330 REDUCED HYPERACUTE REJECTION BY TRIPLE TRANSGENIC EXPRESSION OF HUMAN COMPLEMENT REGULATORY FACTORS (hDAF and hCD59) AND H-TRANSFERASE

2011 ◽  
Vol 23 (1) ◽  
pp. 261
Author(s):  
Y. H. Jeong ◽  
G. H. Jang ◽  
I. S. Hwang ◽  
C. H. Park ◽  
H. J. Lee ◽  
...  
Keyword(s):  
Human Serum ◽  
Cell Line ◽  
Cell Lines ◽  
Expression Vector ◽  
Inhibitory Effect ◽  
Hyperacute Rejection ◽  
Stable Cell Line ◽  
Control Group ◽  
Human Complement ◽  
Transgenic Cell

The present study was conducted to establish a porcine transgenic cell line with human CRPs and HT genes, focused on hyperacute rejection (HAR) considering clinical xenotransplantation as alternative sources of human organs. As a first step towards establishing the stable cell line, the cDNA for 3 genes encoding human DAF, CD59, and H-transferase were cloned and sequenced. A tricistronic expression vector was constructed with the aid of 2 IRES elements (pCMV-hDAF_IRES-hHT_IRES-hCD59). The CMV-based expression vector was then introduced into miniature pig ear fibroblast cells by electroporation. Reverse transcription PCR analysis revealed that cell lines stably expressing human transgene-specific transcripts were established. The inhibitory effect of immune response in the established transgenic cell lines was measured by human serum-mediated cytolysis assay, as measured by ELISA. Under the assay conditions (based on human serum from 10 to 50%), the transgenic cell group showed significantly greater survival rate under various serum concentrations than did the nontransgenic cell control group. Moreover, the transgenic cell lines used as nuclear donors for a subsequent NT experiment were confirmed to be expressing their transgene transcripts in vitro developed preimplantation stage embryos. These results indicated that the established cell lines with human transgenes might have an inhibitory effect against lysis by human complement. It is possible that these transgenic cells could serve as nuclear donors to produce transgenic cloned pigs for xenotransplantation.

184 INHIBITION OF HUMAN COMPLEMENT-MEDIATED CYTOTOXICITY IN MINIPIG CELLS BY EXPRESSION OF hCD59

2008 ◽  
Vol 20 (1) ◽  
pp. 172
Author(s):  
K. W. Park ◽  
E. J. Kim ◽  
K. M. Choi ◽  
S. P. Hong ◽  
G. S. Han ◽  
...  
Keyword(s):  
Cell Lines ◽  
Membrane Attack Complex ◽  
Inhibitory Effect ◽  
Negative Control ◽  
Control Group ◽  
Human Complement ◽  
Transgenic Pigs ◽  
Clonal Cell ◽  
Fetal Fibroblasts ◽  
Clonal Cell Lines

Xenotransplantation of a pig organ to a human is a possible solution for the shortage of donor organs for transplantation. However, hyperacute rejection (HAR) due to natural antibodies (Nab) present major obstacle in pig-to-human xenotransplantation. To overcome this, much effort has been dedicated to preparing transgenic pigs that express human complement regulatory proteins (CRPs). One of CRPs, the human CD59 gene, can prevent the terminal polymerization of the membrane attack complex by complement. In this study, we investigated the inhibitory effect of hCD59 on complement-mediated cytotoxicity in hCD59-transfected minipig cells. To generate cell lines expressing hCD59, we transfected the hCD59 gene into minipig fetal fibroblasts and established seven transgenic clonal cell lines. The integration of hCD59 gene was confirmed by PCR and expression levels were measured by RT-PCR, fluorescence-activated cell sorting (FACS), Western blot, and immunohistochemistry. FACS analysis of hCD59 clonal cell lines demonstrated a substantial increment of hCD59 expression. The level (82% to 95%) of hCD59 protein expression was increased relative to that of the control. Human complement-mediated cytotoxicity was measured using a CytoTox96� (Promega Corp., Sydney, Australia) non-radioactive cytotoxicity (LDH) assay using normal minipig cells as a negative control. In the LDH release assay, human complement-mediated cytotoxicity was also significantly reduced to 39.6 � 17.8% in comparison to that of the control group (73.6 � 19.1%; P < 0.05; n = 6). These results indicate that the expression of hCD59 gene in minipig cells can efficiently control complement-mediated cytotoxicity.

