In vitro Aggregation Ability of Five Commercially Available Aβ42 Peptides

2021 ◽  
Vol 18 ◽  
Author(s):  
Zhaoji Lv ◽  
Xi Du ◽  
Zhongsheng Chen ◽  
Fengjuan Liu ◽  
Rong Zhang ◽  
...  
Keyword(s):  
Western Blot ◽  
Amyloid Β ◽  
Cell Counting ◽  
Basic Material ◽  
Aggregation Kinetics ◽  
Basic Study ◽  
Sds Page ◽  
Sigmoidal Kinetics ◽  
High Cytotoxicity

Background: As the most basic material, synthetic human Amyloid-β (1-42) (Aβ42) pep- tides from different manufacturers have been widely used. Their aggregation ability is vital to the reliability, repeatability and comparability of studies on Aβ42 physiology and pathology. However, it has not been evaluated and compared. Objectives: To analyze the consistency of the aggregation ability of 5 commercially available Aβ42 peptides. Methods: 5 Aβ42 peptides represented as A, B, C, D and E were pretreated by HFIP. The pretreated Aβ42 peptides were dissolved in Thioflavin T (ThT) solution, and their aggregation kinetics was monitored for 30 h with the aggregation kinetics test. Meanwhile, the pretreated peptides were ag- gregated in phosphate buffered saline. After aggregated for 12 h, they were detected by methods of ThT fluorescence, far-UV circular dichroism (CD), SDS-PAGE, western blot, and transmission electron microscopy (TEM), respectively. After aggregation for 8 h and 12 h, their cytotoxicity to SH-SY5Y cells was further evaluated using Cell Counting Kit-8. Results: For aggregation kinetics, peptides A, C and E remained low level curves, while peptides B and D presented typical sigmoidal kinetics curves. In CD measurement, the aggregates of pep- tides B and D showed relatively high negative CD peaks with the height of -8.09 mdeg and -14.37 mdeg, while the height of peptides A, C and E was -1.04, -3.55, and -3.88. In ThT assay, relative fluorescence intensity of the aggregates of peptides B and D were 7.79 and 8.82, higher than 1.19, 1.71, and 2.70 of peptide A, C and E, respectively. In SDS-PAGE, all aggregates contained monomers and eleven polymers. Moreover, peptide B-E presented a trapezoidal distribution from dimers to trimers, and peptide A aggregated to dimers. By western blot, the bands of monomers re- mained in all aggregates. Furthermore, peptides B and D aggregated to dimers and trimers, pep- tides A and C only aggregated to dimers, and peptide E showed a strong band of trimers. By TEM, protofibrils were observed only in peptide B, while substantial spherical aggregates were formed in other peptides. Additionally, peptides B, D and E exhibited higher cytotoxicity after being aggregat- ed for 8 h, whereas peptides A, B and D presented relatively high cytotoxicity after 12-hour aggre- gation. Conclusion: Commercially available Aβ42 peptides showed obvious differences in aggregation abil- ity, which should arouse enough attention in the field of basic study related to Aβ42. The aggrega- tion ability evaluation with the various assay methods has some discrepancies, and it is highly ur- gent to establish a reasonable and uniform measurement strategy.

scholarly journals MYL6B drives the capabilities of proliferation, invasion, and migration in rectal adenocarcinoma through the EMT process

2020 ◽  
Vol 15 (1) ◽  
pp. 522-531
Author(s):  
Jin-Liang Li ◽  
Zai-Qiu Wang ◽  
Xiao-Li Sun
Keyword(s):  
Western Blot ◽  
Cell Apoptosis ◽  
Rectal Adenocarcinoma ◽  
Drug Application ◽  
Biological Significance ◽  
Expression Level ◽  
Cell Counting ◽  
Migration And Invasion ◽  
Promoting Effect

