Expression of tumor endothelial markers (TEMs) in vitro in a canine hemangiosarcoma model of malignant angiogenesis.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e21145-e21145
Author(s):  
Jackie M. Wypij ◽  
Timothy M. Fan ◽  
Holly Pondenis
Keyword(s):  
Flow Cytometry ◽  
Protein Expression ◽  
Western Blotting ◽  
Receptor Expression ◽  
Degenerate Primers ◽  
In Vitro Expression ◽  
Endothelial Markers ◽  
Animal Tumor ◽  
Novel Model

e21145 Background: Expression of tumor endothelial markers (TEMs) in tumor-associated blood vessels presents opportunities for targeted therapy. TEM7 and TEM8 are selectively expressed and are associated with a worse prognosis in cancer patients. TEM expression is conserved across species, however species differences do exist as TEM7 is undetectable in murine tumor endothelium. Thus, further investigation of the role of TEMs in malignant angiogenesis is hindered by the lack of optimal animal tumor models. Canine hemangiosarcoma is relevant spontaneous model of malignant angiogenesis based on archetypal cell markers, endothelial functionality, growth factor/receptor expression, and angiogenic gene expression. The aims of this study were to characterize the in vitro expression of TEM7 and TEM8 in canine hemangiosarcoma as a novel model of malignant angiogenesis. Methods: Two canine hemangiosarcoma cell lines were assessed (Den and Fitz). Total RNA was isolated using standard technique, reverse transcribed to cDNA, and amplified by polymerase chain reaction (PCR) reaction using degenerate primers specific for human and murine TEM7 transcripts with forward and reverse primers. Protein expression of TEM7 and TEM8 in cell lysates was evaluated via Western blotting and cell surface expression was analyzed by flow cytometry. Polyclonal anti-TEM7 and anti-TEM8 antibodies (Sigma-Aldrich) were used with positive and negative controls. Results: Basal in vitro expression of TEM7 mRNA was confirmed in canine hemangiosarcoma, as well as in vitro protein expression of TEM7 and TEM8 via Western blotting and flow cytometry. Conclusions: The validation of TEM expression in canine hemangiosarcoma establishes TEM7 and TEM8 as promising targets for further evaluation in this novel model of malignant angiogenesis, and investigation of TEM-targeting is ongoing. This may represent a novel spontaneous animal tumor model for investigation of tumor vascular targeting.

scholarly journals In Vitro Regulation of FcRIα Expression on Human Basophils by IgE Antibody

Blood ◽  
1998 ◽  
Vol 91 (5) ◽  
pp. 1633-1643 ◽  
Author(s):  
Donald MacGlashan ◽  
Jane McKenzie-White ◽  
Kristine Chichester ◽  
Bruce S. Bochner ◽  
Frances M. Davis ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Western Blotting ◽  
Receptor Expression ◽  
In Vivo Studies ◽  
Whole Cell ◽  
Cell Lysates ◽  
Ige Antibody ◽  
Human Basophils

Abstract In vivo studies suggested the possibility of an IgE-dependent regulation of high-affinity (FcRI) IgE receptor expression on basophils. The current studies extend these observations to in vitro cultures of human basophils. Incubation of basophils for 3 to 4 weeks resulted in a slow dissociation of IgE antibody, during which time FcRI expression decreased, as measured by flow cytometry using the anti-FcRIα monoclonal antibody, 22E7, or by measuring FcRIα mass by Western blotting of whole-cell lysates. Culture of basophils with IgE resulted in upregulation of FcRIα expression by both flow cytometry and Western blotting of whole-cell lysates. Upregulation followed a linear time course during 2 weeks of culture. The relative increase in FcRIα density depended on the starting density; with starting densities of FcRIα of 10,000 to 170,000 per basophil, the upregulation varied 20- to 1.1-fold, respectively. Upregulation occurred in high-purity basophils, was not influenced by IgG at concentrations up to 1 mg/mL, and was inhibited by dimeric IgE. Heat-inactivated IgE was less effective and the monoclonal antibody CGP51901 that prevents IgE binding to FcRIα blocked the ability of IgE to induce upregulation. The dose-response curve for IgE-induced upregulation had an effective concentration50 of 230 ng/mL. Although the receptor through which IgE induces this upregulation is not yet known, several characteristics suggest that the upregulation is mediated by IgE interacting through FcRIα itself.