scholarly journals In vitro cytotoxic study for partially purified resveratrol extracted from grape skin fruit Vitis vinifera

2009 ◽  
Vol 3 (2) ◽  
pp. 40-47
Author(s):  
Zainab Y. Mohammed ◽  
Essam F. Al-Jumaily ◽  
Nahi Y. Yaseen
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Cytotoxic Effect ◽  
Inhibitory Effect ◽  
Mammary Adenocarcinoma ◽  
Dependent Manner ◽  
Grape Skin ◽  
Grape Fruit ◽  
Glass Column

The partial purified resveratrol was obtained from the skin of black grape fruit cultivated in Iraq using 80% ethanolic solution, then an acid hydrolysis with 10% HCl solution for (10–30) min at 60Cº was carried out. The aglycone moiety was taken with an organic solvent (chloroform), then using an open glass column packed with silica gelG 60 as a stationary phase and a mobile phase of; benzene: methanol: actic acid (20:4:1). The study utilized an in vitro evaluation for the cytotoxic effect of the partially purified resveratrol on some cell lines including, the murine mammary adenocarcinoma (Ahmed –Mohammed –Nahi–2003 -AMN -3) cell line; the human laryngeal carcinoma (Hep -2) cell line and the Rat Embryo Fibroblast (REF) cell line at different concentrations and different exposure time of treatment. The partial purified resveratrol extract concentrations ranging (7.8–4000) µg/ml in a two fold serial dilutions were used to treat the three types of cell lines for 48 and 72 hours intervals. AMN-3 cell lines showed highest sensitivity toward the cytotoxic effect of the paritial purified resveratrol than other cell lines after 48 hours in a dose dependent manner. While Hep-2 cell line showed novel behavior, the lowest concentration of cell treatment gave the most significant (P< 0.01) inhibitory effect. Only the highest concentration gave significant inhibitory effect (P< 0.01) with the transformed Ref cell line.

scholarly journals Interaction of Arylidenechromanone/Flavanone Derivatives with Biological Macromolecules Studied as Human Serum Albumin Binding, Cytotoxic Effect, Biocompatibility Towards Red Blood Cells

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3172 ◽  
Author(s):  
Angelika A. Adamus-Grabicka ◽  
Magdalena Markowicz-Piasecka ◽  
Michał B. Ponczek ◽  
Joachim Kusz ◽  
Magdalena Małecka ◽  
...  
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Red Blood Cells ◽  
Human Serum ◽  
Cell Line ◽  
Cell Lines ◽  
Blood Cells ◽  
Cytotoxic Effect ◽  
Human Leukemia ◽  
Ic50 Values

The aim of this study was to determine the cytotoxic effect of 3-arylidenechromanone (1) and 3arylideneflavanone (2) on HL-60 and NALM-6 cell lines (two human leukemia cell lines) and a WM-115 melanoma cell line. Both compounds exhibited high cytotoxic activity with higher cytotoxicity exerted by compound 2, for which IC50 values below 10 µM were found for each cell line. For compound 1, the IC50 values were higher than 10 µM for HL-60 and WM-115 cell lines, but IC50 < 10 µM was found for the NALM-6 cell line. Both compounds, at the concentrations close to IC50 (concentration range: 5–24 µM/L for compound 1 and 6–10 µM/L for compound 2), are not toxic towards red blood cells. The synthesized compounds were characterized using spectroscopic methods 1H- and 13C-NMR, IR, MS, elemental analysis, and X-ray diffraction. The lipophilicity of both synthesized compounds was determined using an RP-TLC method and the logP values found were compared with the theoretical ones taken from the Molinspiration Cheminformatics (miLogP) software package. The mode of binding of both compounds to human serum albumin was assessed using molecular docking methods.

scholarly journals Synthesis and Anticancer Activity Evaluation of Azepinobisindoles; the Isomeric Iheyamine A Derivatives

2019 ◽  
Vol 35 (2) ◽  
pp. 723-731
Author(s):  
Weerachai Phutdhawong ◽  
Sopita Rattanopas ◽  
Jitnapa Sirirak ◽  
Thongchai Taechowisan ◽  
Waya S. Phutdhawong
Keyword(s):  
Anticancer Activity ◽  
Cell Line ◽  
Cell Lines ◽  
Human Cancer ◽  
Docking Study ◽  
Inhibitory Effect ◽  
Molecular Docking Study ◽  
Human Cancer Cell Lines ◽  
Activity Evaluation ◽  
Cancer Line