AbstractObjectiveThis study was designed to explore the biological significance of myosin light chain 6B (MYL6B) in rectal adenocarcinoma.MethodsProfiles on the Oncomine dataset, GEPIA website, and UALCAN-TCGA database were searched to assess the MYL6B expression level in rectal adenocarcinoma tissues and normal tissues. After MYL6B knockdown using siRNA strategy, cell counting kit-8 (CCK-8) and transwell assays were conducted to measure cell proliferation, migration and invasion, respectively. Flow cytometry analysis was conducted to assess cell apoptosis. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot were performed to detect the expression level of mRNAs and proteins.ResultsThe data showed that overexpression of MYL6B was observed in rectal adenocarcinoma tissues and correlated with a poor prognosis of patients. Functional in vitro experiments revealed that MYL6B knockdown could inhibit proliferation, migration, and invasion of rectal adenocarcinoma cells, while promote cell apoptosis. Moreover, western blot analysis suggested that increased expression of E-cadherin and decreased expression of N-cadherin and Vimentin were induced by si-MYL6B.ConclusionIn summary, this study elaborated on the promoting effect of MYL6B in rectal adenocarcinoma progression, thus providing novel insight for strategies of clinical diagnosis and drug application in the future clinical study.

scholarly journals Σύνθεση και μελέτη της δομής και της βιοδραστικότητας οργανοκασσιτερικών ενώσεων με αρυλιμινο υποκατεστημένα βενζοϊκά οξέα

2015 ◽  
Author(s):  
Muvudji Cephas Mwanasaka
Keyword(s):  
Western Blot ◽  
1H Nmr ◽  
13C Nmr ◽  
Sds Page ◽  
Ft Ir

