Pretranslational Level Research Articles

Continuous countercurrent chromatographic twin-column purification of oligonucleotides: The role of the displacement effect.

Oligonucleotides (ONs) are breaking through in the biopharmaceutical industry as a promising class of biotherapeutics. The main success of these molecules is due to their peculiar way of acting in the cellular process, regulating the gene expression and hence influencing the protein synthesis at a pretranslational level. Although the Food and Drug Administration (FDA) already approved a few ON‐based therapeutics, their production cost strongly limits large‐scale manufacturing: a situation that can be alleviated through process intensification. In this study, we address this problem by developing an efficient and continuous chromatographic purification process for ONs. In particular, we considered the chromatographic purification of an ON crude prepared by chemical synthesis using anion exchange resins. We demonstrate that in this system the competitive adsorption of the various species on the same sites of the resin leads to the displacement of the more weakly adsorbing species by the more strongly adsorbing ones. This phenomenon affects the behavior of the chromatographic units and it has been investigated in detail. Then, we developed a continuous countercurrent solvent gradient purification (MCSGP) process, which can significantly improve the productivity and buffer consumption compared to a classical single‐column, batch chromatographic process.

Glucocorticoid Induces Atrial Arrhythmogenesis via Modification of Ion Channel Gene Expression in Rats.

Excess psychological stress is one of the precipitating factors for paroxysmal atrial fibrillation (AF), although the involved mechanisms are still uncertain. To test a hypothesis that one of the stress-induced hormones, glucocorticoid, is involved in the pathogenesis of stress-induced AF, we investigated whether the glucocorticoid could alter the temporal profile of cardiac ion channels gene expression, thereby leading to atrial arrhythmogenesis.Dexamethasone (DEX, 1.0 mg/kg) was injected subcutaneously in Sprague-Dawley rats. At predetermined times after DEX injection (0, 1, 3, 6, 12, and 24 hours), the mRNA levels of cardiac ion channel genes (erg, KvLQT1, Kv4.3, Kv4.2, Kv2.1, Kv1.5, Kv1.4, Kv1.2, SUR2A, Kir6.2, Kir3.4, Kir3.1 Kir2.2, Kir2.1, SCN5A, and α1C) were determined using RNase protection assay. DEX induced immediate and transient increase in the mRNA level of Kv1.5 and Kir2.2 with peaks at 6 (5.0 fold) and 3 hours (3.3 fold) after DEX injection, respectively. Patch-clamp studies revealed a significantly increased current density of the corresponding current, IKur and IK1 at 6 hours after DEX injection. Simultaneously, electrophysiological study in isolated perfused hearts showed significantly increased number of repetitive atrial responses induced by single atrial extrastimulus (3.2 ± 2.4 to 26.7 ± 16.4, P = 0.004) with shorting of the refractory period (36.4 ± 4.6 to 27.4 ± 5.5 ms, P = 0.049) after DEX injection.Glucocorticoid immediately modified Kv1.5 and Kir2.2 gene expression at pretranslational levels, thus leading to effective refractory period shortening that could be arrhythmogenic. These results implied that transient glucocorticoid-induced biochemical modification of cardiac ion channels might be one of the mechanisms underlying the stress-induced paroxysmal AF.

Quantitative measurement of Bax and Bcl2 genes and protein expression in MCF7 cell-line when treated by Aloe Vera extract

Plants always regarded interesting as potential sources of therapeutic agents. Identification of plant ingredients is among the most important research activities worldwide. In this study, Aloe Vera (Aloe barbadensis) extract owing to its low cost and high oral intake possibility is selected to treat breast cancer cell line MCF-7. The MTT test was conducted to evaluate the inhibitory effect of Aloe Vera on the metabolic activity of MCF-7 cells. To evaluate the effect of Aloe Vera on cancer cells apoptosis, the real-time polymerase chain reaction and to identify the protein, Western blot technique were used to assess to protein Bax and Bcl2 at pre-translational and post-translational level. The results of this study demonstrate that Aloe Vera increased Bax expression and decreased bcl-2 expression and inhibited the metabolic activity of MCF-7 cell line in a dose and time dependent manner and induced apoptosis in these cells. Results indicated that the treatment of MCF7 cells with the hydro-alcoholic extract of Aloe Vera and Tamoxifen induced the apoptosis in cells; hence, the herbal extract can be useful in cancer treatment and be used as an adjuvant medicinal agent.

