylethanolamine (DOPE) or cholesterol (Chol)] could effectively condense genes into nanocomplexes, which exhibited significantly higher uptake rates and gene silencing efficiency than 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) liposome. Furthermore, SECL-based liposomes containing cholesterol-mediated IGF-1R siRNA delivery could inhibit tumor growth by the downregulation of Bcl-2 and the upregulation of Bax. Most importantly, these liposomes displayed low cytotoxicity in vitro and in vivo because they could be degraded under tumor acidic pH environments. Our study establishes a novel method for the synthesis of SEs from TGS and demonstrates the applicability of SECLs for gene delivery with the potential for clinical therapy.

PH-sensitive, tail-modified, ester-linked ionizable cationic lipids for gene delivery.

Ionizable cationic lipids have great potential for gene delivery, yet the effect of the molecular structure of such lipids on gene delivery efficiency is an ongoing research challenge. To better understand corresponding structure-function activity relationships, we synthesized four ester-linked, pH-responsive, ionizable cationic lipids. The screened DEDM4 lipid, containing 2-ethylenedimethylamine in the headgroup and a branched-chain tail, exhibited a high delivery efficacy of plasmid DNA and siRNA in A549 cells, which was comparable with that of the commercial reagent lipofectamine 3000 (lipo3000). Moreover, because of its pKa value of 6.35 and pH-sensitivity under acidic conditions, DEDM4 could carry sufficient positive charge in the acidic environment of endosomes and interact with the endosome lumen, leading to destruction of the endomembrane and subsequent release of siRNA into the cytoplasm with endosomal escape. Furthermore, we used DEDM4 to deliver IGF-1R siRNA to induce cancer cell apoptosis, thereby leading to great tumor inhibition. More importantly, it also showed very low toxicity in vivo. These structure-activity data for DEDM4 demonstrate potential clinical applications of DEDM4-mediated gene delivery for cancer.

Structure–activity relationships of pH-responsive and ionizable lipids for gene delivery

Ionizable lipids are the leading vectors for gene therapy. Understanding the effects of molecular structure on efficient gene delivery is one of the most important challenges for maximizing the utility of such lipid vectors. We synthesized an array of pH-responsive and ionizable lipids to investigate the relationship between lipid structure and activity. The optimized lipid (EDM) has double tertiary amines in the headgroup and an ester linker. EDM exhibited efficient DNA and siRNA delivery to, and gene silencing of, A549 cells. EDM has a pKa value of 6.67, which enabled it to quickly escape from the endosome after entering the cell; the ester linkages rapidly degraded and enabled gene release into the cytoplasm. EDM also delivered IGF-1R siRNA to inhibit tumor growth and induce cancer cell apoptosis by efficient inhibition of IGF-1R expression in mice. Our study on the structure–activity relationships of ionizable lipids will facilitate clinical applications.

Tyrphostin AG1024 downregulates aryl hydrocarbon receptor (AhR) expression in an IGF1R and IR-independent manner

The aryl hydrocarbon receptor (AhR) is a receptor-type transcription factor that is crucial for endocrine disruption and carcinogenesis caused by environment chemicals. Previous studies have indicated that certain intracellular signals are involved in AhR activation by their agonists, but the detailed mechanism remains unclear. In this study, we screened for important molecules for AhR activation using SCAD inhibitor kits. Among 164 kinase inhibitors listed in these kits, tyrphostin AG1024, commonly used as an inhibitor of insulin-like growth factor receptor (IGF1R) and insulin receptor (IR), was identified as a potent inhibitor of 3-methylcholanthrene (MC)-mediated AhR activation. We further investigated the mechanism by which AG1024 suppresses MC-mediated AhR activation. AG1024 decreased AhR-dependent luciferase activity, CYP1A1 gene expression, and its protein expression. However, when IGF1R siRNA and IR siRNA were used, AhR activation was slightly increased, in contrast to AG1024 treatment. In addition, AG1024 treatment downregulated the expression of AhR protein but not AhR gene, and decreased both nucleic and cytosolic AhR proteins. Therefore, AG1024 suppressed AhR activation by downregulating AhR protein expression. The molecular target of AG1024 remains unclear, and should be an important target for the regulation of AhR-dependent toxicity.