scholarly journals In Vitro Regulation of FcRIα Expression on Human Basophils by IgE Antibody

Blood ◽  
1998 ◽  
Vol 91 (5) ◽  
pp. 1633-1643 ◽  
Author(s):  
Donald MacGlashan ◽  
Jane McKenzie-White ◽  
Kristine Chichester ◽  
Bruce S. Bochner ◽  
Frances M. Davis ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Western Blotting ◽  
Receptor Expression ◽  
In Vivo Studies ◽  
Whole Cell ◽  
Cell Lysates ◽  
Ige Antibody ◽  
Human Basophils

In vivo studies suggested the possibility of an IgE-dependent regulation of high-affinity (FcRI) IgE receptor expression on basophils. The current studies extend these observations to in vitro cultures of human basophils. Incubation of basophils for 3 to 4 weeks resulted in a slow dissociation of IgE antibody, during which time FcRI expression decreased, as measured by flow cytometry using the anti-FcRIα monoclonal antibody, 22E7, or by measuring FcRIα mass by Western blotting of whole-cell lysates. Culture of basophils with IgE resulted in upregulation of FcRIα expression by both flow cytometry and Western blotting of whole-cell lysates. Upregulation followed a linear time course during 2 weeks of culture. The relative increase in FcRIα density depended on the starting density; with starting densities of FcRIα of 10,000 to 170,000 per basophil, the upregulation varied 20- to 1.1-fold, respectively. Upregulation occurred in high-purity basophils, was not influenced by IgG at concentrations up to 1 mg/mL, and was inhibited by dimeric IgE. Heat-inactivated IgE was less effective and the monoclonal antibody CGP51901 that prevents IgE binding to FcRIα blocked the ability of IgE to induce upregulation. The dose-response curve for IgE-induced upregulation had an effective concentration50 of 230 ng/mL. Although the receptor through which IgE induces this upregulation is not yet known, several characteristics suggest that the upregulation is mediated by IgE interacting through FcRIα itself.

scholarly journals Honeysuckle Aqueous Extracts Induced let-7a Suppress EV71 Replication and Pathogenesis In Vitro and In Vivo and Is Predicted to Inhibit SARS-CoV-2

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 308
Author(s):  
Ying-Ray Lee ◽  
Chia-Ming Chang ◽  
Yuan-Chieh Yeh ◽  
Chi-Ying F. Huang ◽  
Feng-Mao Lin ◽  
...  
Keyword(s):  
Protein Expression ◽  
Western Blotting ◽  
Expression Profiles ◽  
Plaque Assay ◽  
Virus Titer ◽  
Enterovirus 71 ◽  
Luciferase Reporter ◽  
Pathogen Invasion

Honeysuckle (Lonicera japonica Thunb) is a traditional Chinese medicine (TCM) with an antipathogenic activity. MicroRNAs (miRNAs) are small non-coding RNA molecules that are ubiquitously expressed in cells. Endogenous miRNA may function as an innate response to block pathogen invasion. The miRNA expression profiles of both mice and humans after the ingestion of honeysuckle were obtained. Fifteen overexpressed miRNAs overlapped and were predicted to be capable of targeting three viruses: dengue virus (DENV), enterovirus 71 (EV71) and SARS-CoV-2. Among them, let-7a was examined to be capable of targeting the EV71 RNA genome by reporter assay and Western blotting. Moreover, honeysuckle-induced let-7a suppression of EV71 RNA and protein expression as well as viral replication were investigated both in vitro and in vivo. We demonstrated that let-7a targeted EV71 at the predicted sequences using luciferase reporter plasmids as well as two infectious replicons (pMP4-y-5 and pTOPO-4643). The suppression of EV71 replication and viral load was demonstrated in two cell lines by luciferase activity, RT-PCR, real-time PCR, Western blotting and plaque assay. Furthermore, EV71-infected suckling mice fed honeysuckle extract or inoculated with let-7a showed decreased clinical scores and a prolonged survival time accompanied with decreased viral RNA, protein expression and virus titer. The ingestion of honeysuckle attenuates EV71 replication and related pathogenesis partially through the upregulation of let-7a expression both in vitro and in vivo. Our previous report and the current findings imply that both honeysuckle and upregulated let-7a can execute a suppressive function against the replication of DENV and EV71. Taken together, this evidence indicates that honeysuckle can induce the expression of let-7a and that this miRNA as well as 11 other miRNAs have great potential to prevent and suppress EV71 replication.