Azepinobisindole derivatives, the isomeric Iheyamine skeleton, was prepared and its anticancer activity evaluation were investigated against two human cancer cell lines, Hepatocellular carcinoma (HepG2) and human cervical cancer line (Hela) as well as the normal cell line (Vero cell line) using MTT assay. The anticancer activity results indicated that 2-methoxy-5-methyl-5H-azepino[2,3-b:4,5-bʹ]diindole was the most active derivative against tested cell lines. Additionally, molecular docking study in silico the possible inhibitory effect of cyclin-dependent kinase 2 (CDK2) by the azepinoindole revealed that all synthesized compounds fit well in the binding cavity of CDK2.

Cytotoxicity Assessment of Quassinoids Neosergeolide and Isobrucein B toward Hematopoietic and Solid Malignancies Identifies STAT3 Activation To Be Predictive of Antitumor Response.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1394-1394
Author(s):  
Mitsuteru Hiwatari ◽  
Jingqiu Dai ◽  
Wei Liu ◽  
Yu-Dong Zhou ◽  
Dale G. Nagle ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cytotoxic Activity ◽  
Cell Line ◽  
Cell Lines ◽  
Strong Support ◽  
Luciferase Reporter ◽  
Control Group ◽  
Antitumor Effects ◽  
Protein Levels ◽  
Ic50 Values

Abstract Quassinoids are natural product compounds known to possess tumor cytotoxicity and antimalarial activity. Neosergiolide and isobrucein B are two quassinoids previously isolated from roots and stems of Picrolemma sprucei. In screening studies to identify inhibitors that target STAT3, we discovered neosergeolide and isobrucein B as active compounds. Approximately 5000 plant-derived extracts were screened using a cell line that stably expresses a STAT3-dependent luciferase reporter and NPM-ALK, which constitutively induces STAT3 transcriptional activity. Of 25 total hits, a P. sprucei extract was potent and selective for STAT3 inhibition, and bioassay-guided isolation identified neosergeolide and isobrucein B as the inhibitory compounds. Western blot analysis confirmed that neosergeolide and isobrucein B not only inhibit the tyrosine phosphorylation and activation of STAT3 but also decrease total STAT3 protein levels via a mechanism due in part to enhanced proteasome-mediated degradation. Small-molecule proteasome inhibitors such as MG132 and ALLN reversed the ability of the two quassinoids to decrease STAT3 protein levels; furthermore, simultaneous incubation of various hematopoietic malignancy cell lines with either neosergeolide or isobrucein B and MG132 or ALLN antagonized the cytotoxic activity of the quassinoids. Assessment of neosergiolide and isobrucein B antitumor effects using an XTT assay revealed both compounds to possess potent cytotoxic activity across a broad spectrum of hematopoietic malignancies, with T-leukemias/lymphomas being especially responsive. For example, mycosis fungoides (MF)- and Sezary syndrome (SS)-derived cell lines, as well as non-MF/SS cutaneous T-cell lymphoma (CTCL) lines, were potently inhibited by both quassinoids (neosergiolide IC50 values: MAC-1, 11.6 nM; MAC-2A, 6.9 nM; Hut-78, 6.6 nM; HH, 4.3 nM; MJ, 7.0 nM; isobrucein B IC50 values: MAC-1, 31.9 nM; MAC-2A, 72.3 nM; Hut-78, 23.5 nM; HH; 20.3 nM; MJ, 13.5 nM). Non-hematopoietic cell lines representing various solid tumors also exhibited potent cytotoxic responses to the quassinoids (e.g., gastric carcinoma line AGS [neosergiolide IC50: 16.9 nM; isobrucein B IC50: 114.9 nM]). With rare exceptions, the cytotoxicity of the quassinoids against a specific tumor cell line correlated with STAT3 activation status; for example, breast cancer line MCF7 with inactive STAT3 was resistant to both quassinoids even at the maximum concentration tested (6.25 μM), whereas breast cancer lines MDA-MB-468 and MDA-MB-435s with activated STAT3 were inhibited by both compounds at low concentrations (neosergiolide IC50: MDA-MB-435s, 31.3 nM; MDA-MB-468, 29.9 nM; isobrucein B IC50: MDA-MB-435s, 209.3 nM; MDA-MB-468, 356.8 nM). The in vitro antitumor activity of the two quassinoids could also be demonstrated in vivo. For example, isobrucein B (1.0 mg/kg IP once q 3d x 5 doses) could be safely administered and potently inhibited the growth in SCID mice of the CD30+ primary CTCL MAC-1 cell line; mice at treatment day 16 showed average subcutaneous tumor volumes of 3839 ± 863 (s.e.) mm3 in the vehicle-control group and 913 ± 349 (s.e.) mm3 in the isobrucein B group (P=0.008, t-test). These results provide strong support for STAT3 targeting in antitumor drug discovery and suggest that quassinoids may have utility in such an approach.

