The preclinical anti-angiogenic and pro-apoptotic activity of lenalidomide in urothelial carcinoma (UC).

2013 ◽  
Vol 31 (6_suppl) ◽  
pp. 294-294
Author(s):  
Weiguo Jian ◽  
Jonathan M. Levitt ◽  
Keith S. Chan ◽  
Seth P. Lerner ◽  
Guru Sonpavde
Keyword(s):  
Flow Cytometry ◽  
Cell Viability ◽  
Cell Line ◽  
Immune Modulation ◽  
Human Subjects ◽  
Low Grade ◽  
Angiogenic Activity ◽  
Apoptotic Activity

294 Background: Lenalidomide (Len) is an immunomodulatory drug (IMiD) approved for hematologic conditions and demonstrates immune modulation, anti-angiogenic activity and direct anti-tumor cytotoxicity. A rationale can be made to evaluate the preclinical activity of Len in UC. Methods: The in vitro anti-tumor activity of Len was evaluated in 4 human (5637, TCC-SUP, RT4, RT112) and 1 murine (MB49) cell line. Anti-proliferative activity activity (MTT assay), apoptosis (Annexin-FITC immunohistochemistry [IHC], flow cytometry) and cell viability by colony forming assay were measured. In vivo activity of daily oral Len 10 mg/kg or placebo orally for 5 days a week for up to 4 weeks was examined in syngeneic immunocompetent C57BL/6 mice bearing subcutaneous (SC) MB49-Luc25 tumors and RT4 subcutaneous xenografts. Tumors underwent immunohistochemistry (IHC) for microvessel density (CD31), apoptosis (cleaved caspase [cc]-3) and CD3+/CD20+ lymphocyte infiltration. Cereblon, a molecular target of Len was analyzed by IHC. Results: In vitro cultures for 3 days with daily repletion of Len showed significant pro-apoptotic activity (flow cytometry) at low micromolar concentrations attainable in human subjects (2.2 µM) against RT4 cells, a superficially invasive human UC cell line. Long-term cultures of RT4 cells for 2 weeks with daily repletion of Len significantly reduced cell viability and colony forming ability. Cereblon expression was numerically lower in sensitive RT4 cells compared to resistant 5637 cells (p=NS). In the immunocompetent model in vivo, Len did not decrease tumor size, or increase cc-3 and CD3+/CD20+ lymphocytes, but post-Len tumors exhibited decreased CD31 (p<0.05). In RT4 xenografts, Len significantly decreased the size of tumors and CD31, and increased cc-3 (all p<0.05). Cereblon expression increased in Len treated RT4 xenografts (p=0.024). Conclusions: Lenalidomide demonstrated selective preclinical activity against superficially invasive low grade human UC cells attributable to direct tumor cell apoptosis and anti-angiogenic activity. Clinical evaluation in patients with low grade or non-invasive UC and further study of cereblon as a predictive biomarker may be warranted.

The preclinical activity of lenalidomide in urothelial carcinoma (UC).

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e15002-e15002
Author(s):  
Jonathan M. Levitt ◽  
Keith S. Chan ◽  
Weiguo Jian ◽  
Seth P. Lerner ◽  
Guru Sonpavde
Keyword(s):  
Flow Cytometry ◽  
Cell Viability ◽  
Urothelial Carcinoma ◽  
Cell Line ◽  
Immune Modulation ◽  
Non Invasive ◽  
Bcg Therapy ◽  
Anti Tumor Activity