scholarly journals A Novel Mouse Monoclonal Antibody C42 against C-Terminal Peptide of Alpha-1-Antitrypsin

2021 ◽  
Vol 22 (4) ◽  
pp. 2141
Author(s):  
Srinu Tumpara ◽  
Elena Korenbaum ◽  
Mark Kühnel ◽  
Danny Jonigk ◽  
Beata Olejnicka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Western Blotting ◽  
Complex Mixture ◽  
Immunoglobulin M ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359–394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1–0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.

scholarly journals Identification of pluripotent cells in bovine uterus: in situ and in vitro studies

Reproduction ◽  
2015 ◽  
Vol 149 (4) ◽  
pp. 317-327 ◽  
Author(s):  
Martyna Łupicka ◽  
Gabriel Bodek ◽  
Nahum Shpigel ◽  
Ehud Elnekave ◽  
Anna J Korzekwa
Keyword(s):  
Flow Cytometry ◽  
Protein Expression ◽  
Mrna Expression ◽  
Uterine Horn ◽  
Uterine Tissue ◽  
Pluripotent Cells ◽  
Flow Cytometry Assay ◽  
Myometrial Cells ◽  
Gene And Protein Expression

The aim of this study was to identify uterine pluripotent cells both in bovine uterine tissues as well in epithelial, stromal, and myometrial uterine cell populations. Moreover, the relationship of pluripotent markers expression with age and the uterine horn side was considered. Uterine tissue was collected from ipsilateral and contralateral horns (days 8–10 of the estrous cycle). Immunohistostaining for C-KIT, OCT3/4, NANOG, and SOX2 in uterine tissue was determined. mRNA expression of C-KIT, OCT3/4, NANOG and SOX2 was evaluated in uterine tissue relative to the age of the cow and uterine horn side. Gene and protein expression of these markers in the uterine luminal epithelial, stromal, and myometrial cells was evaluated by real-time PCR and western blotting respectively. The expression of pluripotent cell markers OCT3/4, NANOG, and SOX2 was identified by flow cytometry assay in epithelial, stromal, and myometrial cells. Multilineage differentiation of the bovine uterine cells was performed. mRNA expression of OCT3/4, NANOG, and SOX2 in uterine tissue was higher in the ipsilateral horn than in the contralateral horn. Flow cytometry assay revealed positive fluorescence for OCT3/4, NANOG, and SOX2 in all uterine cell types. Results showed the age-dependent expression of pluripotent markers in uterine tissue. Beside, the different expression of pluripotent cells in each horn of uterus suggests the influence of ovarian hormones on these characteristics. The highest mRNA and protein expression for pluripotent markers was observed in stromal cells among uterine cells, which indicates this population of cells as the main site of pluripotent cells in the cow uterus.

Daidzein affects steroidogenesis and oestrogen receptor expression in medium ovarian follicles of pigs

2013 ◽  
Vol 61 (1) ◽  
pp. 85-98 ◽  
Author(s):  
Anna Nynca ◽  
Dominika Słonina ◽  
Olga Jablońska ◽  
Barbara Kamińska ◽  
Renata Ciereszko
Keyword(s):  
Protein Expression ◽  
Granulosa Cells ◽  
Oestrogen Receptor ◽  
Receptor Expression ◽  
Progesterone Production ◽  
Progesterone Secretion ◽  
Mrna And Protein Expression ◽  
Induced Changes ◽  
Reproductive Processes

