scholarly journals Prostatic Acid Phosphatase Is a Progenitor Cell Marker That Persists After Androgen Ablation

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A1030-A1031
Author(s):  
Sudeh Izadmehr ◽  
Alexander Kirschenbaum ◽  
Shen Yao ◽  
Alice C Levine
Keyword(s):  
Acid Phosphatase ◽  
Protein Expression ◽  
Bone Metastases ◽  
Cell Lines ◽  
Gestational Age ◽  
Prostatic Acid Phosphatase ◽  
Scid Mice ◽  
Castration Resistant

Abstract Introduction: Prostatic Acid Phosphatase (PAP), a protein phosphatase and 5’ecto-nucleotidase, is expressed in prostate cancer (PCa) bone metastases and correlates with poor survival. Growing evidence suggests that PAP is not regulated by androgens, but rather by factors in the tumor microenvironment. Hypothesis: We hypothesized that PAP is a marker for a more progenitor type PCa cell and its expression is androgen-independent, persisting in castration-resistant disease. Methods: Protein expression of PAP and three androgen-regulated proteins, the Androgen Receptor (AR), Prostate-Specific Antigen (PSA), and ETS-related gene (ERG) protein, was assessed with immunohistochemistry in human fetal prostate (9.5 - 20 weeks of gestational age), archival human PCa bone metastases, and human PCa cell lines. VCaP cells were treated in vitro with dihydrotestosterone (DHT) and the effects on AR and PAP protein expression determined with Western Blotting. PAP-expressing PCa cell lines (LNCaP, C42B, and VCaP) were inoculated subcutaneously (s.c.) into SCID mice. To model tumor-bone interaction, LNCaP and MC3T3 osteoblast cells were co-inoculated s.c. into SCID mice. A VCaP castration study with surgical or sham castration was performed after tumors were palpable and effects of castration on tumor growth and protein expression determined. Results: PAP expression was observed in the fetal prostate as early as 11.5 weeks of gestational age prior to PSA and AR expression. Strong PAP expression was noted in all human PCa bone metastases examined, both treatment-naive and castrate-resistant (n=10). In vitro, VCaP cells expressed high levels of AR and PAP protein and DHT treatment increased AR and decreased PAP protein expression. In vivo, PAP expression was observed in all tumor models; LNCaP (low PAP expression), C42B (moderate PAP expression) and VCaP (high PAP expression). Castrated VCaP tumors underwent tumor stasis, were significantly smaller compared to intact mice, had decreased AR, PSA and ERG expression but persistent expression of PAP. Double staining of tumors for PAP and AR demonstrated a population of cells that were positive for PAP but negative for AR expression in hypoxic areas near necrosis. Inoculation of LNCaP cells with MC3T3 osteoblastic cells increased PAP expression in vivo. Conclusions: PAP is expressed early in human fetal prostate development prior to the secretion of significant androgens or expression of AR. In mouse xenograft tumors and human PCa bone metastases, androgens did not significantly regulate PAP expression. Both hypoxia and stroma increased PAP expression. These data demonstrate that PAP is a marker of early progenitor cells, is persistently expressed after castration and is upregulated by tumor microenvironmental factors. PAP may be a suitable target for the treatment of castration-resistant metastatic disease.

scholarly journals SUN-143 Prostatic Acid Phosphatase Is Not Regulated by Androgens During Prostate Development and Tumorigenesis

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Sudeh Izadmehr ◽  
Shen Yao ◽  
Alexander Kirschenbaum ◽  
Alice C Levine
Keyword(s):  
Acid Phosphatase ◽  
Bone Metastases ◽  
Mouse Models ◽  
Gestational Age ◽  
Prostatic Acid Phosphatase ◽  
Normal Prostate ◽  
Prostate Development ◽  
Castration Resistant ◽  
Castrate Resistant