e15002 Background: Lenalidomide is approved for multiple myeloma and deletion 5q myelodysplastic syndromes (MDS) and demonstrates immune modulation, anti-angiogenic activity and direct anti-tumor cytotoxicity. A rationale can be made to evaluate the preclinical activity of lenalidomide in urothelial carcinoma (UC) based on the importance of these pathways. Methods: The in vitro anti-tumor activity of lenalidomide was evaluated in 4 human (5637, TCC-SUP, RT4, RT112) and 1 murine (MB49) cell line. Anti-proliferative activity activity (MTT assay), apoptosis (Annexin-FITC immunohistochemistry [IHC], flow cytometry) and cell viability by colony forming assay were measured. In vivo examination of activity of daily oral lenalidomide 10 mg/kg orally once daily or placebo for 4 weeks is examined in a syngeneic immunocompetent mouse model employing MB49-Luc25 cells injected subcutaneously in C57BL/6 mice. Murine tumors will be studied for anti-tumor activity. Results: In vitro activity of lenalidomide was detected at low ~1 µM concentrations (attainable in human subjects) against a non-invasive human UC cell line (RT4). Long-term cultures of RT4 cells for 10 days with daily repletion of lenalidomide reduced cell viability and colony forming ability to 75.6% of controls. Induction of apoptosis in RT4 was demonstrated by Annexin-FITC IHC and flow cytometry compared to control (30.11 vs. 14.74%). Invasive human UC cells and murine MB49 cells did not demonstrate apoptosis with lenalidomide exposure in vitro. Futher, lenalidomide did not inhibit overall tumor growth in the syngeneic immunocompetent murine model; immune activity and stem cell directed activity will be presented. Conclusions: Lenalidomide demonstrated preclinical anti-tumor activity against non-invasive human UC cells. Given its favorable toxicity profile compared to cytotoxic chemotherapy, clinical evaluation in patients with non-muscle-invasive bladder cancer and recurrence after BCG therapy may be warranted.

scholarly journals Libidibia ferreaFruit Crude Extract and Fractions Show Anti-Inflammatory, Antioxidant, and Antinociceptive EffectIn Vivoand Increase Cell ViabilityIn Vitro

2019 ◽  
Vol 2019 ◽  
pp. 1-14 ◽  
Author(s):  
Tamires Rocha Falcão ◽  
Aurigena Antunes de Araújo ◽  
Luiz Alberto Lira Soares ◽  
Iuri Brilhante de Farias ◽  
Wliana Alves Viturino da Silva ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Cell Viability ◽  
Gallic Acid ◽  
Cell Line ◽  
Crude Extract ◽  
Leukocyte Migration ◽  
Total Glutathione ◽  
Anti Inflammatory

Background.Libidibia ferrea(L. ferrea)is found throughout the northeastern region of Brazil, where it has been used in folk medicine with beneficial effects on many inflammatory disorders.Purpose. This study investigated the phytochemical composition of the crude extract and fractions ofL. ferreafruit and evaluated its anti-inflammatory and antinociceptive activitiesin vivoand effect on cell viabilityin vitro.Methods. Characterization of polyphenols present in crude extract (CE), hydroalcoholic fractions of 20-80% ethanol (CE20, CE40, CE60, and CE80), aqueous fraction (AqF), and ethyl acetate (EAF) fractions ofL. ferreafruit was performed by chromatographic analysis.Anti-inflammatory activity was evaluated by using a carrageenan-induced peritonitis model submitted to a leukocyte migration assay and myeloperoxidase activity (MPO) analysis. Total glutathione and malondialdehyde (MDA) levels were assessed to evaluate the oxidative stress level. Antinociceptive activity was evaluated by acetic acid-induced abdominal writhing and hot plate test.In vitrocell viability was determined by using MTT assay in a mouse embryonic fibroblast cell line (3T3 cells).Results. Chromatography revealed the presence of ellagic acid content in EAF (3.06), CE (2.96), and CE40 (2.89). Gallic acid was found in EAF (12.03), CE 20 (4.43), and CE (3.99).L. ferreacrude extract and all fractions significantly reduced leukocyte migration and MPO activity (p<0.001).L. ferreaantioxidant effect was observed through high levels of total glutathione and reduction of MDA levels (p<0.001). Acetic acid-induced nociception was significantly inhibited after administration ofL. ferreacrude extract and all fractions (p<0.001). Crude extract and all fractions significantly increased the viability of the 3T3 cell line (p<0.05).Conclusions. The appropriate extraction procedure preserves the chemical components ofL. ferreafruit, such as gallic acid and ellargic acid. Crude extract and fractions ofL. ferreafruit exhibited anti-inflammatory, antioxidant, antinociceptive activitiesin vivoand enhanced cell viabilityin vitro.