Oligonucleotides: Current Trends and Innovative Applications in the Synthesis, Characterization, and Purification.

Oligonucleotides (ONs) are gaining increasing importance as a promising novel class of biopharmaceuticals. Thanks to their fundamental role in gene regulation, they can be used to develop custom-made drugs (also called N-to-1) able to act on the gene expression at pre-translational level. With recent approvals of ON-based therapeutics by the Food and Drug Administration (FDA), a growing demand for high-quality chemically modified ONs is emerging and their market is expected to impressively prosper in the near future. To satisfy this growing market demand, a scalable and economically sustainable ON production is needed. In this paper, the state of the art of the whole ON production process is illustrated with the aim of highlighting the most promising routes toward the auspicated market-size production. In particular, the most recent advancements in both the upstream stage, mainly based on solid-phase synthesis and recombinant technology, and the downstream one, focusing on chromatographic techniques, are reviewed. Since ON production is projected to expand to the large scale, automatized multicolumn countercurrent technologies will reasonably be required soon to replace the current ones based on batch single-column operations. This consideration is supported by a recent cutting-edge application of continuous chromatography for the ON purification.

Transforming growth factor-β1 suppress pentraxin-3 in human orbital fibroblasts.

Transforming growth factor-β (TGF-β), recognized as a crucial factor in regulating fibrosis and tissue remodeling, plays a role in thyroid-associated ophthalmopathy (TAO). Pentraxin-3 (PTX3), a member of pentraxins, was recently implicated in many autoimmune and fibrotic diseases. Thus, we hypothesize if there is a potential correlation between TGF-β and PTX3 in orbital fibroblasts (OFs). Several strains of OFs obtained from patients with TAO (n = 8) and healthy donors (n = 3) were established as the study model. Recombinant TGF-β1 was exerted as an intervention and the expression of PTX3 was detected. To uncover the underlying mechanism, specific inhibitors of TGF-β and siRNA knockdown of Smads were utilized. We found that TGF-β1 can reduce PTX3 protein expression in OFs. We also demonstrated that this downregulation was mediated at a pretranslational level, and PTX3 mRNA was inhibited in a time- and concentration-dependent manner by TGF-β1. Interestingly, the basic level of PTX3 and the magnitude of suppression were not significantly different between TAO and control groups. Furthermore, the TGF-β receptor complex (type I:type II) and the Smad2/3-Smad4-dependent pathway are essential for TGF-mediated PTX3 repression. These findings indicated that TGF-β1 can inhibit PTX3 expression in human OFs, which may participate in inflammation and fibrosis in patients with TAO and provide a potential target for the antifibrotic treatment.

Clinical Challenge and Call for Research on Keloid Disorder: Meeting Report from The 3rd International Keloid Research Foundation Symposium, Beijing 2019

Clinical Challenge and Call for Research on Keloid Disorder: Meeting Report from The 3rd International Keloid Research Foundation Symposium, Beijing 2019

Widespread pre-translational regulation of the inclusion of signal peptides in human proteins

Signal peptides (SP) are cleavable N-terminal protein motifs used co-translationally for entry of nascent polypeptides into the secretory pathway. Their co-translational cleavage prevents their extensive post-translational regulation and flexibility in their usage is made possible by the control of their inclusion at a pre-translational level. To characterize this regulation on a transcriptome scale, we analyzed the level and mechanisms of inclusion of the 3298 most likely human SP-encoding genes, 47% of which alternatively express their SP. Analysis of RNA-seq data across different normal human tissues indicates that pre-translational regulation of the SP differs depending on tissue-coverage of the gene, with alternative SP genes more likely to be widely expressed than constitutive SP genes. SP inclusion represents a new metric to measure functional gene expression and its deregulation in disease. Our analysis supports the extensive use of pre-translational regulation of SP inclusion, with functional consequences and implications for biomarker discovery.