Στα πλαίσια της παρούσας διδακτορικής διατριβής πραγματοποιήθηκε μια διεπιστημονική ερευνητική εργασία, η οποία είχε ως κεντρική ιδέα τη χημεία και τη βιοδραστικότητα των οργανοκασσιτερικών ενώσεων. Για την επίτευξη της μελέτης αυτής, η ερευνητική εργασία κινήθηκε σε τρεις κεντρικούς άξονες: σχεδιασμός και σύνθεση, ανάλυση και ταυτοποίηση και, τέλος, in vitro μελέτη βιολογικών δράσεων. Αρχικά σχεδιάστηκαν και συντέθηκαν ιμίνες (βάσεις Schiff) παράγωγα του βενζοϊκού οξέος, από τη συμπύκνωση του 3- και του 4-αμινοβενζοϊκου οξέος με π-υποκατεστημένες (Η, ΝΟ2, Cl, MeO, Me2N) βενζαλδεΰδες, καθώς και από τη συμπύκνωση των π-υποκατεστημένων (Η, ΝΟ2, Cl, MeO, Me2N) ανιλίνων με π-φορμυλοβενζοϊκό οξύ. Από τις αντιδράσεις αυτές συντέθηκαν βιολογικά ενεργά αρυλιμηνο υποκατεστημένα βενζοϊκά οξέα, τα οποία χρησιμοποιήθηκαν στη συνέχεια της συνθετικής διαδικασίας ως ligands (L) για τη σύνθεση νέων τριοργανοκασσιτερικών εστέρων γενικού τύπου R3SnL με R= Μe και Ph. Η ταυτοποίηση και ο χαρακτηρισμός των νέων αυτών ligands και των συμπλόκων που συντέθηκαν πραγματοποιήθηκε με συνδυασμό αναλυτικών τεχνικών: φασματοσκοπικές τεχνικές (FT-IR, 1H-NMR, 13C-NMR, UV-vis), στοιχειακή ανάλυση και την κρυσταλλογραφία ακτίνων Χ. Επόμενο στάδιο ήταν η in vitro μελέτη βιοδραστικότητας των ενώσεων αυτών η οποία, χωρίστηκε σε δύο σκέλη. Το ένα βιολογικό σκέλος κινήθηκε σε κυτταρικό επίπεδο, όπου αξιολογήθηκε πρώτα η in vitro αποπτωτική δράση σε τέσσερεις καρκινικές κυτταρικές σειρές, ΗepG2, HeLa, K562 και N2a. Για το σκοπό αυτό χρησιμοποιήθηκε η δοκιμή ΜΤΤ (ΜΤΤ assay), η οποία εκτιμά την επίδραση στη βιωσιμότητα των κυττάρων μετά από έκθεσή τους σε διάφορες συγκεντρώσεις των ουσιών. Στη συνέχεια έγιναν νευροκυτταροτοξικές μελέτες, εκτιμώντας την ενδεχόμενη in vitro αναπτυξιακή νευροτοξικότητα. Για το σκοπό αυτό αξιολογήθηκε η επίδραση των υπό μελέτη ουσιών στη διαδικασία ανάπτυξης αξονικών προεκβολών σε υπό διαφοροποίηση Ν2a κύτταρα, μετά από έκθεση σε υποκυτταροτοξικές συγκεντρώσεις των υπό μελέτη ουσιών. Το άλλο βιολογικό σκέλος κινήθηκε σε μοριακό επίπεδο, στοχεύοντας στην εκτίμηση της επίδρασης των υπό μελέτη ουσιών σε συγκεκριμένους βιοχημικούς παράγοντες με σκοπό τη διαλεύκανση των βιοχημικών μηχανισμών δράσεών τους. Εκτιμήθηκε η δράση τους σε συγκεκριμένες πρωτεΐνες του νευροκυτταρικού σκελετού, χρησιμοποιώντας SDS-PAGE και ανοσοχημική τεχνική ανάλυσης Western blot. Επιπλέον, έγινε εκτίμηση της in vitro ανασταλτικής δράσης τους στη λιποξυγονάση, ένζυμο που είναι γνωστό για το ρόλο του στην παραγωγή ελευθέρων ριζών στη φλεγμονή. Τέλος, εκτιμήθηκε η ενδεχόμενη in vitro αντιοξειδωτική δράση τους, αξιολογώντας την ικανότητά τους στην αναστολή της επαγόμενης από το ΑΑΡΗ λιπιδικής υπεροξείδωσης, καθώς και την ικανότητα αλληλεπίδρασής τους με τη σταθερή ρίζα DPPH. Από τα αποτελέσματα που προέκυψαν προσπαθήσαμε να βγάλουμε συμπεράσματα για το κατά πόσο η δομή των καρβοξυλικών οξέων που χρησιμοποιήθηκαν ως Ligands επηρεάζει τόσο τον τρόπο συναρμογής της καρβοξυλικής ομάδας με το τριοργανοκασσιτερικό κατιόν, όσο και τη βιολογική δράση των υπό μελέτη τριοργανοκασσιτερικών εστέρων στις προαναφερόμενες μελέτες.

Knockdown of BAG3 synergizes with olaparib to kill ovarian cancer cells via repressing autophagy

2020 ◽  
pp. jim-2020-001602
Author(s):  
Kexin Wang ◽  
Jianhua Zheng
Keyword(s):  
Ovarian Cancer ◽  
Western Blot ◽  
Transfection Efficiency ◽  
Parp Inhibitor ◽  
Beclin 1 ◽  
Cell Counting ◽  
Ovarian Cancer Cells ◽  
Western Blot Assay ◽  
Related Proteins