IGF-1R stimulation alters microglial polarization via TLR4/NF-κB pathway after cerebral hemorrhage in mice

Intracerebral hemorrhage (ICH) is a common neurological disease, causing severe disability and even deaths. Stem cell transplantation has been increasingly used in stroke therapy, and neural stem cells (NSCs) are a good source for stem cell transplantation. However, the regulatory mechanism of NSCs remains unclear. In this study, we examined the impact of insulin-like growth factor-1 (IGF-1) secreted by NSCs on microglial polarization following ICH in adult C57BL/6 mice. Mouse models of ICH were established by collagenase injection. ICH mice received NSC transplantation 1 h after model establishment. Firstly, the changes of microglial polarization in cerebral tissues of ICH mice were detected by immunofluorescence and ELISA. Secondly, the molecular mechanism underlying the microglial polarization was evaluated repeatedly with the application of IGF-1R siRNA and IGF-1R-mediated inhibition. We assessed the brain water content and behavioral deficits of ICH mice 12, 24, 48, and 72 h after surgery. The survival of neurons in the brain was examined using Nissl staining and TUNEL staining at 72 h. The TLR4/NF-κB pathway implicated in ICH-induced inflammation was profiled by Western blot analysis and immunohistochemistry. Finally, the changes in microglia after ICH in mice were re-examined under different doses of rhIGF-1. In summary, NSC transplantation changed microglial polarization in ICH mice. IGF-1R inhibition and knockdown reversed the therapeutic effect of NSCs, and rhIGF-1 had an anti-inflammatory effect. The results of this study suggest that IGF-1R stimulation is a potential target for stem cell-based treatment of cerebral hemorrhage and may attenuate inflammatory response in the brain after bleeding.

Liposome-based co-delivery of 7-O-geranyl-quercetin and IGF-1R siRNA for the synergistic treatment of non-small cell lung cancer

The combination of siRNAs and chemical drugs for the treatment of tumors has received more and more attention. This study investigated the synergistic effect of 7-O-geranyl quercetin (GQ) and IGF-1R siRNA (siIGF-1R) co-delivered by a liposome on human non-small cell lung cancer (NSCLC). GQ was firstly loaded with cationic liposome CDO14 to form CDO14-GQ which then combined with siIGF-1R to form co-delivery liposome CDO14-GQ-siIGF-1R. CCk-8 assay indicated that CDO14-GQ-siIGF-1R enhanced the anti-proliferation effect of CDO14-GQ or CDO14-siIGF-1R in NCI–H460 and A549 cells, AO/EB and AV/PI staining assays showed that the co-delivery liposome increased the pro-apoptosis effect of CDO14-GQ or CDO14-siIGF-1R. The growth inhibition effect of CDO14-GQ-siIGF-1R on the xenograft of NCI–H460 and A549 cells in mice was much stronger than that of CDO14-GQ or CDO14-siIGF-1R. Western blot assay indicated that CDO14-GQ-siIGF-1R down-regulated the expression levels of siIGF-1R in cells and in tumor tissues, and its effect on the expression of apoptosis-related proteins Bcl-2 and Bax was stronger than that of CDO14-GQ or CDO14-siIGF-1R. These results demonstrated that co-delivery of GQ and siIGF-1R by liposome improved the antitumor effect of either of them. Our study highlight that siRNAs combined with chemotherapeutic drugs is a promising strategy for the treatment of NSCLC.