Rational Combinations Including a Novel Syk Inhibitor, Fostamatinib Disodium (FosD) in Diffuse Large B Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 283-283
Author(s):  
Randall M Rossi ◽  
Valerie Grose ◽  
Polly Pine ◽  
Richard I Fisher ◽  
Craig T. Jordan ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Cell Viability ◽  
B Cell ◽  
Cell Line ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
In Vivo Studies

Abstract Abstract 283 Certain malignant B-cells rely upon B-cell receptor-mediated survival signals. Spleen tyrosine kinase (Syk) initiates and amplifies the B-cell receptor-mediated signal. We and others have demonstrated that fostamatinib disodium (FosD: a prodrug of R406, a potent and specific inhibitor of Syk) induces apoptosis in lymphoma cell lines and primary tumors. A recent clinical trial has demonstrated significant clinical activity of FosD in relapsed/refractory B-cell non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia, and minimal overlap in toxicities with conventional agents. Given this background, future development in B-cell NHL will include rational combinations of FosD and currently available therapies. Therefore, we conducted in vitro and in vivo studies of rational combinations including FosD, in anticipation of clinical trial development. First, using a human DLBCL cell line of GCB genotype, (OCI-Ly19), we analyzed in vitro the combination of R406 with the following agents: fludarabine, rapamycin, rituximab, bendamustine and bortezomib. Increased cytotoxicity was observed using in vitro culture assays with the addition of fludarabine, rapamycin, or rituximab to R406. Cell viability at 72 hours was 25% with R406 alone, 27% for fludarabine alone, and only 9% for the fludarabine/R406. At 48 hours, cell viability was 49% using R406 alone, 31% using rituximab alone, and 21% for rituximab/R406. At 120 hours using primary lymphoma cells (DLCL27), there were no viable cells treated with the rapamycin/FosD combination, compared with rapamycin alone (7%) or FosD alone (25%) The addition of bortezomib or bendamustine to FosD resulted in only a minimal additive increase in cytotoxicity. Results with all combinations were similar with the OCI-Ly10 human DLBCL line of ABC genotype. We then performed in vivo studies by subcutaneous transplantation of the DLBCL cell line OCI-Ly19, (engineered to express luciferase allowing for real time in vivo imaging) into immune deficient NOD/SCID mice which reproducibly formed tumors. Recipient animals were separated into uniform cohorts when the tumors were less than or equal to 500 mm3 in size. The animals were then simultaneously treated with FosD (n=7; 3 gm/kg ad. lib.; translates into 2-5 micromolar R406 systemically throughout the 24h period) and either bortezomib, (n=6; 0.4 mg/kg weekly IP), or rituximab, (n=13; 3 mg/kg, 2x weekly IP). Analysis of the OCI-Ly19 tumor volumes at day 46 showed a median of 2364 mm3 with bortezomib alone compared with 1823 mm3 with bortezomib and FosD. When FosD was combined with rituximab the most significant cytotoxicity was observed: (p=0.01; median tumor volume of 497 mm3 following the combination) in comparison to either FosD alone (3150 mm3) or rituximab alone (1764 mm3). We conclude that the addition of FosD appears to increase activity against NHL of several drugs, including fludarabine and rapamycin. These agents have significant activity in indolent and mantle cell NHL as well as CLL. Moreover, there is no evidence that FosD impedes rituximab responses in vitro or in vivo; in fact we have suggested possible synergy with the combination of rituximab and FosD. Based upon the documented single agent activity of FosD in humans, and this data, clinical trials are now indicated using these promising combinations in NHL and CLL. Disclosures: Pine: Rigel: Employment. Friedberg:Rigel: Research Funding.

scholarly journals Preliminary study of the toxicity and radioprotective effects of zymosan in vitro and in vivo