Daidzein, a phytoestrogen present in soybean products used in swine feed, has been demonstrated to affect both reproductive and endocrine functions. The aims of this study were to examine the in vitro effects of daidzein on (1) progesterone (P4) and oestradiol (E2) secretion by porcine luteinised granulosa cells harvested from medium follicles, and (2) the mRNA and protein expression of oestrogen receptors α and β (ERα and ERβ) in these cells. The influence of E2 on P4 secretion and ERα and ERβ expression in the granulosa cells of pigs was also investigated. It was found that daidzein inhibited progesterone secretion by luteinised granulosa cells isolated from medium follicles. In contrast, E2 did not affect progesterone production by these cells. Moreover, daidzein did not alter the granulosal secretion of E2. Both daidzein and E2 decreased mRNA expression of ERα in the cells examined. The expression of ERβ mRNA was not affected by daidzein but was inhibited by E2. ERα protein was not detected while ERβ protein was found in the nuclei of the cells. Daidzein and E2 upregulated the expression of ERβ protein in the cells. In summary, the phytoestrogen daidzein directly affected the porcine ovary by inhibiting progesterone production and increasing ERβ protein expression. Daidzein-induced changes in follicular steroidogenesis and granulosal sensitivity to oestrogens may disturb reproductive processes in pigs.

scholarly journals COX-2 mediates angiotensin II-induced (pro)renin receptor expression in the rat renal medulla

2014 ◽  
Vol 307 (1) ◽  
pp. F25-F32 ◽  
Author(s):  
Fei Wang ◽  
Xiaohan Lu ◽  
Kexin Peng ◽  
Li Zhou ◽  
Chunling Li ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Protein Expression ◽  
Renal Medulla ◽  
Receptor Expression ◽  
Ang Ii ◽  
Cox 2 ◽  
Renin Content

(Pro)renin receptor (PRR) is predominantly expressed in the distal nephron where it is activated by angiotensin II (ANG II), resulting in increased renin activity in the renal medulla thereby amplifying the de novo generation and action of local ANG II. The goal of the present study was to test the role of cycloxygenase-2 (COX-2) in meditating ANG II-induced PRR expression in the renal medulla in vitro and in vivo. Exposure of primary rat inner medullary collecting duct cells to ANG II induced sequential increases in COX-2 and PRR protein expression. When the cells were pretreated with a COX-2 inhibitor NS-398, ANG II-induced upregulation of PRR protein expression was almost completely abolished, in parallel with the changes in medium active renin content. The inhibitory effect of NS-398 on the PRR expression was reversed by adding exogenous PGE2. A 14-day ANG II infusion elevated renal medullary PRR expression and active and total renin content in parallel with increased urinary renin, all of which were remarkably suppressed by the COX-2 inhibitor celecoxib. In contrast, plasma and renal cortical active and total renin content were suppressed by ANG II treatment, an effect that was unaffected by COX-2 inhibition. Systolic blood pressure was elevated with ANG II infusion, which was attenuated by the COX-2 inhibition. Overall, the results obtained from in vitro and in vivo studies established a crucial role of COX-2 in mediating upregulation of renal medullary PRR expression and renin content during ANG II hypertension.

scholarly journals 125I Low Dose Rate Radiation Increases Radiotherapy Sensitivity on Esophageal Cancer Cells via Multiple Mechanisms

2020 ◽  
Author(s):  
Hui Xue ◽  
Lifa Du ◽  
Chunxiao Li ◽  
Hao Wang ◽  
Lixiang Xue ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Esophageal Cancer ◽  
Single Dose ◽  
Protein Expression ◽  
Dose Rate ◽  
Western Blotting ◽  
Low Dose ◽  
Caspase 3 ◽  
Low Dose Rate