Anti-Mucin1 Aptamer-Conjugated Chitosan Nanoparticles for Targeted Co-Delivery of Docetaxel and IGF-1R siRNA to SKBR3 Metastatic Breast Cancer Cells

Anti-Mucin1 Aptamer-Conjugated Chitosan Nanoparticles for Targeted Co-Delivery of Docetaxel and IGF-1R siRNA to SKBR3 Metastatic Breast Cancer Cells

Anti-Mucin1 Aptamer-Conjugated Chitosan Nanoparticles for Targeted Co-Delivery of Docetaxel and IGF-1R siRNA to SKBR3 Metastatic Breast Cancer Cells

Background:Targeted co-delivery of siRNA and a chemotherapeutic drug is an attractive approach to cancer drug design and treatment. This study was carried out to design an anti-Mucin1 aptamer (Apt)-conjugated chitosan nanoparticle (NP) for targeted co-delivery of insulin-like growth factor receptor 1 (IGF-1R) Silencer siRNA and docetaxel (DTX) to SKBR3 cells.Methods:Characterization of nano-drugs, cellular uptake of NPs, cell viability, and gene expression studies were evaluated based on metastatic breast cancer cells.Results:The results of this study showed that NPs had spherical and smooth morphology with 110-118 nm in size and had positive zeta potential (12-14 mV). siRNA and DTX were considerably loaded into NPs. The appropriate conjugation of the Apt to the NPs was affirmed by gel electrophoresis. The Apt-conjugated NPs were observed to enhance the cellular uptake of NPs into the SKBR3 cells. Although the combination treatment significantly decreased the cell viability of SKBR3 cells, the augmentative effect was observed when Apt was conjugated to NPs. Furthermore, Apt-conjugated NPs dramatically reduced the genetic expression of IGF-1R, signal transducers and activators of transcription 3 (STAT3), matrix metalloproteinases (MMP9), and vascular growth factor (VEGF).Conclusion:The targeted NPs may augment the targeting of pathways involved in tumorigenesis and metastasis of breast cancer. Therefore, more animal model experiments are needed to further clarify the efficacy and safety of this functionalized nanodrug.

Effects of sucrose ester structures on liposome-mediated gene delivery

Sucrose esters (SEs) have great potential applications in gene delivery because of their low toxicity, excellent biocompatibility, and biodegradability. By using tripeptide-based lipid (CDO) as a model lipid and SEs as helper lipids, a series of liposomes were prepared. The SEs with hydrophilic–lipophilic balance (HLB) values of 1, 6, 11, or 16 and the fatty acids of laurate, stearate, or oleate were used in the liposomes. We investigated the effect of HLB values of SEs and fatty acid types on gene transfection efficiency and toxicity of liposomes. The results showed that transfection efficiencies of the liposomes containing SEs with HLB value of 6 were superior to other liposomes in HeLa, MCF-7, NCI-H460, and A549 tumor cells. For the same HLB value, liposomes of laurate SEs were preferable to transfect cells compared to SEs of stearate and oleate. The liposomes with SEs showed higher cellular uptake than liposome without SEs (LipoCDO). LipoL12-6/Luc-siRNA treatment on tumor-bearing mice exhibited about 60% in vivo gene silencing of luciferase, and LipoL12-6 could mediate IGF-1R siRNA to greatly inhibit tumor growth. Moreover, liposomes with SEs revealed remarkably low toxicity in vitro and in vivo. The illustration of SE structures on gene delivery will promote the use of SEs for clinical trials of liposomes. Statement of SignificanceThis article is the first to study the effects of various chain lengths and hydrophilic–lipophilic balance (HLB) of sucrose esters (SEs) on gene transfection efficiency and safety of liposomes for gene delivery. The in vitro delivery of pDNA and siRNA by lipoplexes against HeLa, MCF-7, NCI-H460, and A549 tumor cells showed that the lipoplexes could lead to better transfection and lower cytotoxicity after the addition of SEs. SEs with shorter chain and a median HLB value could provide the liposomes with much higher gene transfection efficiency than others. The in vivo delivery of siRNA to tumor-bearing mice further confirmed that liposome containing laurate SE (LipoL12-6) could be a potential therapeutic vector, as it delivered siRNA to silence nearly 60% of the luciferase in tumors and also greatly inhibited the tumor growth. Therefore, the addition of SEs to liposomes proved to be relatively safe in vitro and in vivo. These preliminary results demonstrated that SEs show great potential for constructing controlled-release systems for gene delivery. The readers will get insights into a series of gene vectors and deepen their understanding about gene delivery.