2020 ◽  
Author(s):  
Yue-zhi Zhang ◽  
Shu-jing Ge ◽  
Qing-zhen Leng ◽  
Jian-jun Ma ◽  
Hanchen Liu
Keyword(s):  
Flow Cytometry ◽  
Protective Effect ◽  
Cell Viability ◽  
Treatment Time ◽  
Positive Control ◽  
Radioprotective Effect ◽  
X Rays ◽  
Spleen Index

Abstract Background: This study aimed to confirm the cytotoxicity of zymosan in AHH-1 cells and HIECs and to determine the treatment time and dose of zymosan at which it exerts radioprotective effects.Methods: AHH-1 cells and HIECs were administered 0, 20, 40, 80 or 160 μg/mL zymosan. The CCK-8 assay and flow cytometry were used to evaluate cell viability and apoptosis 24 h, 48 h, and 72 h after administration. Furthermore, 12 h before irradiation, the cells were treated with 0, 5, 10, or 20 μg/mL zymosan and then irradiated with 4 Gy X-rays. Cell viability and apoptosis were measured by the CCK-8 assay and flow cytometry at 24 h. In addition, the protective effect of zymosan against radiation in vitro was compared to that of 20 μg/mL LPS as a positive control. In vivo, weight, the spleen index and the thymus index were measured to evaluate the toxicity of 0, 5, 10, 20 and 10 mg/kg zymosan. In addition, rats were treated with 0, 2, 4, 8 or 10 mg/kg zymosan and then irradiated with 7 Gy X-rays. The survival rate, spleen index and thymus index were evaluated. The protective effect of zymosan against radiation in vivo was compared to that of 10 mg/kg LPS a positive control. Results: The viability and apoptosis of cells treated with different doses of zymosan for different treatment times were not different from those of control cells (p<0.05). Furthermore, cell viability and apoptosis were clearly improved after zymosan preadministration (p<0.05). The radioprotective effect of zymosan was dose-dependent. In addition, the viability of cells pretreated with zymosan was higher than that of cells pretreated with LPS, and the apoptosis rate of zymosan-treated cells was lower than that of cells pretreated with LPS (p<0.05). In vivo, weight, the spleen index and the thymus index were significantly decreased by zymosan at a concentration of 20 mg/kg (p<0.05). Further experiments showed that the concentration at which zymosan exerted radioprotective effects was 10 mg/kg. The radioprotective effect of zymosan was better than that of LPS pretreatment (p<0.05). Conclusion: Zymosan is nontoxic to cells and exerts a better radioprotective effect than LPS.

scholarly journals Construction and Identification of New Molecular Markers of Triple-Negative Breast Cancer Stem Cells

2021 ◽  
Vol 11 ◽  
Author(s):  
Tingting Liu ◽  
Hongyue Wang ◽  
Zhiyong Liu ◽  
Jing Zhang ◽  
Yan Liu ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Stem Cells ◽  
Flow Cytometry ◽  
Cancer Stem Cells ◽  
Cell Line ◽  
Screening Method ◽  
Breast Cancer Stem Cells ◽  
Positive Phage

Objective: We screened the TNBC stem cells using phage display (PD) and acquired the specific binding clones; and then the positive phage DNAs were amplified and extracted, synthesized with specific polypeptides, and labeled with fluorescein isothiocyanate (FITC). Finally, we identified the specificity of the polypeptides in vitro and in vivo.Methods: Human breast cancer cell line MDA-MB-231 and human mammary gland cell line hs578bst were chosen in our study, and MDA-MB-231 breast cancer stem cells (BCSCs) were cultured and identified by flow cytometry. The phage peptide library was screened using MDA-MB-231 BCSCs, the positive phage clones were identified by ELISA, and the DNA of the positive phages was extracted and sent to a biotechnology company for sequencing. According to the sequencing results, a specific polypeptide was synthesized and labeled with FITC. In the end, the specificity of a polypeptide to BCSCs was identified in vivo and in vitro.Results: The MDA-MB-231 BCSCs were cultured and enriched with the “serum and serum-free alternate” method. The BCSCs were found to have characteristics of CD44+/CD24−/low epithelial surface antigen (ESA) and ALDH+ with flow cytometry. The phage was enriched to 200-fold after three rounds of screening for MDA-MB-231 BCSCs. The positive phages were sequenced; then a polypeptide named M58 was synthesized according to sequencing results. Polypeptide M58 has a specific affinity to MDA-MB-231 BCSCs in vivo and in vitro.Conclusion: Specific polypeptides binding to MDA-MB-231 BCSCs were screened out by PD screening method, which laid a theoretical foundation for the targeted therapy and further research of BCSCs.