Autophagic and Apoptotic Effects of Tyrosine Kinase Inhibitors in Myeloid Leukemia: Comparison of Three Generation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4915-4915
Author(s):  
Cagla Kayabasi ◽  
Cigir Biray Avci ◽  
Sunde Yilmaz Susluer ◽  
Tugce Balci ◽  
Yusuf Baran ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
Tyrosine Kinase Inhibitors ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Fluorescent Protein ◽  
Control Group ◽  
Green Fluorescent

Abstract Abstract 4915 The aim of the study was to evaluate the differences in cytotoxicity, apoptosis and autophagy levels in myeloid leukemia cell lines treated with tyrosine kinase inhibitors compared to cell line resistant to imatinib and control group. Chronic myeloid leukemia model was created by using cell lines as K-562 cell line for Ph+ chronic myeloid leukemia model, HL-60 cell line for acute promyelocytic Ph- leukemia model. NCI-BL2171 normal cell line was used as a control group while K562/ima3 cell line was used as an imatinib resistant model. Imatinib (STI571), Dasatinib (BMS-354825), Ponatinib (AP24534) were used as tyrosine kinase inhibitors in this study. Cytotoxicity analysis was conducted by WST-1 analysis. Apoptotis was evaluated by AnnexinV-enhanced green fluorescent protein (EGFP) and by Mitoprobe JC-1 for Mitochondrial Potential Detection. Autophagy was analyzed by The Premo Autophagy Tb/GFP TR-FRET LC3B assay which measures autophagy in cells expressing green fluorescent protein (GFP)-tagged LC3B using a Tb-based TR-FRET immunoassay approach. By using IC50 doses of tyrosine kinase inhibitors, autophagic effect of these drugs on cell lines were examined at 24th hours. Cells not treated with the active substance or chloroquine were considered as control groups. Chloroquine-treated cells were used as positive control for autophagy. LC3B-II increase is an indicator of autophagic suppression. Cells treated with chloroquine were compared with cells treated with active substances and concentrations of BacMam that displayed the highest LC3B-II increase were selected. Autophagic suppression ratio of the drugs was evaluated among the control group. Cytotoxicity, apoptosis and autophagy analysis results were provided in Table. Compared to control group, 30 μM chloroquine repressed autophagy 1. 93, 1. 48, 2. 74 and 1. 54 fold in K562, HL-60, K562/ima3 and NCI-BL 2171 cells, respectively. In HL-60 cells while Imatinib represented 0. 77 fold autophagy, it repressed autophagy 1. 77 and 3. 49 fold in K562 and K562/ima3 cells respectively. Dasatinib repressed autophagy 2. 11, 1. 95 and 4. 62 fold and Ponatinib repressed autophagy 2. 09, 1. 60 and 9. 15 fold in K562, HL-60, K562/ima3 cells respectively. Imatinib, Dasatinib and Ponatinib did not repressed autophagy in NCI-BL 2171 cells. In conclusion, apoptosis and autophagy paradox was illuminated in myeloid leukemia cells via tyrosine kinase inhibitors and autophagy may be a new strategy for targeted therapy in myeloid leukemia after clarifying responsible genes and proteins in signal transduction pathways. Cytotoxicity Apoptosis Autophagy WST-1 IC50 (nM) Annexin V JC-1 Premo Autophagy Ýmatinib Dasatinib Ponatinib Ýmatinib Dasatinib Ponatinib Ýmatinib Dasatinib Ponatinib Ýmatinib Dasatinib Ponatinib K562 24th hour 1.70 3.65 3.05 3.07 1.37 1.35 1.43 1.77 2.11 2.09 48th hour 650.00 0.24 2.67 2.51 2.32 2.03 2.35 2.06 72nd hour 4.53 4.81 3.00 2.97 3.07 2.50 HL-60 24th hour 1.33 1.26 1.32 1.29 1.22 1.34 0.77 1.95 1.60 48th hour 18000.00 1.39 1.23 1.41 1.61 1.92 1.96 72nd hour 896.00 607.00 2.21 1.80 2.82 1.58 1.73 2.23 K562/ima3 24th hour 1.33 0.76 1.69 1.51 1.36 1.59 3.49 4.62 9.15 48th hour 18350.00 1830.00 9.87 1.80 1.94 2.03 2.82 1.22 1.40 72nd hour 1.34 1.44 1.41 2.61 1.40 1.56 NCI-BL 2171 24th hour 48.00 2.48 2.79 2.62 3.99 4.04 4.25 1.01 0.88 0.90 48th hour 274.00 30.00 4.11 4.33 4.15 5.05 2.75 3.11 72nd hour 6.14 6.04 6.03 8.27 3.71 3.95 Disclosures: No relevant conflicts of interest to declare.