Co-delivery of insulin-like growth factor 1 receptor specific siRNA and doxorubicin using chitosan-based nanoparticles enhanced anticancer efficacy in A549 lung cancer cell line

Here, we investigated the effects of dual delivery of IGF-1R siRNA and doxorubicin by chitosan nanoparticles on viability of A549 lung cancer cells line by utilization of MTT and qRT-PCR. Furthermore apoptosis and migration of treated cells were assessed by Annexin-PI and wound healing assays, respectively. The chitosan nanoparticles had about 176 nm size with zeta potential and polydispersive index about 11 mV and 0.3, respectively. The IGF-1R siRNA had synergistic effect on DOX-induced cytotoxicity and apoptosis in tumour cells. In addition, siRNA/DOX-loaded chitosan nanoparticles could significantly decrease migration and expressions of mmp9, VEGF and STAT3 in A549 cells.

A Convenient In Vivo Model Using Small Interfering RNA Silencing to Rapidly Assess Skeletal Gene Function.

It is difficult to study bone in vitro because it contains various cell types that engage in cross-talk. Bone biologically links various organs, and it has thus become increasingly evident that skeletal physiology must be studied in an integrative manner in an intact animal. We developed a model using local intraosseous small interfering RNA (siRNA) injection to rapidly assess the effects of a target gene on the local skeletal environment. In this model, 160-g male Sprague-Dawley rats were treated for 1–2 weeks. The left tibia received intraosseous injection of a parathyroid hormone 1 receptor (Pth1r) or insulin-like growth factor 1 receptor (Igf-1r) siRNA transfection complex loaded in poloxamer 407 hydrogel, and the right tibia received the same volume of control siRNA. All the tibias received an intraosseous injection of recombinant human parathyroid hormone (1–34) (rhPTH (1–34)) or insulin-like growth factor-1 (IGF-1). Calcein green and alizarin red were injected 6 and 2 days before euthanasia, respectively. IGF-1R and PTH1R expression levels were detected via RT-PCR assays and immunohistochemistry. Bone mineral density (BMD), microstructure, mineral apposition rates (MARs), and strength were determined by dual-energy X-ray absorptiometry, micro-CT, histology and biomechanical tests. The RT-PCR and immunohistochemistry results revealed that IGF-1R and PTH1R expression levels were dramatically diminished in the siRNA-treated left tibias compared to the right tibias (both p<0.05). Using poloxamer 407 hydrogel as a controlled-release system prolonged the silencing effect of a single dose of siRNA; the mRNA expression levels of IGF-1R were lower at two weeks than at one week (p<0.01). The BMD, bone microstructure parameters, MAR and bone strength were significantly decreased in the left tibias compared to the right tibias (all p<0.05). This simple and convenient local intraosseous siRNA injection model achieved gene silencing with very small quantities of siRNA over a short treatment period (≤7 days).

Effects of Type 1 Insulin-Like Growth Factor Receptor Silencing in a Human Adrenocortical Cell Line.