CD40 Expression in Fibrocytes Is Induced by TSH: Potential Synergistic Immune Activation

ContextFibrocytes appear to participate in inflammation and tissue remodeling in patients with thyroid-associated ophthalmopathy (TAO). These patients have increased frequencies of circulating TSH receptor (TSHR)- and CD40-positive fibrocytes, suggesting TSHR and CD40 may play roles in proinflammatory cytokine production, which ultimately leads to orbital inflammation and tissue remodeling.ObjectiveTo investigate the potential interactions between the TSHR and CD40 signaling pathways and their roles in IL-6 and TNF-α production.Design and Outcome MeasuresCD40 expression on fibrocytes was assessed using flow cytometry; IL-6 and TNF-α protein release using Luminex technology; increased IL-6 and TNF-α mRNA abundance, using real-time PCR; TSH- and CD40 ligand (CD40L)-stimulated Akt phosphorylation in fibrocytes, by western blot analysis; TSHR-CD40 protein-protein interaction, using co-immunoprecipitation, and CD40-TSHR co-localization, using immunocytochemistry.ResultsTSH enhances CD40 expression at a pre-translational level in fibrocytes. Production of IL-6 and TNF-α after costimulation with TSH and CD40L was greater than that after TSH or CD40L stimulation alone. TSH and CD40L costimulation also resulted in greater Akt phosphorylation. Akt and nuclear factor (NF)-κB inhibitors significantly reduced cytokine production after TSH and CD40L costimulation. TSHR and CD40L are colocalized on the cell surface and form a complex.ConclusionsTSHR and CD40 in fibrocytes appear to be physically and functionally related. TSH stimulates CD40 production on the fibrocyte surface. Cytokine expression upon simultaneous stimulation of TSHR and CD40 is greater than levels achieved with TSH or CD40L alone. Increased expression of CD40 by TSH is a potential mechanism for this process.

Kisspeptin Induces Dynamic Chromatin Modifications to Control GnRH Gene Expression.

In vitro studies have demonstrated an increase in GnRH gene expression associated with an elevated secretory response to kisspeptin administration, suggesting that kisspeptin mediates GnRH expression at both the secretory and pretranslational levels. However, the kisspeptin-mediated intracellular mechanisms associated with the dynamic chromatin modifications modulating GnRH gene expression are unclear. The studies in this manuscript describe specific histone modifications on the enhancer and promoter of the mouse GnRH (mGnRH) gene induced by kisspeptin in GnRH neuronal cell lines (GT1-7 cells). ChIP assays followed by quantitative real-time PCR (qPCR) demonstrate that 15 and 45min of 10(-9)M kisspeptin significantly increased histone 3 acetylation (H3Ac) at the kisspeptin response element (KsRE) contained between -3446 and -2806bp of the mGnRH enhancer (GnRHen) in GT1-7 cells, while no changes were observed in the downstream neuron-specific element (NSE). Moreover, kisspeptin specifically induced acetylation of H3AcK14 and K27 and trimethylation of H3 lysine 4 at the KsRE (markers of active chromatin) and no changes in dimethylation of H3K9 (a marker associated with gene repression). Occupancy of RNA Pol II (RNAPII) and a differential carboxyl-terminal domain (CTD) phosphorylation pattern was observed. An interaction between the NSE and the KsRE via a chromatin loop in the mGnRH gene by kisspeptin was detected by the chromosome conformation capture assay (3C). In conclusion, these results demonstrate that kisspeptin induces histone acetylation/methylation and consequently enhances the formation of a chromatin loop in the mGnRH gene which results in known increase in kisspeptin-dependent mGnRH expression.

Commentary: "The Role of T3 Surface Molecules in the Activation of Human Cells: A Two-Stimulus Requirement for IL-2 Production Reflects Events Occurring at a Pretranslational Level".

The human T cell leukemia Jurkat was used as a model to examine the requirements of T cell activation. These studies demonstrated that antibodies reactive with the T cell-specific T3 antigen were insufficient to result in the activation of Jurkat cells, determined by the secretion of IL 2. IL 2 production occurred only in the presence of a second stimulus, the phorbol ester PMA. With the use of an IL 2-specific cDNA probe, the appearance of IL 2 RNA, similarly, occurred only when cells were stimulated with both anti-T3 antibodies and PMA. These results demonstrate a two-stimulus requirement for gene expression in human T cells.