Abstract INTRODUCTION: Prostatic acid phosphatase (PAP) is a soluble factor secreted by prostate luminal epithelial cells. PAP expression correlates with prostate cancer (PCa) bone metastases and poor survival. The androgenic regulation of PAP in prostate development and tumorigenesis is not fully understood. We investigated the relationship between PAP and androgens in human prostate specimens and in vivo. HYPOTHESIS AND OBJECTIVES: We hypothesized that PAP expression was independent of androgens. Our objectives were to determine the immunohistochemical expression of PAP in human fetal prostate tissue, human PCa bone metastases, and xenograft and surgical castration mouse models. METHODS: Immunohistochemical staining for PAP and three androgen-regulated proteins, the Androgen Receptor (AR), Prostate-Specific Antigen (PSA), and ETS-related gene (ERG) protein, was carried out on human fetal prostate (9.5, 11.5, 13, 16.5, 18 and 20 weeks of gestational age), archival human PCa bone metastases, and PCa mouse models. For xenograft studies, PAP-expressing PCa cell lines, LNCaP, C42B, and VCaP cells, were inoculated subcutaneously into SCID mice. A castration study with surgical or sham castration was performed after VCaP tumors were palpable. Mouse tumor growth and weight were measured biweekly, and tumor tissue isolated after mouse sacrifice. RESULTS: PAP expression was observed in the fetal prostate as early as 11.5 weeks of gestational age. Strong PAP expression was noted in all human PCa bone metastases examined, both treatment-naive and castrate-resistant (n=10). However, AR and ERG expression was absent in two of four castrate-resistant specimens. PSA was weakly expressed in human castration-resistant bone metastatic prostate specimens. In vivo, PAP expression was observed in all tumor models; however, the expression of PAP differed among androgen-sensitive models; LNCaP (low PAP), C42B (moderate PAP) and VCaP (high PAP). Castrated VCaP tumors underwent tumor stasis and were significantly smaller compared to intact mice. Strong expression of PAP was observed after castration. In contrast, AR, PSA, and ERG expression were reduced in castrated VCaP tumors compared to tumors from intact mice. Double staining of tumors for PAP and AR demonstrated a population of cells that were positive for PAP but negative for AR expression located in hypoxic areas near necrosis. CONCLUSIONS: Our findings demonstrated that PAP is expressed early in normal human fetal prostate development prior to the secretion of significant androgens or expression of AR. In mouse xenografts and human PCa bone metastases, androgens did not significantly regulate PAP expression. These data demonstrate that PAP is a marker of early progenitor cells in the normal prostate and is persistently expressed after castration. PAP may be a suitable target for the treatment of castration-resistant metastatic disease.

scholarly journals Novel Caprine Adeno-Associated Virus (AAV) Capsid (AAV-Go.1) Is Closely Related to the Primate AAV-5 and Has Unique Tropism and Neutralization Properties

2005 ◽  
Vol 79 (24) ◽  
pp. 15238-15245 ◽  
Author(s):  
Alejandra E. Arbetman ◽  
Michael Lochrie ◽  
Shangzhen Zhou ◽  
Jennifer Wellman ◽  
Ciaran Scallan ◽  
...  
Keyword(s):  
Protein Expression ◽  
Neutralization Assay ◽  
Biological Properties ◽  
Scid Mice ◽  
Factor Ix ◽  
In Vivo Model ◽  
Adeno Associated Virus ◽  
Human Factor Ix

ABSTRACT Preexisting humoral immunity to adeno-associated virus (AAV) vectors may limit their clinical utility in gene delivery. We describe a novel caprine AAV (AAV-Go.1) capsid with unique biological properties. AAV-Go.1 capsid was cloned from goat-derived adenovirus preparations. Surprisingly, AAV-Go.1 capsid was 94% identical to the human AAV-5, with differences predicted to be largely on the surface and on or under the spike-like protrusions. In an in vitro neutralization assay using human immunoglobulin G (IgG) (intravenous immune globulin [IVIG]), AAV-Go.1 had higher resistance than AAV-5 (100-fold) and resistance similar to that of AAV-4 or AAV-8. In an in vivo model, SCID mice were pretreated with IVIG to generate normal human IgG plasma levels prior to the administration of AAV human factor IX vectors. Protein expression after intramuscular administration of AAV-Go.1 was unaffected in IVIG-pretreated mice, while it was reduced 5- and 10-fold after administration of AAV-1 and AAV-8, respectively. In contrast, protein expression after intravenous administration of AAV-Go.1 was reduced 7.1-fold, similar to the 3.8-fold reduction observed after AAV-8administration in IVIG-pretreated mice, and protein expression was essentially extinguished after AAV-2 administration in mice pretreated with much less IVIG (15-fold). AAV-Go.1 vectors also demonstrated a marked tropism for lung when administered intravenously in SCID mice. The pulmonary tropism and high neutralization resistance to human preexisting antibodies suggest novel therapeutic uses for AAV-Go.1 vectors, including targeting diseases such as cystic fibrosis. Nonprimate sources of AAVs may be useful to identify additional capsids with distinct tropisms and high resistance to neutralization by human preexisting antibodies.