scholarly journals Preliminary study of the toxicity and radioprotective effects of zymosan in vitro and in vivo

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yue-zhi Zhang ◽  
Shu-jing Ge ◽  
Qing-zhen Leng ◽  
Jian-jun Ma ◽  
Han-chen Liu
Keyword(s):  
Flow Cytometry ◽  
Protective Effect ◽  
Cell Viability ◽  
Treatment Time ◽  
Survival Rates ◽  
Positive Control ◽  
X Rays ◽  
Spleen Index

Abstract Background This study aimed to confirm the cytotoxicity of zymosan in vitro and in vivo and determine the appropriate treatment time and the dose of zymosan. Methods AHH-1 cells and HIECs were administered by 0, 20, 40, 80 or 160 μg/mL zymosan. The CCK-8 assay and flow cytometry were used to evaluate the cell viability and apoptosis 24 h, 48 h, and 72 h after administration. Furthermore, 12 h before irradiation, the cells were treated with 0, 5, 10, or 20 μg/mL zymosan and then irradiated with 4 Gy X-rays. Cell viability and apoptosis were measured by the CCK-8 assay and flow cytometry at 24 h. In addition, the protective effect of zymosan against radiation in vitro was compared to that of 20 μg/mL LPS. In vivo, weight, the spleen index, and the thymus index were measured to evaluate the toxicity of 0, 5, 10, 20, and 10 mg/kg zymosan. In addition, rats were treated with 0, 2, 4, 8, or 10 mg/kg zymosan and then irradiated with 7 Gy X-rays. The survival rate, organ index were evaluated. The protective effect of zymosan against radiation in vivo was compared to that of 10 mg/kg LPS a positive control. Results The viability and apoptosis of cells treated with different doses and treatment times of zymosan were not different from those of control cells (p < 0.05). Furthermore, cell viability and apoptosis were clearly improved after zymosan preadministration (p < 0.05). The radioprotective effect of zymosan was dose-dependent. In addition, the viability of cells pretreated with zymosan was higher than that of cells pretreated with LPS, and the apoptosis rate of zymosan-treated cells was lower than that of cells pretreated with LPS (p < 0.05). In vivo, weight, the spleen index and the thymus index were significantly decreased by zymosan at a concentration of 20 mg/kg (p < 0.05). Further experiments showed that the concentration at which zymosan exerted radioprotective effects was 10 mg/kg. The survival curves in the irradiated rats were barely separated between the LPS treatment and zymosan treatment. Conclusion Zymosan administration before radiation exposure significantly increased cell viability and the survival rates of rats.

scholarly journals Piperazine-Derived α1D/1A Antagonist 1- Benzyl-N- (3-(4- (2-Methoxyphenyl) Piperazine-1-yl) Propyl) -1H- Indole-2- Carboxamide Induces Apoptosis in Benign Prostatic Hyperplasia Independently of α1-Adrenoceptor Blocking

2021 ◽  
Vol 11 ◽  
Author(s):  
Qing Xiao ◽  
Qi-Meng Liu ◽  
Ru-Chao Jiang ◽  
Kai-Feng Chen ◽  
Xiang Zhu ◽  
...  
Keyword(s):  
Benign Prostatic Hyperplasia ◽  
Cell Viability ◽  
Cell Line ◽  
Prostate Volume ◽  
Prostatic Hyperplasia ◽  
Subtype Selectivity ◽  
Prostate Growth ◽  
Apoptotic Induction