Abstract Objective: To determine the biological effectiveness of iodine 125 (125I) radioactive seeds continuous low dose rate radiation on the human esophageal cancer cell line KYSE150 compare to single dose radiation and explore its potential cellular mechanisms.Method: Three groups of KYSE150 cells were explored: control group, single dose irradiation (SDR) and 125I seeds continuous low dose rate irradiation group (125I-CLDR). Dose-survival curves was obtained by colony formation assay. MTT proliferation assay was used to measure KYSE150 cell vibility. KYSE150 cell apoptosis was analysis by Annexin V-FITC/PI staining, meanwhile, cell cycle analysis was performed by flow cytometry. Bim, Bcl-2, caspase-3 and cleaved caspase-3 protein expression were measured by western blotting to vertify the apoptosis level, as well as CyclinB1 protein expression which values the fuction of G2/M check point.Cell morphology changes were observated under phase contrast microscope. Endoplasmic reticulum stress (ER stress) was measured by GPR78/Bip1 and PERK changes in gene expression, which was detected by Real-time PCR. Reactive oxygen species(ROS) changes and mitochondria were meaured by flow cytometry. ATP detection kit was used to measured ATP level afer two modes of irradiation. DNA-Pks, Ku70 and Ku80 expression were measured by western blotting to represent the damage of DNA damage and repair capabilities. Acridine orange (AO) staining was used to detect level of autophagy and quantitative was measured by flow cytometry. LC3-II, ATG5, Beclin1, p-Akt, p-mTOR, mTOR and p-S6 proteins expression were detectd by western blotting.Results: KYSE150 cells were more radiosensitive to 125I-CLDR than SDR. Two modes of irradiation could both inhibit the proliferation vability of KYSE150 cells, while the inhibition effects in 125I-CLDR was significantly stronger compared with SDR. Compared with SDR, 125I-CLDR showed more proportions of the early and late apoptosis rate as well as cells at G2/M phase. Apoptosis related proteins, such as Bim, caspase-3 and cleaved caspase-3, were elevated, while CyclinB1 expression was decreased in 125I-CLDR group. Cells became bigger and grainy in 125I-CLDR group, meanwhile, ROS levels and ER stress signal was elevated except ATP concentration. Cells stained by AO was anaysised by flow cytometry, results showed that red: green ratio in 125I-CLDR group increased as well as LC3 and ATG5 protein expression detected by western blotting. p-Akt, p-mTOR and p-S6 protein expression were decreased to some extent in 125I-CLDR group cells, while total mTOR protein showed no significant changes.Conclusion: Our results confirmed that 125I-CLDR could strong inhibite KYSE150 cancer cells. Furthermore, we revealed that cell cycle arrest and apoptosis were not the only fate after 125I-CLDR, autophagy might be another important choice which mTOR pathway might be involed in. These findings can support the application of esophageal stent loaded with 125I seeds in clinic.

163 Treatment of Growth Differentiation Factor 8 (GDF8) During Porcine Oocyte In Vitro Maturation Changed Transcriptional Pattern and AKT Signaling

2018 ◽  
Vol 30 (1) ◽  
pp. 221
Author(s):  
J. D. Yoon ◽  
E. Lee ◽  
S.-H. Hyun
Keyword(s):  
Protein Expression ◽  
Western Blotting ◽  
Gene Transcription ◽  
In Vitro Maturation ◽  
Specific Gene ◽  
Pcr Array ◽  
Maternal Factors ◽  
Realtime Pcr ◽  
Differentiation Factor

Growth differentiation factor 8 (GDF8) is a member of the transforming growth factor-β that has been identified as a strong physiological regulator. The purpose of this study was to investigate the effects of GDF8 on porcine oocytes during in vitro maturation (IVM). We investigated specific gene transcription levels in oocytes and cumulus cells (CC) after IVM by realtime PCR array, and specific protein expression and activation levels in matured CC by Western blotting. To collect IVM oocytes, the cumulus–oocyte complexes (COC) were aspirated and matured in 500 μL of M199 containing 10% porcine follicular fluid and eCG and hCG for 22 h, and then cultured in M199 without hormones for 22 h. Each concentration (0, 1, 10, and 100 ng mL−1) of GDF8 was included in M199 during whole process of in vitro maturation. Data were analysed by ANOVA followed by Duncan using SPSS (IBM/SPSS, Armonk, NY, USA). Data are presented as means, and differences were considered significant at P < 0.05. After 44 h of IVM, oocytes were mechanically denuded from CC with 0.1% of hyaluronidase, and then the separated oocytes and CC were sampled following each group. To assess the effect of GDF8 on specific gene transcription level changes as a dose response during IVM, a realtime PCR array was performed. In CC, the 1 and 10 ng mL−1 GDF8 supplement groups showed that transcription levels of transcription co-factors CBP and SP1, cell metabolic regulator MAPK1, and cumulus expansion-related genes Has2, Cox-2, Ptx3, and Areg were significantly different from control when hierarchically clustered by euclidean distance with average linkage method after IVM. In matured oocytes, the 10 and 100 ng mL−1 GDF8 supplement groups showed the maternal factors JMJD3 and Zar1, transcriptional regulator FOXO1, Sirt1, and Sirt2, mitochondrial activity factor Sirt3, ACSL3, and ACADL, anti-apoptosis gene BCL-2, and oocyte secrete factor BMP15 mRNA transcription levels were significantly different compared with control. To determine the effect of GDF8 supplement during IVM, the oocyte maturational regulator AKT protein expression and phosphorylation levels analysed in CC by Western blotting. The 10 and 100 ng mL−1 supplement groups showed significantly increase phosphorylated (P)-AKT per total (T)-AKT (1.22 and 1.18 times higher than control) protein levels (P < 0.05). In conclusion, supplement of GDF8 during IVM activates FOXO homologue transcription and induces cumulus cell expansion via activation of AKT signalling in CC. During the process of IVM, the changes in transcriptional landscape in CC may result in accumulation of maternal factors and mitochondrial activation in oocytes. This work was supported, in part, by a grant from the ‘the Next-Generation BioGreen 21 Program (Project No. PJ011288, PJ011077)’ Rural Development Administration and the ‘National Research Foundation of Korea Grant funded by the Korean Government (NRF-2016R1D1A1B03933191, NRF-2017R1A2B4002546)’, Republic of Korea.