This study aimed at expounding the synergistic effect of Bcl-2-associated athanogene 3 (BAG3) knockdown and poly ADP-ribose polymerase (PARP) inhibitor on ovarian cancer (OC) cells and the potential mechanism. Short hairpin RNA (shRNA) targeting BAG3 (sh-BAG3) was transfected into SK-OV-3 (SKOV-3 ;SKOV3) and A2780 cells, and western blot assay was used to detect transfection efficiency. Cell proliferation and apoptosis were detected by the cell counting kit-8 method, 5-Bromodeoxyuridine (BrdU) experiment and flow cytometry analysis, respectively. The expressions of apoptosis-related proteins Bax and Bcl-2, as well as the expressions of autophagy-related proteins LC3-I, LC3-II and Beclin-1, were examined by western blot assay. Additionally, the cells were treated with autophagy activator rapamycin to investigate whether the tumor-suppressive function of BAG3 knockdown+PARP inhibitor was dependent on autophagy. In this work, we demonstrated that BAG3 knockdown further sensitized OC cells to olaparib treatment, reducing cellular viability and promoting apoptosis. Both sh-BAG3 and olaparib decreased the expression of Beclin-1 and the LC3-Ⅱ:LC3-I ratio, and their synergism further inhibited the process of autophagy. However, the aforementionede effects were reversed after the cells were treated with rapamycin. Based on these results, we concluded that BAG3 knockdown synergizes with olaparib to kill OC cells in vitro by repressing autophagy.

Mediation of multiple pathways regulating cell proliferation, migration, and apoptosis in the human malignant glioma cell line U87MG via unphosphorylated STAT1

2013 ◽  
Vol 118 (6) ◽  
pp. 1239-1247 ◽  
Author(s):  
Haitao Ju ◽  
Xin Li ◽  
Hong Li ◽  
Xiaojuan Wang ◽  
Hongwei Wang ◽  
...  
Keyword(s):  
Cell Growth ◽  
Western Blot ◽  
Caspase 3 ◽  
Apoptotic Cell ◽  
Glioma Cell Line ◽  
Significant Inhibition ◽  
Cell Counting ◽  
Nuclear Antigen ◽  
Normal Brain

Object Signal transducer and activator of transcription 1 (STAT1) is thought to be a tumor suppressor protein. The authors investigated the expression and role of STAT1 in glioblastoma. Methods Immunohistochemistry was used to detect the expression of STAT1 in glioblastoma and normal brain tissues. Reverse transcription–polymerase chain reaction and Western blot analysis were used to detect mRNA and protein expression levels of STAT1. Cell growth, proliferation, migration, apoptosis, and the expression of related genes and proteins (Bcl-2, Bax, cleaved caspase-3, caspase-9, p21, and proliferating cell nuclear antigen) were examined in vitro via cell counting kit-8, wound-healing, flow cytometry, Rhodamine B, TUNEL, and Western blot assays. Results Human glioblastoma had decreased expression of STAT1 proteins. Transfection of the U87MG cells with STAT1 plasmid in vitro demonstrated significant inhibition of cell growth and an increase in apoptotic cell death compared with cells transfected with vector or mock plasmids. These effects were associated with the upregulation of cleaved caspase-3, Bax, and p21 and the downregulation of Bcl-2 expression. Conclusions The results of this study suggest that increased expression of STAT1 by transfection with STAT1 plasmid synergistically inhibits human U87MG glioblastoma cell growth in vitro.

scholarly journals Hypoxia-Related Gene FUT11 Promoted Pancreatic Cancer Progression Via Maintaining The Stabilization of PDK1

2021 ◽  
Author(s):  
Wenpeng Cao ◽  
Zhirui Zeng ◽  
Runsang Pan ◽  
Zhiwei He ◽  
Hao Wu ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cell Growth ◽  
Western Blot ◽  
Cancer Progression ◽  
Pyruvate Dehydrogenase Kinase ◽  
Cell Counting ◽  
Luciferase Assay ◽  
Migration And Invasion