CD40 ligand induces expression of vascular cell adhesion molecule 1 and E-selectin in orbital fibroblasts from patients with Graves' orbitopathy.

The aim of this study was to detect the effect of the CD40 ligand (CD40L) on the expression of vascular cell adhesion molecule 1 (VCAM-1) and E-Selectin in orbital fibroblasts (OFs) from patients with Graves' orbitopathy (GO), as well as the signaling pathways involved in this effect. OFs were isolated from orbital tissues obtained from patients with severe GO who were undergoing orbital decompression surgery. VCAM-1 and E-selectin RNA and protein expression levels were quantified in OFs stimulated with soluble CD40L (sCD40L). RNA and protein quantification was performed with real-time polymerase chain reaction (PCR) and western blot analysis. Cytoplasmic and nuclear fractions were isolated in order to detect the nuclear translocation of nuclear factor-κB (NF-κB). Signaling pathway inhibitors were applied to determine the pathways involved. Compared to unstimulated OFs, the mRNA and protein levels of VCAM-1 and E-selectin in OFs incubated with sCD40L were significantly increased. This was observed in dose- and time-course experiments, and the inductive effects of sCD40L were much weaker in OFs from healthy donors. At the same time, we observed that CD40L induced nuclear translocation of NF-κB, also in a dose- and time-dependent manner. The up-regulation of VCAM-1 and E-selectin, as well as the NF-κB nuclear translocation induced by CD40L, was significantly attenuated by inhibitors targeting mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3K), and NF-κB. CD40L demonstrated the ability to up-regulate the expression of VCAM-1 and E-selectin at the pre-translational level in OFs from patients with GO. The MAPK and PI3K pathways and NF-κB may play important roles in CD40L-induced VCAM-1 and E-selectin expression.

Transcriptional regulation of hypoxia inducible factors alpha (HIF-α) and their inhibiting factor (FIH-1) of channel catfish (Ictalurus punctatus) under hypoxia

Hypoxia inducible factors (HIFs) are considered to be the master switch of oxygen-dependent gene expression with mammalian species. In most cases, regulation of HIF has been believed at posttranslational levels. However, little is known of HIF regulation in channel catfish, a species highly tolerant to low oxygen condition. Here we report the identification and characterization of HIF-1α, HIF-2αa, HIF-2αb, HIF-3α, and FIH-1 genes, and their mRNA expression under hypoxia conditions. The transcripts of the five genes were found to be regulated temporally and spatially after low oxygen challenge, suggesting regulation of HIF-α genes at pre-translational levels. In most tissues, the HIF-α mRNAs were down-regulated 1.5h but up-regulated 5h after hypoxia treatment. Of these HIF-α mRNAs, the expression of HIF-3α mRNA was induced in the most dramatic fashion, both in the speed of induction and the extent of induction, compared to HIF-1α and HIF-2α genes, suggesting its importance in responses to hypoxia.

P.1.g.058 The influence of brain serotonergic system on the expression and activity of cytochrome P450 in the liver

The aldehyde dehydrogenase inhibitor disulfiram (DS) has been used to deter drinking in alcoholics, but it also precipitates pharmacokinetic interactions with coadministered drugs. From previous experiments conducted in vitro, it has been proposed that the ethanol-inducible cytochrome P450 2E1 (CYP2E1) is the major target for inhibition by DS, but the inference from reported drug interactions is that the drug inhibits multiple CYPs. The aim of the present study was to evaluate the inhibition of major constitutive CYPs in rat liver by DS. Thus, the effects of DS on activities mediated by CYPs 2A1/2, 2C11, 2E1, and 3A, which constitute ∼80% of total CYPs in male rat liver, were evaluated. It was found that CYP2E1-mediated aniline 4-hydroxylase activity was weakly inhibited by DS in vitro, but that preincubation of the drug with NADPH-supplemented microsomes to generate metabolites of DS enhanced the extent of inhibition somewhat. In contrast, constitutive testosterone hydroxylases were inhibited effectively at low concentrations of DS (20 μM decreased the activities of all hydroxylation pathways to 40–60% of control), and a preincubation step between DS and NADPH-fortified microsomes enhanced the inhibition of CYP2C11 and 3A2 activities. In vivo studies were undertaken in which a single dose of DS (100 mg/kg, i.p.) was administered to rats; 24 hr later, CYP2E1-mediated aniline 4-hydroxylase activity was decreased to about 50% of the activity in untreated control rats. CYP2E1 apoprotein and mRNA were also decreased to 38% of the respective control, and CYP3A apoprotein and CYP3A2 mRNA responded similarly. In contrast, CYP2C11 apoprotein was decreased to 66% of control after DS administration, and CYP2A1 expression was unchanged. These findings establish that multiple CYPs are targets for inhibition by DS and provide a basis for clinically significant drug interactions involving CYPs other than 2E1. In addition, the in vivo modulation of CYP function by DS administration is not restricted to enzyme inactivation and may also include down-regulatory effects mediated at a pretranslational level.