Type 1 insulin-like growth factor receptor (IGF-1R) is overexpressed in a variety of human cancers, including adrenocortical tumors. The aim of the work was to investigate the effects of IGF-1R downregulation in a human adrenocortical cell line by small interfering RNA (siRNA). The human adrenocortical tumor cell line NCI H295R was transfected with 2 specific IGF1R siRNAs (# 1 and # 2) and compared with untreated cells and a negative control siRNA. IGF1R expression was determined by quantitative reverse-transcription PCR (qRTPCR) and Western blot. The effects of IGF-1R downregulation on cell proliferation and apoptosis were assessed. IGF-1R levels were significantly decreased in cells treated with IGF-1R siRNA # 1 or # 2. Relative expression of IGF1R mRNA decreased approximately 50% and Western blot analysis revealed a 30% of reduction in IGF-1R protein. Downregulation of this gene resulted in 40% reduction in cell growth in vitro and 45% increase in apoptosis using siRNA # 2. These findings demonstrate that decreasing IGF-1R mRNA and protein expression in NCI H295R cells can partially inhibit adrenal tumor cell growth in vitro. Targeting IGF1R is a promising therapy for pediatric malignant adrenocortical tumor and can still be an option for adult adrenocortical cancer based on personalized genomic tumor profiling.

Interaction between Esophageal Squamous Cell Carcinoma and Adipose Tissue in Vitro

Esophageal squamous cell carcinoma (ESCC) develops within the squamous epithelial layer and invades the submucosa to the subadventitia that has adipose tissue (AT). AT seems critical to ESCC progression, but the underlying mechanism is unknown. We aimed to address the association between ESCC and AT invitro. ESCC cells were cultured on rat or human subcutaneous AT-embedded or -non-embedded collagen gel. AT promoted the growth of ESCC cells and inhibited their apoptosis. AT promoted the expression of the squamous differentiation marker involucrin in ESCC cells. AT accelerated the expression of invasion-related factors in poorly differentiated ESCC cells only. AT promoted the expression of phosphorylated-insulin-like growth factor-1 receptor in ESCC cells, whereas it inhibited that of the human epidermal growth factor receptor 2. Insulin-like growth factor-1, but not leptin, adiponectin, or resistin, promoted and inhibited the growth and apoptosis of ESCC cells, respectively. In turn, ESCC cells decreased the production of these adipokines in AT and the number of preadipocytes and mesenchymal stem cell-like cells, which developed from AT. These results suggest that i) AT may influence the progression of ESCC with increased growth or invasion and decreased apoptosis through insulin-like growth factor-1/insulin-like growth factor-1 receptor signaling, ii) AT may affect human epidermal growth factor receptor 2-targeted therapy; and iii) the cancer cells may affect adipokine production in AT.

Synthesis of a stabilized 177Lu-siRNA complex and evaluation of its stability and RNAi activity.

Serum and intracellular instability limits the therapeutic applications of short interfering RNA (siRNA) as a radiopharmaceutical. Chemical modifications like phosphorothioate (PS) substitution and 2'-O-methoxy (2'-O-Me) modifications could eliminate such limitations. In this study, the effects of PS and 2'-O-Me modifications at the backbone of siRNA on serum stability and RNA interference activity were investigated. Fully PS and 2'-O-Me-modified type 1 insulin-like growth factor receptor (IGF-1R) siRNA was radiolabeled with lutetium-177 ((177)Lu) through p-SCN-Bn-DTPA as a chelator. After purification with Vivaspin and PD-10 columns, the radiolabs were examined for stability in serum by instant thin-layer chromatography and polyacrylamide gel electrophoresis. The level of IGF-1R in response to the modified and labeled IGF-1R siRNA was examined using RT-PCR and ELISA assay in colon cancer cells. The effects of such siRNA on the prevention of proliferation of colon cancer cells and its apoptosis were investigated using MTT assay and Annexin-V/propidium iodide double staining, respectively. Cellular accumulation quantities of the labeled and modified IGF-1R siRNA were determined using a γ-counter by taking advantage of (177)Lu as a γ-emitter. Both the modified (177)Lu-siRNA complex and the modified nonlabeled siRNA showed significant stability in serum. The levels of IGF-1R mRNA and protein significantly decreased with both the labeled and nonlabeled IGF-1R siRNAs, but no such reduction in IGF-1R was observed with luciferase siRNA (P<0.01). Proliferation decreased significantly and apoptosis increased in the cells treated with modified (177)Lu-IGF-1R siRNA in comparison with either (177)Lu or labeled luciferase siRNA (P<0.001). Uniform chemically modified siRNAs can form stable complexes with Lu that pronounce its cytotoxic effect through apoptosis in colon cancer cells.