Abstract Background: Hypoxia participated in the occurrence and development of pancreatic cancer (PC). However, genes associated with hypoxia respond and their regulated mechanism in PC cells were unclear. The current research was aimed to illuminate the role and hypoxia regulated mechanism of fucosyltransferase 11 (FUT11) in the progression of PC.Methods: After predicting FUT11 as a key hypoxia associated gene in PC using bioinformatics analysis. The expression of FUT11 in PC using quantitative real-time fluorescent PCR, western blot and immunohistochemistry. The effects of FUT11 on PC cells proliferation, migration and invasion under normoxia and hypoxia were detected using Cell Counting Kit 8, 5-ethynyl-2’-deoxyuridine assay, colony formation assay and transwell assay. Spleen capsule injected liver metastasis and subcutaneously injected model were performed to confirm the effects of FUT11 in vivo. Furthermore, western blot, luciferase assay and immunoprecipitation were performed to explore the regulated relationship among FUT11, hypoxia-inducible factor 1α (HIF1α) and pyruvate dehydrogenase kinase 1 (PDK1) in PC.Results: FUT11 was markedly increased of PC cells in hypoxia, up-regulated in the PC clinical tissues, and predicted a poor outcome. Inhibition of FUT11 reduced PC cell growth and mobility of PC cells under normoxia and hypoxia conditions in vitro, and growth and mobility in vivo. FUT11 bind with PDK1 and regulated the expression PDK1 under normoxia and hypoxia. FUT11 knockdown significantly increased the degradation rate of PDK1 under hypoxia, while treatment with MG132 can relieve the degradation of PDK1 induced by FUT11 knockdown. Overexpression of PDK1 in PC cells under hypoxia conditions reversed the suppressiv impacts of FUT11 knockdown on PC cell growth and mobility. In addition, HIF1α bound to the enhancer of FUT11 and increased its expression, as well as co-expressing with FUT11 in PC tissues. Furthermore, overexpress of FUT11 partially rescued the suppressiv effects of HIF1α knockdown on PC cell growth and mobility in hypoxia conditions.Conclusion: Our data further implicate that hypoxia-induced FUT11 in PC contributes to proliferation and metastasis by maintaining the stability of PDK1, and suggest FUT11 maybe a novel and effective target for treatment of pancreatic cancer.

scholarly journals The Protective Effects of Κ-Opioid Receptor Stimulation in Hypoxic Pulmonary Hypertension Involve Inhibition of Autophagy Through the AMPK-MTOR Pathway

2017 ◽  
Vol 44 (5) ◽  
pp. 1965-1979 ◽  
Author(s):  
Yaguang Zhou ◽  
Yuanbo Wang ◽  
Xu Wang ◽  
Xin Tian ◽  
Shumiao Zhang ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Western Blot ◽  
Opioid Receptor ◽  
Mtor Pathway ◽  
Cell Counting ◽  
Protective Effects ◽  
Receptor Stimulation ◽  
Hypoxic Pulmonary Hypertension ◽  
Pulmonary Arterial

Background/Aims: In a previous study, we showed that κ-opioid receptor stimulation with the selective agonist U50,488H ameliorated hypoxic pulmonary hypertension (HPH). However, the roles that pulmonary arterial smooth muscle cell (PASMC) proliferation, apoptosis, and autophagy play in κ-opioid receptor-mediated protection against HPH are still unknown. The goal of the present study was to investigate the role of autophagy in U50,488H-induced HPH protection and the underlying mechanisms. Methods: Rats were exposed to 10% oxygen for three weeks to induce HPH. After hypoxia, the mean pulmonary arterial pressure (mPAP) and the right ventricular pressure (RVP) were measured. Cell viability was monitored using the Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis was detected by flow cytometry and Western blot. Autophagy was assessed by means of the mRFP-GFP-LC3 adenovirus transfection assay and by Western blot. Results: Inhibition of autophagy by the administration of chloroquine prevented the development of HPH in the rat model, as evidenced by significantly reduced mPAP and RVP, as well as decreased autophagy. U50,488H mimicked the effects of chloroquine, and the effects of U50,488H were blocked by nor-BNI, a selective κ-opioid receptor antagonist. In vitro experiments showed that the inhibition of autophagy by chloroquine was associated with decreased proliferation and increased apoptosis of PASMCs. Under hypoxia, U50,488H also significantly inhibited autophagy, reduced proliferation and increased apoptosis of PASMCs. These effects of U50,488H were blocked by nor-BNI. Moreover, exposure to hypoxic conditions significantly increased AMPK phosphorylation and reduced mTOR phosphorylation, and these effects were abrogated by U50,488H. The effects of U50,488H on PASMC autophagy were inhibited by AICAR, a selective AMPK agonist, or by rapamycin, a selective mTOR inhibitor. Conclusion: Our data provide evidence for the first time that κ-opioid receptor stimulation protects against HPH by inhibiting PASMCs autophagy via the AMPK-mTOR pathway.