EB10, an Anti-FLT3 Monoclonal Antibody, Selectively Targets ALL Cell Lines and Primary ALL Blasts without Interfering with Normal Hematopoiesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2744-2744 ◽  
Author(s):  
Obdulio Piloto ◽  
Patrick Brown ◽  
Li Li ◽  
Bao Nguyen ◽  
Kyu-Tae Kim ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Lines ◽  
Scid Mice ◽  
Cytotoxic Agents ◽  
Radioactive Isotopes ◽  
Leukemic Cells ◽  
Class Iii ◽  
Antibody Treatment

Abstract The class III receptor tyrosine kinase, FLT3, is expressed by >90% of B-lineage acute lymphoblastic leukemias (ALL) blasts. In addition, it is expressed at extremely high levels in ALL patients with MLL-rearrangements or hyperdiploidy and sometimes mutated in these same patients. In this report, we investigated the effects of EB10, an anti-human FLT3 monoclonal antibody capable of preventing binding of FLT3 ligand (FL), on ALL cell lines and primary cells. In vitro studies, examining the ability of EB10 to inhibit FLT3 activation and downstream signaling in ALL cell lines and primary blasts, yielded variable results. In some cell lines FLT3 phosphorylation was inhibited and with it, downstream activation of pathways involving MAPK, AKT, and STAT5 phosphorylation. However, several cell lines actually exhibited FLT3 activation upon antibody treatment, possibly because of antibody-mediated receptor dimerization, and subsequent activation of downstream pathways. Nevertheless, through antibody-mediated cellular cytotoxicity (ADCC) such an antibody could still prove efficacious against leukemia cells in vivo. In fact, EB10 treatment significantly prolongs survival and/or reduces engraftment of ALL cell lines and primary ALL blasts in NOD/SCID mice. This effect might be even more pronounced in a host that was less immune compromised than are NOD/SCID mice. The leukemic cells surviving EB10 treatment in the mice were characterized by FACS analysis and found to express low levels or no FLT3. In contrast to the reduction in engraftment of human ALL primary blasts, EB10 treatment of NOD/SCID mice did not reduce engraftment of human hematopoietic CD34+ cells. Taken together, these data demonstrate that EB10 is selectively cytotoxic to ALL blasts while having little effect on normal hematopoiesis. Such an antibody, either naked or conjugated to radioactive isotopes or cytotoxic agents, may prove useful in the therapy of infant ALL as well as childhood and adult ALL patients whose blasts typically express FLT3.

The Multi-Targeted Receptor Tyrosine Kinase Inhibitor, ABT-869, Induces Apoptosis of AML Cells Both In Vitro and In Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 616-616 ◽  
Author(s):  
Deepa B. Shankar ◽  
Jenny C. Chang ◽  
Bertrand Parcells ◽  
Salemiz Sandoval ◽  
Junling Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Receptor Tyrosine Kinase ◽  
Scid Mice ◽  
Parp Cleavage