Previous studies have indicated that α1D/1A antagonist naftopidil (NAF) suppresses prostate growth by decreasing cell proliferation without affecting apoptosis and prostate volume in benign prostatic hyperplasia (BPH). A NAF-derived α1D/1A antagonist 1- benzyl-N-(3-(4-(2-methoxyphenyl) piperazine-1-yl) propyl)-1H-indole-2- carboxamide (HJZ-12) has been reported from our laboratory, which exhibits high subtype-selectivity to both α1D- and α1A- AR (47.9- and 19.1- fold, respectively) with respect to a1B-AR in vitro. However, no further study was conducted. In the present study, a pharmacological evaluation of HJZ-12 in BPH was performed on an estrogen/androgen-induced rat BPH model and human BPH-1 cell line. In vivo, HJZ-12 exhibited better performance than NAF in preventing the progression of rat prostatic hyperplasia by not only decreasing prostate weight and proliferation (similar to NAF) but also, shrinking prostate volume and inducing prostate apoptosis (different from NAF). In vitro, HJZ-12 exhibited significant cell viability inhibition and apoptotic induction in BPH-1 cell line, without presenting cell anti-proliferation properties. Intriguingly, the role of HJZ-12 on cell viability and apoptosis was an α1-independent action. Furthermore, RNA-Seq analysis was applied to screen out six anti-apoptotic genes (Bcl-3, B-lymphoma Mo-MLV insertion region 1 [Bmi-1], ITGA2, FGFR3, RRS1, and SGK1). Amongst them, Bmi-1 was involved in the apoptotic induction of HJZ-12 in BPH-1. Overall, HJZ-12 played a remarkable role in preventing the progression of prostatic hyperplasia through α1-independent apoptotic induction, indicating that it will be a multi-target effective candidate for BPH treatment.

scholarly journals WT1 and DNMT3A Play an Essential Function and Represent Therapeutic Vulnerabilities in Certain AML Samples, As Shown By CRISPR/Cas9 Mediated Knockout in PDX Models In Vivo

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 377-377
Author(s):  
Maryam Ghalandary ◽  
Yuqiao Gao ◽  
Martin Becker ◽  
Diana Amend ◽  
Klaus H. Metzeler ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Line ◽  
Cell Lines ◽  
Target Gene ◽  
Target Genes ◽  
Nsg Mice ◽  
Essential Function ◽  
Pdx Models