scholarly journals Expression of Grapevine leafroll-associated virus 3 coat protein gene in Escherichia coli and production of polyclonal antibodies

2007 ◽  
Vol 32 (6) ◽  
pp. 496-500 ◽  
Author(s):  
Thor V.M. Fajardo ◽  
Danielle R. Barros ◽  
Osmar Nickel ◽  
Gilmar B. Kuhn ◽  
F. Murilo Zerbini
Keyword(s):  
Escherichia Coli ◽  
Coat Protein ◽  
Western Blot ◽  
Coat Protein Gene ◽  
Protein Gene ◽  
Polyclonal Antibodies ◽  
Yield And Quality ◽  
Sds Page ◽  
Grapevine Leaves

Grapevine leafroll-associated virus 3 (GLRaV-3), the main viral species of the grapevine leafroll complex, causes yield and quality reduction in grapes (Vitis spp.). The coat protein gene was RT-PCR-amplified from total RNA extracted from infected grapevine leaves and the amplified fragment was cloned and completely sequenced. The fragment was subsequently subcloned into the pRSET-C expression vector. The recombinant plasmid was used to transform Escherichia coli BL21:DE3 and express the capsid protein. The coat protein, fused to a 6 His-tag, was purified by affinity chromatography using an Ni-NTA resin. The identity of the purified protein was confirmed by SDS-PAGE and Western blot. The in vitro-expressed protein was quantified and used for rabbit immunizations. The antiserum was shown to be sensitive and specific for the detection of GLRaV-3 in grapevine extracts in Western blot and DAS-ELISA assays, with no unspecific or heterologous reactions against other non-serologically related viruses being observed.

scholarly journals Producción y evaluación inmunoquímica de extractos alergénicos del ácaro del polvo doméstico Blomia tropicalis (Acari: Echymyopodidae)

2018 ◽  
Vol 21 ◽  
Author(s):  
Luis Acuña-Cantillo ◽  
Dary Luz Mendoza-Meza ◽  
Gloria Garavito ◽  
Ana Sofía Moreno-Woo ◽  
Eduardo Egea-Bermejo
Keyword(s):  
Western Blot ◽  
Blomia Tropicalis ◽  
Sds Page

Los alérgenos de Blomia tropicalis son un factor de riesgo para el desarrollo de alergias en países tropicales. Los extractos alergénicos son reactantes valiosos para el diagnóstico de la hipersensibilidad alérgica a los ácaros y su control biológico. El objetivo fue evaluar las propiedades inmunoquímicas de extractos de B. tropicalis producidos desde cultivos in vitro. Este trabajo no tuvo como propósito producir extractos de aplicación clínica. Los ejemplares de B. tropicalis se establecieron a partir de ácaros aislados del polvo doméstico en la ciudad de Barranquilla, Colombia. Los ácaros libres de medio de cultivo se usaron para la obtención de los extractos. El rendimiento de la extracción se determinó mediante ensayo Bradford y el perfil electroforético por SDS-PAGE. La presencia de enzimas hidrolasas se determinó por Api® Zym y la inmunogenicidad se evaluó mediante Western blot donde se identificaron proteínas de unión a IgE sérica. La producción máxima de ácaros en los cultivos fue de 0,9 g/300 g medio. El rendimiento de extracción de proteínas fue de 3,61 % (± 0,42). Todos los extractos presentaron actividad enzimática tripsina, alfa-amilasa y quimotripsina. Western blot confirmó la presencia de proteínas de unión a IgE sérica. Presentamos un método sencillo para obtener extractos del ácaro B. tropicalis con actividad enzimática y alergénica.