DLK1 Protein Expression during Mouse Development Provides New Insights into Its Function

Delta-like 1 homolog (DLK1) is a noncanonical ligand in the Delta-Notch signalling pathway. Although Dlk1 mRNA is abundantly present embryonically and declines rapidly just before birth, Dlk1 knockouts display a relatively mild phenotype. To assess whether this mild phenotype was due to posttranscriptional regulation, we studied the expression of DLK1 protein in mouse embryos and found abundant expression in liver, lung, muscle, vertebrae, pancreas, pituitary, and adrenal gland(s). DLK1 expression was absent in heart, stomach, intestine, kidney, epidermis, and central nervous system. DLK1 protein expression, therefore, correlates well with the reported Dlk1 mRNA expression pattern, which shows that its expression is mainly regulated at the pretranslational level. The comparison of the reported expression patterns of Notch mRNA and those of DLK1 in organs where lineage commitment and branching morphogenesis are important developmental processes suggests that DLK1 is a ligand that prevents premature Notch-dependent differentiation, possibly by competing with canonical ligands.

Both Estrogen Receptor α and β Stimulate Pituitary GH Gene Expression

Although sex steroids have been implicated in the control of mammalian growth, their direct effect on GH synthesis is less clear. The aim of this study was to establish whether estradiol (E2) directly affects GH synthesis in somatotrophs. Somatotroph GH3 and MtT/S cells were used as in vitro models. At physiological doses of E2 stimulation, GH mRNA levels were increased and the ER antagonist ICI 182,780 completely abolished this effect. Estrogen receptor (ER) α- and ERβ-selective agonists, propylpyrazole triol (PPT), and 2,3-bis(4-hydroxyphenyl) propionitrile (DPN), respectively, augmented GH mRNA expression and secretion, whereas E2 and PPT, but not DPN increased prolactin (PRL) mRNA levels. E2, PPT, and DPN stimulated expression of the pituitary transcription factor Pou1f1 and increased its binding to the GH promoter. In vivo evidence of E2 effects on GH synthesis was obtained from the generation of the somatotroph-specific ERα knockout (sERα-KO) mouse model. Basal pituitary GH, PRL, POU1F1, and ERα mRNA expression levels were lower in sERα-KO mice compared with those in controls; whereas ERβ mRNA levels remained unchanged. E2 and DPN stimulated pituitary GH mRNA expression and serum GH levels in control and sERα-KO ovariectomized mice; however, serum GH levels were unchanged in PPT-treated ovariectomized sERα-KO mice. In these animal models, PRL mRNA levels increased after either E2 or PPT, but an increase was not seen after DPN treatment. Thus, we propose a mechanism by which estrogen directly regulates somatotroph GH synthesis at a pretranslational level. In contrast to the predominant effect of ERα in the lactotroph, these results support a role for both ERα and ERβ in the transcriptional control of Gh in the somatotroph and illustrate important differences in ER isoform specificity in the anterior pituitary gland.