Nanoparticle-facilitated Intratumoral Delivery of Bcl-2/IGF-1R siRNAs and p53 Gene Synergistically Inhibits Tumor Growth in Immunocompetent Mice

Breast cancer, the leading cause of cancer-related deaths in women, is usually treated with surgery, radiation therapy, hormone-blocking/chemotherapy drugs, monoclonal antibodies or combinations of these approaches and agents depending on the cancer stage and the existence of specific receptors, with accompanying various adverse effects. Silencing of the genes responsible for cancer development and progression with small interfering RNA (siRNA) in combination with overexpression of the genes with tumor suppressing activities could be an attractive concept for precisely treating breast cancer with minimal side effects. However the short half-life in plasma due to nuclease-mediated degradation and renal clearance, and the inefficiency in penetrating the plasma membrane limit the applications of siRNA and DNA as therapeutic molecules. Recently, we have developed pH-sensitive carbonate apatite nano-carrier to efficiently deliver siRNA or DNA across the cell membrane facilitating them to escape endosomal acidic compartment and specifically cleave a cytoplasmic mRNA transcript or enable gene expression after nuclear translocation, respectively. Moreover, we demonstrated nanoparticle-accelerated delivery of the siRNAs targeting cyclin B1, PLC-gamma-2/ calmodulin1, HER2/ErbB2, ABCG2/ABCB1 and cROS1 mRNAs sensitizes cervical adenocarcinoma and breast cancer cells towards conventional anti-cancer drugs. Here, we report that co-delivery of the siRNAs targeting IGF-1R and Bcl-2 gene transcripts and the pasmid DNA containing p53 gene with the help of carbonate apatite nanoparticles synergistically induces inhibition of growth/proliferation of breast cancer cell lines as well as regression of the breast tumor induced in Balb/c mice. Additionally, concerted delivery of nanoparticle-associated IGF-1R/Bcl-2 siRNAs and p53 gene apparently slows down the growth of the established tumor in presence of doxorubicin or paclitaxel compared with the individual free drugs. Thus, the combination of IGF-1R/Bcl-2 knockdown and restoring of normal p53 function could be a highly promising approach that should be further investigated through pre-clinical trials to establish the therapeutic role of this combination therapy for breast cancer.

Silencing of insulin-like growth factor-1 receptor enhances the radiation sensitivity of human esophageal squamous cell carcinoma in vitro and in vivo

BackgroundEsophageal squamous cell carcinoma (ESCC) is a prevalent fatal cancer worldwide, and the number of deaths due to this disease is increasing. Due to ESCC resistance to chemotherapy and radiation treatment, new therapies are urgently needed for the improvement of ESCC patient clinical outcomes.MethodsEca-109 and TE-1 cells were transfected with 100 nM IGF-1r siRNA, and a combination of IGF-1r siRNA and radiation therapy was tested in vitro and in vivo. The effects of IGF-1r siRNA were determined through Western blotting and flow cytometry experiments.ResultsAfter radiotherapy, the number of IGF-1r siRNA-transfected Eca-109 cells decreased by approximately 67.3%, and a 78.9% reduction was observed in the transfected TE-1 cells. In addition, the Eca-109 and TE-1 cells that were irradiated following IGF-1r knockdown contained 16.2% and 20.3% apoptotic cells, respectively.ConclusionsThe results of the current study suggest that IGF-1r knockdown may enhance the radiation sensitivity of ESCC and increase the therapeutic effects of radiation both in vitro and in vivo. These results provide strong evidence that the targeted application of siRNA will enable the development of new therapeutic strategies for the clinical treatment of ESCC patients.