scholarly journals Disulfram/Copper Complex May Enhance Its Cytotoxic Effect on CD34+CD38- KG1-Alpha Cells By Down Regulating the Expression of HIF1-Alpha in the Co-Cultured MSCs

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5143-5143
Author(s):  
Guo Xutao ◽  
Haiqing Zheng ◽  
Shi Pengcheng ◽  
Wang Yan ◽  
Liang Jiabao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Western Blot ◽  
Copper Complex ◽  
Cell Apoptosis ◽  
Untreated Control ◽  
Cytotoxic Effect ◽  
Cultured Cells ◽  
Cell Counting

Abstract Backgroud We had reported that Disulfram/copper complex (DS/ Cu) had a potent and selective anti-leukemia property in vitro against leukemia stem-like cells (e.g., CD34+/CD38- KG1α and Kasumi-1 cells and primary CD34+ cells isolated from AML patients) as well as was highly effective in vivo in CD34+/CD38- leukemic cell-derived xenograft mouse models. Here, we report the in vitro activity of DS/Cu against CD34+/CD38- leukemia stem-like cells sorted from KG1α AML cell line by affecting the co-cultured AML bone marrow mesenchymal stromal cells. Methods KG1α cells were cultured and stained with hCD34-APC, hCD38- PE, and hCD123-PE-Cy7 to determine the percentage of cells expressing CD34, CD38, and CD123. To isolate CD38- cells, CD38 microbeads were used for the depletion of CD38+ cells. First, we examined the cytotoxic effect of DS/Cu on CD34+CD38- KG1α cells and MSCs using the Cell Counting Kit-8 (CCK-8) and monitor expression of HIF-1α expression by western blot. Next, the sorted CD34+CD38- KG1α cells were co-cultured with primary AML bone marrow MSCs isolated from a de novo AML patient in the hypoxic condition induced by Cobalt Chloride (CoCl2) for 24 hr. The co-cultured cells were treated with DS/CU with/without YC1(HIF-1α inhibitor)、IDA(idarubicin) and then separated. CCK8 assay, fow cytometry were used to determine the cell proliferation and apoptosis, respectively. Finally, we used western bolting to explore the underlying mechanism of the cytotoxicity of DS/CU in the AML cells and the sensitizated effect in AML MSCs. Results First, we examined the cytotoxic effect of DS/Cu on CD34+CD38- KG1α cells and MSCs using the Cell Counting Kit-8 (CCK-8). As shown in Figure 1, DS/Cu (DS 1umol/L,Cu 0.5umol/l) administrated alone was unable to induce apoptosis in MSCs (P>0.05 vs untreated control; see below Figure 1). However, DS in combination with 0.5 μM Cu for 24 hrs resulted in signifcantly increased apoptosis in CD34+CD38- KG1α cells.( P<0.001 vs untreated control; see Figure 1) .The co-cultured cells were treated with DS/CU、IDA with/without YC1 and then separated. CCK8 assay, fow cytometry were used to determine the cell apoptosis, respectively. Treatment with DS/CU resulted in signifcantly increased apoptosis in the co-cultured CD34+/CD38- KG1α cells (Figure 2; P<0.05, DS/Cu vs either untreated control). Comparable phenomena were observed in co-cultured CD34+/CD38- KG1α cells treated with idarubicin. However, while treatment with YC1 and DS/CU had no signifcant effect on cell apoptosis (P>0.05 vs untreated control). Taken together, these results indicated that DS/CU was active against co-cultured CD34+/CD38- KG1α cells and the effect can be reversed by HIF1-α inhibitor(see Figure 2).We examined the cytotoxic effect of DS/Cu on CD34+/CD38- KG1α cells in different culture systems using the CCK-8. As shown in Figure 4, while treatment with DS/CU had significant inhibited proliferation on cell proliferation of the co-cultured CD34+/CD38- KG1α cells (P<0.05 vs un-co-cultured control). However, while treatment with DS/CU and CoCl2 (HIF-1α promoter) had no signifcant effect on cell proliferation of the co-cultured CD34+/CD38- KG1α cells (P>0.05 vs un-co-cultured control,see Figure 3).CD34+CD38- KG1α cells and MSCs separated from the co-cultured system were treated with DS/CU with/without YC1 and Cocl2, followed by Western blot analysis to monitor expression of HIF-1α. As shown at the figure 4, the expressions of HIF-1α and SDF-1α between the two cells were clearly down-regulated treated with DS/CU alone. And the expressions of HIF-1α and SDF-1α were up-regulated treated with DS/CU and CoCl2. However, when co-treated with YC1, the down-regulation of HIF-1α was reverse. DS/Cu down redulated the expressions of HIF1-α、SDF1-α、CXCR4 and VLA4 in the co-cultured CD34+CD38- KG1α cells and these effect could be reserved by HIF1-α inhibitor.Conclusion DS/Cu preferentially induces apoptosis of CD34+CD38- KG1α cells, but not MSCs. DS/Cu exhibited cytotoxicity in CD34+/CD38- KG1α cells, and MSCs enhances this effect. The mechanism may be related to the HIF1α/SDF1/CXCR4 and VLA4 pathway. DS/CU may enhance its inhibitory effect on CD34+CD38- KG1α cells by down regulating the expression of HIF1-α in MSCS. Disclosures No relevant conflicts of interest to declare.