Genetic Mechanisms Mediating Kisspeptin Regulation of GnRH gene Expression

Kisspeptins (Kiss) have been shown to be key components in the regulation of gonadotropin-releasing hormone (GnRH) secretion. In vitro studies have demonstrated an increase in GnRH gene expression by Kiss suggesting regulation of GnRH at both the secretory and pretranslational levels. Here, we define genetic mechanisms that mediate Kiss action on target gene expression. In vitro, sequential deletions of the mouse GnRH (mGnRH) gene promoter fused to the luciferase (LUC) reporter gene localized at kisspeptin-response element (KsRE) between -3446 and -2806 bp of the mGnRH gene. In vivo, transgenic mice bearing sequential deletions of the mGnRH gene promoter linked to the LUC reporter localized an identical KsRE. To define the mechanism of regulation, Kiss was first shown to induce nucleosome-depleted DNA within the KsRE, and a potential binding site for the transcription factor, Otx-2, was revealed. Furthermore, increased Otx-2 mRNA, protein, and binding to the KsRE after Kiss treatment were demonstrated. In conclusion, this work identified elements in GnRH-neuronal cell lines and in transgenic mice that mediate positive regulation of GnRH by Kiss. In addition, we show for the first time that Otx-2 is regulated by Kiss, and plays a role in mediating the transcriptional response of mGnRH gene.

Interleukin-6 Production in CD40-Engaged Fibrocytes in Thyroid-Associated Ophthalmopathy: Involvement of Akt and NF-κB

CD40-CD40 ligand (CD40L) interactions appear to play pathogenic roles in autoimmune disease. Here we quantify CD40 expression on fibrocytes, circulating, and bone marrow-derived progenitor cells. The functional consequences of CD40 ligation are determined since these may promote tissue remodeling linked with thyroid-associated ophthalmopathy (TAO). CD40 levels on cultivated fibrocytes and orbital fibroblasts (GOFB) from patients with Graves' disease (GD), as well as fibrocyte abundance, were determined by flow cytometry. CD40 mRNA expression was evaluated by real-time PCR, whereas response to CD40 ligation was measured by Luminex and RT-PCR. Protein kinase B (Akt) and nuclear factor (NF)-kappa B (NF-κB) signaling were determined by Western blot and immunofluorescence. Basal CD40 expression on fibrocytes is greater than that on GOFB. IFN-γ upregulates CD40 in both cell types and its actions are mediated at the pretranslational level. Fibrocytes produce high levels of cytokines, including interleukin-6 (IL-6), TNF-α, IL-8, MCP-1, and RANTES (Regulated on Activation, Normal T Cell Expressed and Secreted) in response to CD40L. IL-6 induction results from an increase in steady state IL-6 mRNA, and is mediated through Akt and NF-κB activation. Circulating CD40(+)CD45(+)Col1(+) fibrocytes are far more frequent in vivo in donors with TAO compared with healthy subjects. Particularly high levels of functional CD40 are displayed by fibrocytes. CD40L-provoked signaling results in the production of several cytokines. Among these, IL-6 expression is mediated through Akt and NF-κB pathways. The frequency of circulating CD40(+) fibrocytes is markedly increased in patients with TAO, suggesting that this receptor might represent a therapeutic target for TAO.

Butyrate increases IL-23 production by stimulated dendritic cells

The gut microbiota is essential for the maintenance of intestinal immune homeostasis and is responsible for breaking down dietary fiber into short-chain fatty acids (SCFAs). Butyrate, the most abundant bioactive SCFA in the gut, is a histone deacetylase inhibitor (HDACi), a class of drug that has potent immunomodulatory properties. This characteristic of butyrate, along with our previous discovery that conventional dendritic cells (DCs) are required for the development of experimental colitis, led us to speculate that butyrate may modulate DC function to regulate gut mucosal homeostasis. We found that butyrate, in addition to suppressing LPS-induced bone marrow-derived DC maturation and inhibiting DC IL-12 production, significantly induced IL-23 expression. The upregulation of mRNA subunit IL-23p19 at the pretranslational level was consistent with the role of HDACi on the epigenetic modification of gene expression. Furthermore, the mechanism of IL-23p19 upregulation was independent of Stat3 and ZBP89. Coculture of splenocytes with LPS-stimulated DCs pretreated with or without butyrate was performed and showed a significant induction of IL-17 and IL-10. We demonstrated further the effect of butyrate in vivo using dextran sulfate sodium (DSS)-induced colitis and found that the addition of butyrate in the drinking water of mice worsened DSS-colitis. This is in contrast to the daily intraperitoneal butyrate injection of DSS-treated mice, which mildly improved disease severity. Our study highlights a novel effect of butyrate in upregulating IL-23 production of activated DCs and demonstrates a difference in the host response to the oral vs. systemic route of butyrate administration.