Demonstration of dose dependent cytotoxic activity in SW480 colon cancer cells by 177Lu-labeled siRNA targeting IGF-1R

The radiolabeling of targeting biomolecules with gamma emitter radionuclides for tracing and beta emitters for therapy involves the conjugation of such biomolecules to the chelating agents to form complexes with the radionuclide of interest. In this study, radioconjugate of IGF-1R siRNA with lutetium-177 ((177)Lu) was produced, and the anti-proliferation and apoptosis effects elicited by this (177)Lu-siRNA complex in the SW480 colon cancer cells were evaluated. IGF-1R and Luciferase siRNAs were conjugated with p-SCN-Bn-DTPA, and then radiolabeled with (177)Lu. The effects of labeled and non-labeled IGF-1R siRNAs on IGF-1R expression were assessed with RT-PCR analysis and ELISA assay. IGF-1R siRNAs induced cell death and apoptosis were investigated using MTT assay and Annexin-V/propidium iodide (PI) double staining, respectively. Combined purification using Vivaspin and PD-10 columns resulted in a radiochemical purity of 97.32% ± 1.97%. Knockdown effect of the labeled IGF-1R siRNA was not significantly different from the non-labeled duplex of the same sequence (P<0.05), but it was significant compared to the Luciferase siRNAs (P<0.001). Proliferation decreased significantly, but apoptosis increased in the cells treated with the (177)Lu-IGF-1R siRNA in comparison with either (177)Lu or IGF-1R siRNA (P<0.001). Radioconjugate of IGF-1R siRNA, p-SCN-Bn-DTPA and (177)Lu was successfully produced and characterized as radiopharmaceutical. The present study demonstrates the involvement of (177)Lu-labeled IGF-1R siRNA in the inhibition of cell growth and induction of apoptosis in colon cancer cells.

Depletion of the type 1 IGF receptor delays repair of radiation-induced DNA double strand breaks

Background and purposeIGF-1R depletion sensitizes prostate cancer cells to ionizing radiation and DNA-damaging cytotoxic drugs. This study investigated the hypothesis that IGF-1R regulates DNA double strand break (DSB) repair. MethodsWe tested effects of IGF-1R siRNA transfection on the repair of radiation-induced DSBs by immunoblotting and immunofluorescence for γH2AX, and pulsed-field gel electrophoresis. Homologous recombination (HR) was quantified by reporter assays, and cell cycle distribution by flow cytometry. ResultsWe confirmed that IGF-1R depletion sensitized DU145 and PC3 prostate cancer cells to ionizing radiation. DU145 control transfectants resolved radiation-induced DSBs within 24h, while IGF-1R depleted cells contained 30–40% unrepaired breaks at 24h. IGF-1R depletion induced significant reduction in DSB repair by HR, although the magnitude of the repair defect suggests additional contributory factors. Radiation-induced G2-M arrest was attenuated by IGF-1R depletion, potentially suppressing cell cycle-dependent processes required for HR. In contrast, IGF-1R depletion induced only minor radiosensitization in LNCaP cells, and did not influence repair. Cell cycle profiles were similar to DU145, so were unlikely to account for differences in repair responses. ConclusionsThese data indicate a role for IGF-1R in DSB repair, at least in part via HR, and support use of IGF-1R inhibitors with DNA damaging cancer treatments.