Abstract Children with acute myeloid leukemia (AML) have less than 60% overall survival despite aggressive chemotherapy and bone marrow transplantation. Only one third of the adult patients diagnosed with AML will be cured. AML blast cells from up to 30% of patients express a constitutively active receptor tyrosine kinase, FLT3-ITD, which contains an internal tandem duplication in the juxtamembrane domain. Patients with FLT3-ITD have a worse prognosis. ABT-869 is a novel multi-targeted small molecule inhibitor of receptor tyrosine kinases and is a potent inhibitor of FLT3, c-Kit, and all members of the VEGF and PDGF receptor families. To determine the effects of ABT-896 on AML cells, we treated AML cell lines, primary cells, and tumors in xenograft models with varying concentrations of the drug. In vitro viability assays showed that ABT-869 inhibited the growth of two different cell lines, MV-4-11 (human AML cell line that expresses FLT3-ITD) and BAF3-ITD (murine B-cell line stably transfected with the FLT3-ITD) at an IC50 of 10nM. ABT-869 was also effective against another mutation of FLT3, D835V, but at higher concentrations (IC50 of 100nM). Phosphorylation of FLT3 and activation of downstream signaling molecules, STAT5 and ERK, were inhibited by ABT-869 in a concentration-dependent manner. Cells were also stained with Annexin V-FITC and Propidium Iodide, and analyzed using FACS. ABT-869 induced apoptosis, caspase-3 activation, and PARP cleavage after 48 hours. To examine the in vitro effects of ABT-869 on normal hematopoietic progenitor cells, we performed methylcellulose-based colony assays with human bone marrow. No significant difference was observed in the number and type of colonies formed using BM cells treated with ABT-869 or control, up to a concentration of 1 micromolar. These results suggest that ABT-869 is not toxic to normal bone marrow progenitor cells at concentrations that are effective against AML cells. To examine the effects of ABT-869 in vivo, we treated SCID mice injected with MV-4-11, Baf3-ITD, Baf3-D835V, or Baf3-WT cells, with oral preparations of ABT-869. Complete regression of MV-4-11 tumors was observed in mice treated with ABT-869 at 20 and 40 mg/kg/day. No adverse effects were detected in the peripheral blood counts, bone marrow, spleen or liver. Histology of the tumors from the control-treated group showed a high degree of proliferation by Ki-67 staining, increased mitotic figures, and a well-defined tumor mass. In contrast, the tumors from mice treated with ABT-869 showed a number of apoptotic bodies by TUNEL staining and the presence of reactive, inflammatory cells. Interestingly, we also observed that mice that received ABT-869 the day after injection of AML cells remained tumor-free for over 2 months in contrast to the mice receiving the vehicle alone. Inhibition of FLT3 phosphorylation was demonstrated in the tumors from mice treated with ABT-869. We are evaluating the activity of ABT-869 treatment of SCID mice injected with Baf3-ITD, Baf3-D835V, or Baf3-WT cells. NOD-SCID mouse models are currently being used to analyze the effects of ABT-869 on primary AML cells in vivo. Our preclinical studies demonstrate that ABT-869 is effective and nontoxic, and provide rationale for the treatment and prevention of relapse in AML patients.

FLT3 Immunotherapy Can Eliminate Engraftment of ALL Cells in NOD/SCID Mice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 869-869
Author(s):  
Obdulio Piloto ◽  
Bao Nguyen ◽  
Patrick Brown ◽  
Kyu-Tae Kim ◽  
David Huso ◽  
...  
Keyword(s):  
Cell Lines ◽  
Scid Mice ◽  
Cytotoxic Agents ◽  
Radioactive Isotopes ◽  
Leukemic Cells ◽  
Pcr Analysis ◽  
Facs Analysis ◽  
Antibody Treatment

Abstract The class III receptor tyrosine kinase, FLT3, is expressed by over 90% of B-lineage acute lymphoblastic leukemias (ALL) blasts. In addition, it is expressed at extremely high levels in ALL patients with MLL-rearrangements or hyperdiploidy and sometimes mutated in these same patients. In this report, we investigated the effects of EB10, an anti-human FLT3 monoclonal antibody capable of preventing binding of FLT3 ligand (FL), on ALL cell lines and primary cells. In vitro studies, examining the ability of EB10 to inhibit FLT3 activation and downstream signaling in ALL cell lines and primary blasts, yielded variable results. In some cell lines FLT3 phosphorylation was inhibited and with it, downstream activation of pathways involving MAPK, AKT, and STAT5 phosphorylation. However, several cell lines actually exhibited FLT3 activation upon antibody treatment, possibly because of antibody-mediated receptor dimerization, and subsequent activation of downstream pathways. Nevertheless, through antibody-mediated cellular cytotoxicity (ADCC) such an antibody could still prove efficacious against leukemia cells in vivo. In fact, EB10 treatment significantly prolongs survival and/or reduces engraftment of several ALL cell lines and some primary ALL samples in NOD/SCID mice, even when EB10 treatment results in FLT3 activation of those cell lines in vitro. Moreover, FACS and PCR analysis of EB10 treated NOD/SCID mice surviving 150 days post leukemic cell injection revealed that FLT3 immunotherapy eliminated leukemic engraftment. The leukemic cells surviving EB10 treatment in the mice were characterized by FACS analysis and found to express lower levels of FLT3. To assess for resistance, cells surviving EB10 treatment were injected into NOD/SCID mice and treated with a single dose of EB10. FACS analysis revealed that these cells remain sensitive to EB10 treatment. Taken together, these data demonstrate that EB10 is cytotoxic to ALL blasts in vivo and EB10 treatment did not select for resistant clones. Such an antibody, either naked or conjugated to radioactive isotopes or cytotoxic agents, may prove useful in the therapy of infant ALL as well as childhood and adult ALL patients whose blasts typically express FLT3.