YM155 Exerts Potent Cytotoxic Activity Against Quiescent (G0/G1) Multiple Myeloma and Bortezomib Resistant Cells Via Inhibition of Mcl-1 and Survivin

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3023-3023
Author(s):  
Miyuki Ookura ◽  
Tatsuya Fujii ◽  
Shinji Kishi ◽  
Hiroko Shigemi ◽  
Naoko Hosono ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Protein Expression ◽  
Cytotoxic Activity ◽  
Cell Line ◽  
Cell Lines ◽  
Inhibitory Effect ◽  
Myeloma Cells ◽  
Ic50 Value ◽  
Induced Apoptosis ◽  
Resistant Cells

Abstract Multiple myeloma (MM) is a molecularly heterogeneous hematologic malignancy and remains mostly incurable despite the recent improvement of treatment strategies by several novel agents. Therefore, it is important to develop more efficacious drug against MM. YM155, a novel small molecule suppressant of survivin, shows anti-proliferative activities against various human cancer cells. YM155 was identified in a survivin gene promoter assay by high throughput screening of chemical libraries. In the present study, we investigated the cytotoxic mechanism of YM155 against human myeloma cells including bortezomib (BTZ) resistant cells (U266/BTZ). Three myeloma cell lines, U266, KMS-11 and KMS-12, were employed. YM155 inhibited the cell growth of these cell lines with the IC50 value of below 5 nM. YM155 suppressed the expression of mRNA and protein of survivin. We also found that YM155 inhibited the protein expression of Mcl-1, as an essential anti-apoptotic protein for survival of myeloma cells. In addition, we observed that YM155 markedly suppressed the phosphorylation of STAT3, which is known as transcription factor of Mcl-1. When KMS-12 cells were incubated with IL-6, phosphorylation of STAT3 and upregulation of Mcl-1 protein were observed. Treatment of KMS-12 with YM155 inhibited these events and eventually induced apoptosis in KMS-12 cells. Interestingly, inhibitory effect of YM155 on Mcl-1 protein expression was much stronger than that on survivin. RQ-PCR analysis indicated that the level of Mcl-1 mRNA was not affected after YM155 treatment. Immunoblot analysis showed that proteasome inhibitor MG-132 blocked the inhibition of Mcl-1 expression by YM155, suggesting that proteasome-mediated degradation is involved in YM155-induced Mcl-1 downregulation. MM is a low-growth-fraction disease and low proliferation of MM seems to contribute to resistance to various anticancer drugs. To determine whether YM155 shows cytotoxic effect against quiescent (G0/G1) MM cells, U266 were cultured in low-serum medium to enrich the G0/G1 population. Dual-parametric flow cytometric analysis using Hoechest33342 and the RNA specific dye pyronin Y revealed that YM155 potently induced cell death of quiescent (G0/G1) MM cells. In quiescent MM cells, inhibitory effect of YM155 on Mcl-1 protein expression was much stronger than that on survivin. We also examined whether similar effect of YM155 could be observed in primary MM cells. The majority of primary MM cells from patients was found to be in quiescent phase by cell-cycle analysis. YM155 showed similar cytotoxic activity against primary MM cells. In contrast, Ara-C, the S-phase specific anticancer drug, never killed quiescent primary MM cells. We established BTZ-resistant MM cell line (U266/BTZ). The IC50 value was 45-fold higher than its parental cell line. DNA sequencing data indicated that U266/BTZ cells possess a point mutation, G322A, in the gene encoding the proteasome beta-5 subunit. YM155 almost equally exhibited cytotoxic activity against U266/BTZ compared with parental cells. U266/BTZ displayed significantly lowered amounts of bcl-2, survivin and aurora-B kinase proteins. Interestingly, U266/BTZ overexpressed the Mcl-1 protein. Treatment with YM155 rapidly suppressed Mcl-1 protein expression and induced apoptosis. These data suggest that overexpression of Mcl-1 may contribute to bortezomib resistance and downregulation of Mcl-1 by YM155 could overcome it. In conclusion, our data indicate that YM155 may exert robust cytotoxic activity against quiescent (G0/G1) MM and bortezomib resistant cells via inhibition of Mcl-1 and survivin. Disclosures No relevant conflicts of interest to declare.