Arsenic Trioxide Targets the Interleukin-6 Cascade in Multiple Myeloma by Promoting Lysosomal Degradation of the Interleukin-6 Receptor Complex.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5159-5159
Author(s):  
Wai Chung Cheung ◽  
Yok Lam Kwong
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Line ◽  
Arsenic Trioxide ◽  
Western Blotting ◽  
Interleukin 6 ◽  
Receptor Complex ◽  
Lysosomal Degradation ◽  
Dose Dependent

Abstract Introduction. Treatment of multiple myeloma (MM), a B-cell neoplasm characterized by clonal expansion of plasma cells in the bone marrow, remains unsuccessful in a significant proportion of patients, so that innovative strategies are needed. Arsenic trioxide (As2O3) has shown notable efficacy against MM in vitro and in clinical studies. Multiple cellular pathways in MM are targeted by As2O3. As cellular growth of MM cells is interleukin-6 (IL-6) dependent, we investigated if As2O3 also targeted the IL-6 cascade. Materials and methods. The IL-6-dependent MM cell line U266 was used as an in vitro model. Cell growth was measured by MTT assay, and apoptosis by flow cytometry. Protein phosphorylation was studied by Western blotting with specific antibodies. Expression of IL-6 receptor (IL-6R) was investigated by Western blotting and flow cytometry. Gene expression was detected by quantitative polymerase chain reaction (Q-PCR). Results. As2O3 showed a time and dose related inhibition of U266 cellular proliferation by induction of apoptosis. At clinically achievable concentrations (2 – 4 μmol/L), As2O3-induced apoptosis was associated with inhibition of constitutive tyrosine phosphorylation of JAK2 and STAT3, in a time and dose-dependent manner. Furthermore, pre-treatment of U266 cells with As2O3 prevented rescue of phosphorylation of JAK2 and STAT3 by exogenous IL-6, implying that the IL-6 cascade was targeted. Using Western blot analysis, we showed that As2O3 induced a time and dose-dependent down-regulation of both components of the IL-6R complex: IL-6R alpha subunit (IL-6Rα) and gp130 signal transducer. These results were confirmed by flow cytometry, showing that As2O3 treatment led to a down-regulation of surface expression of the IL-6Rα. Interestingly, Q-PCR did not reveal any change in the mRNA levels of the two genes with As2O3 treatment, suggesting that As2O3 downregulated IL-6R complex via a post-transcriptional mechanism. It is known that under physiological conditions, the IL-6R complex is internalized upon ligand binding and is targeted to lysosomes for degradation. Treatment of the U266 cell line with the lysosome inhibitor ammonium chloride totally abrogated As2O3-induced degradation of IL-6Rα and gp130. These results suggested that As2O3 might promote lysosomal degradation of IL-6Rα and gp130 by inducing a ligand-independent internalization of the receptor complex. Conclusion. Our results demonstrated that As2O3 suppressed IL-6-induced JAK/STAT3 signaling pathway in MM cells and this might be, at least partly, mediated by promoting ligand-independent internalization and lysosomal degradation of the IL-6R complex. These results have significant implications on the use of As2O3 in the treatment of patients with MM and other malignancies that are IL-6 dependent.

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