A Humanized Anti-Ganglioside GM2 Antibody, BIW-8962, Exhibits ADCC/CDC Activity against Multiple Myeloma Cells and Potent Anti- Tumor Activity in Mouse Xenograft Models.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1718-1718 ◽  
Author(s):  
Toshihiko Ishii ◽  
Asher Alban Chanan-Khan ◽  
Jazur Jafferjee ◽  
Noreen Ersing ◽  
Takeshi Takahashi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Phase 1 ◽  
Xenograft Model ◽  
Surface Expression ◽  
Scid Mice ◽  
Subcutaneous Xenograft ◽  
Anti Tumor Activity

Abstract BIW-8962 is a humanized anti-ganglioside GM2 (GM2) monoclonal antibody, produced by Poteligent technology to enhance ADCC activity. GM2 is expressed on many cancer cells including multiple myeloma (MM), small cell lung cancer and glioma cells. In this study, we evaluated the anti-myeloma activity of BIW-8962 in preclinical myeloma models both in vitro and in vivo. Expression of GM2 was analyzed in 15 human MM cell lines by FCM. Eleven out of 15 MM cell lines had positive surface expression of GM2. GM2 as a potential target was then verified in primary MM samples obtained from patients. Eleven out of 15 samples were positive for GM2. We then used two GM2 positive MM cell lines (U266B1 and KMS-11) and evaluated ADCC and CDC activity of BIW-8962 in vitro. BIW-8962 exhibited a potent ADCC and less potent CDC activity. In vivo anti-tumor activity of BIW-8962 was then examined using the standard subcutaneous xenograft model; KMS-11 was inoculated in the flank of SCID mice. BIW-8962 (intravenously administered biweekly for 3 weeks) exhibited a potent anti-tumor activity from as low a dose level as 0.1 mg/kg. Furthermore, in a more clinically relevant model, in which OPM-2/GFP (GM2 positive MM cell line) cells were intravenously inoculated into SCID mice with preferentially tumor growth within the bone marrow microenvironment, BIW-8962 (intravenously administered biweekly for 4 weeks, 10 mg/kg) suppressed OPM-2/GFP cell growth and serum M protein elevation, demonstrating in vivo anti-myeloma effect of BIW-8962. Our preclinical investigations rationalize clinical evaluation of BIW-8962 in patients with MM. Currently BIW-8962 is being investigated in a Phase 1 study in patients with multiple myeloma.

Effect of inducing HR deficiency using AVB500, a receptor tyrosine kinase AXL inhibitor, on response to olaparib in uterine serous cancer.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e18108-e18108
Author(s):  
Michael Driscoll Toboni ◽  
Barbara Blachut ◽  
Mary M Mullen ◽  
Jo'an Tankou ◽  
Hollie M Noia ◽  
...  
Keyword(s):  
Treatment Period ◽  
Cell Lines ◽  
Day Treatment ◽  
Parp Inhibitor ◽  
Tumor Burden ◽  
Scid Mice ◽  
In Vivo Studies ◽  
Treatment Groups