scholarly journals Isolation, cDNA cloning, and overexpression of a 33-kD cell surface glycoprotein that binds to the globular "heads" of C1q.

1994 ◽  
Vol 179 (6) ◽  
pp. 1809-1821 ◽  
Author(s):  
B Ghebrehiwet ◽  
B L Lim ◽  
E I Peerschke ◽  
A C Willis ◽  
K B Reid
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Human Serum ◽  
Cell Line ◽  
Cell Lines ◽  
Cdna Cloning ◽  
Binding Protein ◽  
Terminal Sequence ◽  
Raji Cells ◽  
Sheep Erythrocytes

This work describes the functional characterization, cDNA cloning, and expression of a novel cell surface protein. This protein designated gC1q-R, was first isolated from Raji cells and was found to bind to the globular "heads" of C1q molecules, at physiological ionic strength, and also to inhibit complement-mediated lysis of sheep erythrocytes by human serum. The NH2-terminal amino acid sequence of the first 24 residues of the C1q-binding protein was determined and this information allowed the synthesis of two degenerate polymerase chain reaction primers for use in the preparation of a probe in the screening of a B cell cDNA library. The cDNA isolated, using this probe, was found to encode a pre-pro protein of 282 residues. The NH2 terminus of the protein isolated from Raji cells started at residue 74 of the predicted pre-pro sequence. The cDNA sequence shows that the purified protein has three potential N-glycosylation residues and is a highly charged, acidic molecule. Hence, its binding to C1q may be primarily but not exclusively due to ionic interactions. The "mature" protein, corresponding to amino acid residues 74-282 of the predicted pre-pro sequence, was overexpressed in Escherichia coli and was purified to homogeneity. This recombinant protein was also able to inhibit the complement-mediated lysis of sheep erythrocytes by human serum and was shown to be a tetramer by gel filtration in nondissociating conditions. Northern blot and RT-PCR studies showed that the C1q-binding protein is expressed at high levels in Raji and Daudi cell lines, at moderate levels in U937, Molt-4, and HepG2 cell lines, and at a very low level in the HL60 cell line. However, it is not expressed in the K562 cell line. Comparison of gC1q-R NH2-terminal sequence with that of the receptor for the collagen-like domain of C1q (cC1q-R) showed no similarity. Furthermore, antibodies to gC1q-R or an 18-amino acid residue-long NH2-terminal synthetic gC1q-R peptide did not cross-react with antibodies to cC1q-R. Anti-gC1q-R immunoblotted a 33-kD Raji cell membrane protein, whereas anti cC1q-R recognized a molecule of approximately 60 kD. The NH2-terminal sequence of gC1g-R appears to be displayed extracellularly since anti-gC1g-R peptide reacted with surface molecules on lymphocytes, polymorphonuclear leukocytes, and platelets, as assessed by flow cytometric and confocal laser scanning microscopic analyses.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals A Glucosamine-Specific Lectin from Green Dragon No. 8 Beans (Phaseolus vulgaris) Induced Apoptosis on Nasopharyngeal Carcinoma Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-7
Author(s):  
Yau Sang Chan ◽  
Lixin Xia ◽  
Tzi Bun Ng
Keyword(s):  
Phaseolus Vulgaris ◽  
Cell Line ◽  
Cell Lines ◽  
Antiproliferative Activity ◽  
Normal Skin ◽  
Nasopharyngeal Cancer ◽  
Inhibitory Effect ◽  
Size Exclusion ◽  
M Phase ◽  
Induced Apoptosis