Abstract Background: The prognosis of patients with acute myeloid leukemia (AML) remains poor and novel therapeutic options are intensively needed. Targeted therapies specifically address molecules with essential function for AML and deciphering novel essential target genes is of utmost importance. Functional genomics via CRISPR\Cas9 technology paves the way for the systematic discovery of novel essential genes, but was so far mostly restricted to studying cell lines in vitro, lacking features of, e.g., primary tumor cells and the in vivo tumor microenvironment. To move closer to the clinical situation in patients, we used the CRISPR\Cas9 technology in patient-derived xenograft (PDX) models of AML in vivo. Methods: Primary tumor cells from seven patients with AML were transplanted into immunocompromised NSG mice and serially transplantable PDX models derived thereof. PDX models were selected which carry the AML specific mutations of interest at variant allele frequencies close to 0.5. PDX cells were lentivirally transduced to express the Cas9 protein and a sgRNA; successfully transduced PDX cells were enriched by flow cytometry gating on a recombinant fluorochrome or by puromycin. The customized sgRNA library was designed using the CLUE (www.crispr-clue.de) platform and cloned into a lentiviral vector with five different sgRNAs per target gene, plus positive and negative controls (Becker et al., Nucleic Acids Res. 2020). PDX cells were lentivirally transduced with the CRISPR/Cas9 sgRNA library, transplanted into NSG mice, grown in vivo and cells re-isolated at advanced AML disease. sgRNA distribution was measured by next generation sequencing and compared to input control using the MAGeCK pipeline. Interesting dropout hits from PDX in vivo screens were validated by fluorochrome-guided competitive in vivo experiments in the PDX models, comparing growth of PDX AML cells with knockout of the gene of interest versus control knockout in the same mouse. PDX cells were transduced with lentiviral vectors expressing a single sgRNA, using in parallel three different sgRNAs per target gene. Targeting and control sgRNAs were marked by different fluorochromes; PDX cells expressing targeting or control sgRNA were mixed at a 1:1 ratio, injected into NSG mice and PDX models competitively grown until advanced disease stage, when cell distributions was determined by flow cytometry. Human AML cell lines were studied in vitro for comparison. Results: In search for genes with essential function in AML, we cloned a small customized sgRNA library targeting 34 genes recurrently mutated in AML and tested the library in two PDX AML models in vivo. From the dropouts, we validated most interesting target genes using fluorochrome-guided competitive in vivo assays. Knockout of NPM1 abrogated in vivo growth in all PDX AML models tested, reproducing the known common essential function of NPM1. KRAS proved an essential function in PDX AML models both with and without an oncogenic mutation in KRAS, although with a stronger effect upon KRAS mutation, suggesting that patients with tumors both with and without KRAS mutation might benefit from treatment inhibiting KRAS. Surprising results were obtained for WT1 and DNMT3A. Both genes are frequently mutated in AML, but most AML cell lines tested in vitro do not show an essential function of any of the two genes, in published knockdown or knockout data, including from the Cancer Dependency Map database. On the contrary, knockout of either WT1 or DNMT3A was shown to enhance growth of AML cell lines and increase leukemogenesis in certain models. In PDX models in vivo, we found a clearly essential function for DNMT3A in all AML samples and WT1 in most samples tested and PDX in vivo results were discordant to cell line in vitro data, suggesting that cell line inherent features and/or the in vivo environment influence the function of WT1 and DNMT3A. Conclusion: We conclude that functional genomics in PDX models in vivo allows discovering essentialities hidden for cell line in vitro approaches. WT1 and DNMT3A harbor the potential to represent attractive therapeutic targets in AML under in vivo conditions, warranting further evaluation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Preliminary study of the toxicity and radioprotective effects of zymosan in vitro and in vivo

2021 ◽  
Author(s):  
Yue-zhi Zhang ◽  
Shu-jing Ge ◽  
Qing-zhen Leng ◽  
Jian-jun Ma ◽  
Hanchen Liu
Keyword(s):  
Flow Cytometry ◽  
Protective Effect ◽  
Cell Viability ◽  
Treatment Time ◽  
Survival Rates ◽  
Positive Control ◽  
X Rays ◽  
Spleen Index

Abstract Background: This study aimed to confirm the cytotoxicity of zymosan in vitro and in vivo and determine the appropriate treatment time and dose of zymosan.Methods: AHH-1 cells and HIECs were administered by 0, 20, 40, 80 or 160 μg/mL zymosan. The CCK-8 assay and flow cytometry were used to evaluate the cell viability and apoptosis 24 h, 48 h, and 72 h after administration. Furthermore, 12 h before irradiation, the cells were treated with 0, 5, 10, or 20 μg/mL zymosan and then irradiated with 4 Gy X-rays. Cell viability and apoptosis were measured by the CCK-8 assay and flow cytometry at 24 h. In addition, the protective effect of zymosan against radiation in vitro was compared to that of 20 μg/mL LPS. In vivo, weight, the spleen index and the thymus index were measured to evaluate the toxicity of 0, 5, 10, 20 and 10 mg/kg zymosan. In addition, rats were treated with 0, 2, 4, 8 or 10 mg/kg zymosan and then irradiated with 7 Gy X-rays. The survival rate, organ index were evaluated. The protective effect of zymosan against radiation in vivo was compared to that of 10 mg/kg LPS a positive control. Results: The viability and apoptosis of cells treated with different doses and treatment times of zymosan were not different from those of control cells (p<0.05). Furthermore, cell viability and apoptosis were clearly improved after zymosan preadministration (p<0.05). The radioprotective effect of zymosan was dose-dependent. In addition, the viability of cells pretreated with zymosan was higher than that of cells pretreated with LPS, and the apoptosis rate of zymosan-treated cells was lower than that of cells pretreated with LPS (p<0.05). In vivo, weight, the spleen index and the thymus index were significantly decreased by zymosan at a concentration of 20 mg/kg (p<0.05). Further experiments showed that the concentration at which zymosan exerted radioprotective effects was 10 mg/kg. The survival curves in the irradiated rats were barely separated between the LPS treatment and zymosan treatment. Conclusion: Zymosan administration before radiation exposure significantly increased cell viability and the survival rates of rats.