scholarly journals Circ_0002060 enhances doxorubicin resistance in osteosarcoma by regulating miR-198/ABCB1 axis

2020 ◽  
Author(s):  
Jun Liu ◽  
Wenshuai Zhu ◽  
Jianqin Ji
Keyword(s):  
Western Blot ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Western Blot Assay ◽  
Doxorubicin Resistance ◽  
Kaplan Meier ◽  
Rna Immunoprecipitation ◽  
Resistance To Doxorubicin

Abstract Background Osteosarcoma (OS) is a common aggressive primary sarcoma of bone. Drug resistance is a huge obstacle to chemotherapy for cancer. This study aimed to investigate the role and mechanism of circ_0002060 in OS resistance to doxorubicin (DOX). Methods The levels of circ_0002060, miR-198 and ATP binding cassette subfamily B member 1 (ABCB1) were measured by quantitative real-time polymerase chain reaction or western blot assay. Kaplan-Meier analysis was performed to determine the relationship between circ_0002060 expression and overall survival. The half inhibition concentration (IC50) of doxorubicin was calculated by Cell Counting Kit-8 (CCK-8) assay. Cell proliferation was assessed by colony formation assay. Cell apoptosis was monitored by flow cytometry. The levels of apoptosis-related proteins were measured by western blot assay. Xenograft assay was utilized to analyze the effect of circ_0002060 on DOX resistance in vivo . The interaction among circ_0002060, miR-198 and ABCB1 were confirmed by dual-luciferase reporter assay, RNA immunoprecipitation assay or RNA pull-down assay. Results Circ_0002060 and ABCB1 were up-regulated, while miR-198 was down-regulated in OS tissues and DOX-resistant OS cells. Circ_0002060 silence reduced DOX resistance in vitro and in vivo . Moreover, circ_0002060 enhanced DOX resistance via sponging miR-198. Besides, miR-198 decreased DOX resistance by binding to ABCB1. In addition, circ_0002060 sponged miR-198 to up-regulate ABCB1 expression. Conclusion Circ_0002060 enhanced doxorubicin resistance of OS by regulating miR-198/ABCB1 axis, which provides potential therapeutic targets for OS therapy.

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