A lectin exhibiting antiproliferative activity on tumor cell lines but devoid of antifungal activity has been purified fromPhaseolus vulgariscv. Green Dragon no. 8 seeds. The lectin was a 60 kDa dimeric protein with two 30 kDa subunits. It was a glucosamine-specific lectin as implied from the inhibitory effect of glucosamine on hemagglutinating activity of the lectin. The steps for isolation of the lectin involved Affi-gel blue gel (affinity gel), Mono Q (anion exchanger), and Superdex 75 column (size exclusion). The lectin was purified 20.8-fold from the crude extract of the beans. The purified lectin showed antiproliferative activity on breast cancer MCF7 cell line and nasopharyngeal cancer HONE1 and CNE2 cell lines, but a low activity on normal skin fibroblast HSF98 cell line. The lectin was shown to induce apoptosis on HONE1 cells, as indicated by increased phosphatidylserine externalization and mitochondrial depolarization. It also blocked HONE1 cell division and kept the cells at the G2/M phase of the cell cycle.

scholarly journals Heat Shock Protein 70 Inhibitor, Alone or in Combination with Bortezomib, Prevented Plasmacytoma Development in Immunodeficient Mice Transplanted with Myeloma Cell Lines

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5658-5658
Author(s):  
Mariana Bleker de Oliveira ◽  
Angela Isabel Eugenio ◽  
Veruska Lia Fook Alves ◽  
Daniela Zanatta ◽  
Mihoko Yamamoto ◽  
...  
Keyword(s):  
Cell Line ◽  
Tumor Cells ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Conflicts Of Interest ◽  
Control Group ◽  
Immunodeficient Mice ◽  
Late Apoptosis

Abstract Introduction: HSP70 has an integrative role in protein degradation due to the interaction with many pathways, such as ubiquitin proteasome (UPS), unfolded protein response (UPR) and autophagy. In multiple myeloma (MM) HSP70 is overexpressed and helps to prevent proteotoxic stress and cell death caused by overload of unfolded/misfolded proteins produced by tumor cells. Aims: To explore the role of HSP70 inhibition, isolated or in association with proteasome inhibitor, as therapeutic strategy for MM through in vitro and in vivo analyses. Methods: RPMI8226-LUC-PURO and U266-LUC-PURO bioluminescent cell lines were treated with HSP70 inhibitor (VER155008- 50 μM or 80μM) and proteasome inhibitor (bortezomib 100nM) for evaluation of apoptosis induction by flow cytometry using annexin V and propidium iodide. NOD.Cg-rkdcscid Il2rgtm1Wjl/SzJ immunodeficient mice were used for plasmacytoma xenograft model and treated with intravenous VER155008 (40mg/kg) and bortezomib (1mg/kg), immediately after transplant of RPMI8226-LUC-PURO and U266-LUC-PURO bioluminescent cell lines (N=3 for each group, including controls, bortezomib, VER155008, and combination of bortezomib and VER155008). Bioluminescence was measured in IVIS Kinetic (Capiler Life Science) once a day for seven days. Results: Bortezomib used as single treatment was able to induce apoptosis in RPMI8226-LUC-PURO cell line: the best result for in vitro studies RPMI8226-LUC-PURO was 65% of late apoptosis after treatment with bortezomib. On the other hand, U266-LUC-PURO cell line presented higher percentage of apoptosis when treated with bortezomib and VER155008 combination: U266-LUC-PURO cell line presented more than 60% of late apoptosis after VER155008 (80μM) combined with bortezomib, showing that inhibition of HSP70 could overcome U266-LUC-PURO resistance to bortezomib alone. Mice treated with VER155008, alone or in combination with bortezomib, showed complete inhibition of tumor growth (absence of bioluminescence) for both cell lines when compared with control group after one week of treatment (p<0.001, Two-way ANOVA). Therefore, in vivo studies using mice treated with VER155008, alone or in combination with bortezomib, prevented tumor development after one week of treatment, independent of the cell line used in the xenotransplant. Conclusion: Our study shows that HSP70 and proteasome inhibitors combination induced apoptosis in tumor cells in vivo for both MM cell lines. Since HSP70 is overexpressed in MM and connects several signaling pathways that maintain cell survival, such as UPS, UPR and autophagy, it can represent a key role to establish a new approach for the treatment of MM. Financial support: FAPESP 2010/17668-6 and CNPq (155272/2013-6). UNIFESP Ethics Committee (0219/12). Disclosures No relevant conflicts of interest to declare.

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