Degradation of Epidermal Growth Factor Receptor Mediates Dasatinib-Induced Apoptosis in Head and Neck Squamous Cell Carcinoma Cells

Epidermal growth factor receptor (EGFR) is an important oncoprotein that promotes cell growth and proliferation. Dasatinib, a bcr-abl inhibitor, has been approved clinically for the treatment of chronic myeloid leukemia and demonstrated to be effective against solid tumors in vitro through Src inhibition. Here, we disclose that EGFR degradation mediated dasatinib-induced apoptosis in head and neck squamous cell carcinoma (HNSCC) cells. HNSCC cells, including Ca9-22, FaDu, HSC3, SAS, SCC-25, and UMSCC1, were treated with dasatinib, and cell viability, apoptosis, and underlying signal transduction were evaluated. Dasatinib exhibited differential sensitivities against HNSCC cells. Growth inhibition and apoptosis were correlated with its inhibition on Akt, Erk, and Bcl-2, irrespective of Src inhibition. Accordingly, we found that down-regulation of EGFR was a determinant of dasatinib sensitivity. Lysosome inhibitor reversed dasatinib-induced EGFR down-regulation, and c-cbl activity was increased by dasatinib, indicating that dasatinib-induced EGFR down-regulation might be through c-cbl-mediated lysosome degradation. Increased EGFR activation by ligand administration rescued cells from dasatinib-induced apoptosis, whereas inhibition of EGFR enhanced its apoptotic effect. Estrogen receptor α (ERα) was demonstrated to play a role in Bcl-2 expression, and dasatinib inhibited ERα at the pretranslational level. ERα was associated with EGFR in dasatinib-treated HNSCC cells. Furthermore, the xenograft model showed that dasatinib inhibited HSC3 tumor growth through in vivo down-regulation of EGFR and ERα. In conclusion, degradation of EGFR is a novel mechanism responsible for dasatinib-induced apoptosis in HNSCC cells.

Abstract 2880: Dasatinib induces apoptosis in head and neck squamous cell carcinoma cells through down-regulation of epidermal growth factor receptor (EGFR) in vitro and in vivo

Abstract Background: Head and neck squamous cell carcinoma (HNSCC) is a worldwide disease with aggressive course and dismal outcome. Dasatinib, a bcr-abl inhibitor, has been approved clinically for the treatment of chronic myeloid leukemia and demonstrated to be effective against solid tumors in vitro through Src inhibition. Here, we disclose that dasatinib induced apoptosis of HNSCC cells in vitro and in vivo through down-regulation of EGFR. Methods: HNSCC cells, including Ca9-22, FaDu, HSC3, SAS, SCC-25, and UMSCC1 were treated with dasatinib. Cell viability, apoptosis, and underlying signal transduction were evaluated. Results: Dasatinib exhibited differential sensitivities against HNSCC cells. The growth inhibition and apoptosis were correlated with its inhibition on Akt, Erk, and bcl-2, but irrespective of Src inhibition. Accordingly, we found that down-regulation of epidermal growth factor receptor (EGFR) was a determinant of dasatinib sensitivity. Dasatinib inhibited EGFR through lysosome degradation. Increased EGFR activation by ligand administration rescued cells from dasatinib-induced apoptosis, whereas inhibition of EGFR enhanced its apoptotic effect. Estrogen receptor (ER) was demonstrated to play a role in bcl-2 expression, and dasatinib inhibited ER at pre-translational level. ER worked in concert with EGFR to regulate dasatinib-induced apoptosis. Furthermore, xenograft model showed that dasatinib inhibited HSC3 tumor growth through in vivo down-regulation of EGFR and ER. Conclusion: Down-regulation of EGFR is a novel mechanism responsible for dasatinib-induced apoptosis in HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2880. doi:1538-7445.AM2012-2880