PAPP-A negatively regulates ABCA1, ABCG1 and SR-B1 expression by inhibiting LXRα through the IGF-I-mediated signaling pathway

Pregnancy-associated plasma protein-A (PAPP-A) has been involved in the atherosclerotic process through regulation of local expression of IGF-1 that mediates the activation of the phosphatidylinositol-3 (PI3-K) and Akt kinase (Akt) signaling cascades which lead to constitutive nitric oxide formation, with its attending vasodilator, antiplatelet and insulin-sensitizing actions. In addition, IGF-1 may decreased cholesterol efflux through reductions of expression in ABCA1 and SR-B1 by the PI3-K/Akt signaling pathway. In the current study, we examined whether PAPP-A was involved in LXRα regulation and in expression of ABCA1, ABCG1 or SR-B1 through the IGF-I-mediated signaling pathway (IGF/PI3-K/Akt). Results showed that PAPP-A significantly decreased expression of ABCA1, ABCG1 and SR-BI at both transcriptional and translational levels in a dose-dependent and time-dependent manner. Cellular cholesterol content was increased while cholesterol efflux was decreased by PAPP-A treatment. Moreover, LXRα which can regulate the expression of ABCA1, ABCG1 and SR-B1, was also down-regulated by PAPP-A treatment. LXRα-specific activation by LXRα agonist almost rescued the down-regulation of ABCA1, ABCG1 and SR-B1 expression by PAPP-A. In addition, PAPP-A can induce the IGF-1/PI3-K/Akt pathway in macrophages. Furthermore, our results indicate that the decreased levels observed in LXRα, ABCA1, ABCG1 and SR-B1 mRNA and protein levels upon treating cells with PAPP-A were strongly impaired with the PI3-K inhibitors or IGF-1R siRNA while the MAPK cascade inhibitor did not execute this effect, indicating that the process of ABCA1, ABCG1 and SR-BI degradation by PAPP-A involves the IGF-1/PI3-K/Akt pathway. In conclusion, PAPP-A may first down-regulate expression of LXRα through the IGF-1/PI3-K/Akt signaling pathway and then decrease expression of ABCA1, ABCG1, SR-B1 and cholesterol efflux in THP-1 macrophage-derived foam cells. Therefore, our study provided one of the mechanisms for understanding the critical effect of PAPP-A in pathogenesis of atherosclerosis.

R1507, an Anti-Insulin-Like Growth Factor-1 Receptor (IGF-1R) Antibody, and EWS/FLI-1 siRNA in Ewing's Sarcoma: Convergence at the IGF/IGFR/Akt Axis

A subset of patients with Ewing's sarcoma responds to anti-insulin-like growth factor-1 receptor (IGF-1R) antibodies. Mechanisms of sensitivity and resistance are unknown. We investigated whether an anti-IGF-1R antibody acts via a pathway that could also be suppressed by small interfering (si) RNA against the EWS/FLI-1 fusion protein, the hallmark of Ewing's sarcoma. The growth of two Ewing's sarcoma cell lines (TC-32 and TC-71) was inhibited by the fully human anti-IGF-1R antibody, R1507 (clonogenic and MTT assays). TC-32 and TC-71 cells express high levels of IGF-2, while RD-ES and A4573 Ewing's cell lines, which were less responsive to R1507 in our assays, express low or undetectable IGF-2, respectively. TC-71 cells also expressed high levels of IGF-1R, and R1507 decreased steady-state levels of this receptor by internalization/degradation, an effect which was associated with a decrease in p-IGF-1R, p-IRS-1, and p-Akt. EWS/FLI-1 siRNA also decreased p-Akt, due to its ability to increase IGF-BP3 levels and subsequently decrease IGF-1 and IGF-2 levels, thus inhibiting signaling through p-IGF-1R. This inhibition correlated with growth suppression and apoptosis. The attenuation of Akt activation was confirmed in TC-71 and HEK-293 (human embryonic kidney) cells by transfecting them with IGF-1R siRNA. We conclude that antibodies and siRNA to IGF-1R, as well as siRNA to EWS/FLI-1, act via intersecting IGF/IGF-1R signals that suppress a common point in this pathway, namely the phosphorylation of Akt.