The PI3K/Akt Pathway Is an Important Regulator of Homing and Adhesion in Waldenstrom’s Macroglobulinemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2417-2417 ◽  
Author(s):  
Xavier Leleu ◽  
Hai Ngo ◽  
Judy Runnels ◽  
Costas Pitsillides ◽  
Joel Spencer ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Confocal Microscopy ◽  
Treated Mouse ◽  
Akt Pathway ◽  
Low Grade ◽  
Dependent Manner ◽  
Surface Adhesion ◽  
Number Of Cells

Abstract Background: Waldenstrom Macroglobulinemia (WM) is a low-grade lymphoma characterized by widespread involvement of the bone marrow (BM) and involvement of lymph nodes and hepatosplenomegaly (HSM) in about 20% of the patients. We have recently demonstrated that the presence of HSM is one of the most important adverse prognostic factors in WM. The mechanisms of trafficking of WM cells to and from the BM and lymphoid organs is not well defined. The PI3k/Akt pathway is constitutively activated in WM, and regulates migration/homing in cancer cells and B-cells. We hypothesized that the Akt inhibitor perifosine (NSC 639966; Keryx Biopharmaceuticals, NY) modulates homing of WM cells to the BM. Methods: WM cell line (BCWM.1) was treated with perifosine 2 to 5uM for 2 hours. Surface adhesion receptors were studied using flow cytometry. The adhesion assay coated with fibronectin, a ligand of VLA-4 (EMD Biosciences, CA) was used to test in vitro adhesion. Migration was determined using the transwell migration assay (Costar, NY). We then studied homing of WM into the BM niches using in vivo flow cytometry and confocal microscopy in Balb/c mice. In brief, BCWM.1 were incubated with 5uM perifosine for 2 hrs (or control PBS). The cells were fluorescently labeled by incubation with the dialkylcarbocyanine membrane dye, “DiD” (Molecular Probes), 0.5uM dye for 30 minutes. Cells were then injected in the tail vein of the Balb/c mice, and in vivo confocal flow cytometry was performed on an artery from the ear lobe of the mice. Cell counts were obtained every 5 min. from the time of injection. In vivo confocal microscopy and two-photon microscopy was performed to study cells homing to BM vasculature of the skull (BM niches). Results: WM cells expressed very high levels of VLA-4, with a median 95% expression. BCWM.1 demonstrated increased adhesion to fibronectin-coated wells as compared to BSA-coated wells. Perifosine inhibited adhesion in a dose dependent manner with 50% decrease in adhesion at 2uM. Perifosine 10uM did not change the level of surface expression of VLA-4 after 6 and 24 hrs, indicating an effect on intracellular signaling but not on surface adhesion molecules. Perifosine 5uM significantly inhibited migration of BCWM.1 in response to SDF-1, a ligand that induces migration of WM cells. Perifosine also demonstrated significant inhibition of in vivo homing of WM cells to the BM niches. The number of cells in the peripheral circulation decreased dramatically (75% decrease) after 1 hr in the control, indicating homing, whereas there was a 40% reduction in the cells at 1hr after perifosine treatment (p=0.001). We then looked at the images obtained from the BM niches. The number of cells that homed and adhered to these areas was lower in the perifosine-treated mouse compared to the control mouse, indicating that fewer cells homed to the BM of the treated mouse. Conclusion: These results confirm that the PI3K/Akt pathway is important for migration, adhesion and homing of WM in vitro and in vivo.

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