e18108 Background: Evidence suggests DNA repair is a therapeutic target in endometrial cancer (EC). Given this, we determined whether combination therapy with AVB500, an AXL inhibitor, could improve response in a uterine serous cancer (USC) model. Methods: Two USC cell lines (ARK1 & ARK4) were treated with AVB500 (Aravive Biologics, Houston, TX) in combination with the poly ADP ribose polymerase (PARP) inhibitor, olaparib. Colony forming assays were assessed after 4 days of treatment with either AVB500 alone, olaparib alone or combination treatment (olaparib + AVB500); colonies were stained and absorbance was obtained to calculate relative cell viability using Graph Pad Prism. Baseline homologous recombination (HR) status was determined after radiating cells with 10Gy and identifying RAD51 foci by immunofluorescence (IF). Cell lines were considered to be HR proficient if over 30% of the cells expressed RAD51 ( > 5 foci per cell). IF was conducted using a Leica confocal microscope and foci were quantified using FociCounter. In vivo studies were performed using NOD-SCID mice injected with 1 x 107 ARK1 cells intraperitoneally followed by treatment q3 days for a 14 and 21 day treatment period. Treatment groups were vehicle control, AVB500 alone, olaparib alone and olaparib with AVB500. Results: The absorbance for olaparib + AVB500 was significantly less than the olaparib only group in two assays involving ARK1s (0.417nm vs 0.756nm, p = 0.001; 0.320nm vs 0.620nm, p = 0.008) as well as in ARK4s (0.186nm vs 0.641nm, p = 0.003). The HR assay indicated both cell lines were HR proficient. After baseline HR proficiency was established, the cell lines were pretreated with AVB500 prior to radiation. When compared to cells without treatment with AVB500, IF showed a decrease in RAD51 foci per cell in ARK1 (2.7 vs 7.3, p = 0.0003) and ARK4 (6.3 vs 13.0, p = 0.0054). The proportion of ARK1 cells expressing RAD51 decreased to 21%, indicating HR deficiency. Lastly, NOD-SCID mice receiving olaparib + AVB500 had less tumor weight than those treated with olaparib alone (0.008g vs 0.138g, p = 0.002) and AVB500 alone (0.008g vs 0.145g, p = 0.0006) in a 14 day and a 21 day treatment period (0.212g vs 0.586g, p = 0.027 and 0.212 vs 0.494g, p = 0.005, respectively). Conclusions: HR proficient USC cell lines treated in vitro and in vivo with the combination of AVB500 and olaparib demonstrate an improved response to olaparib or AVB500 alone with a greater decrease in tumor burden. AVB500 appears to induce HR deficiency. Additional therapeutic and mechanistic experiments are ongoing.

scholarly journals RUNX1/CBFβ Dosage Is Critical for MLL Leukemias Development

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2187-2187
Author(s):  
Xiaomei Yan ◽  
Yoshihiro Hayashi ◽  
Xinghui Zhao ◽  
Aili Chen ◽  
Yue Zhang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Lines ◽  
Research Funding ◽  
Leukemia Cell ◽  
Down Regulation ◽  
Leukemia Cell Lines ◽  
Flanking Regions ◽  
The Impact

Abstract Transcription factors RUNX1/CBFβ play critical roles in hematopoiesis. Both of them are frequently involved in chromosomal translocations, point mutations, or deletions in acute leukemia. The mixed lineage leukemia (MLL) gene is also frequently involved in chromosomal translocations or partial tandem duplication in acute leukemia. We have previously shown that MLL, RUNX1, and CBFβ interact and form a regulatory complex to regulate downstream target genes. However, the functional consequence of MLL fusions on RUNX1/CBFβ activity remains unknown. To determine the impact of MLL fusion protein on RUNX1/CBFβ, we introduced either MLL, MLL-BP (longer N-terminal Flag-tagged MLL construct which contains CXXC domain; 1-1406), or MLL-fusions together with RUNX1, CBFβ, or both RUNX1 and CBFβ into 293T cells. MLL-BP and MLL fusions significantly decreased RUNX1 levels compared with controls (empty vector and MLL). CBFβ protein was mildly decreased by MLL-BP and MLL-fusions when expressed alone. However, when CBFβ was co-expressed with RUNX1, it was significantly decreased compared with controls. The expression levels of RUNX1 and CBFβ proteins in LSK cells from Mll-Af9 knock-in mice were significantly lower than those from wild-type (WT) mice. To confirm these findings in human acute myeloid leukemia (AML), we measured the expression of RUNX1 and CBFβ at both mRNA and protein levels in various leukemia cell lines. The expression levels of RUNX1 and CBFβ proteins were significantly decreased in AML cells with MLL fusion and MLL partial tandem duplication (MLL-PTD) compared with those in AML cells without MLL aberrations. MLL fusions still have CXXC domain. In MLL-PTD, the CXXC domain is duplicated. Our data showed that RUNX1 protein is not only down-regulated by MLL fusion proteins, but also by MLL-BP. Thus, to determine which region is involved in the down-regulation of RUNX1, we introduced a series of MLL deletion mutants into 293T cells and measured RUNX1 protein expression. MLL deletion mutants without CXXC domain had no effect on RUNX1 stability. The construct which contains point mutations in CXXC domain also lacked the ability to reduce RUNX1 expression. Furthermore, overexpression of only CXXC domain and flanking regions could down-regulate RUNX1 protein expression. These results suggest that MLL fusion proteins and the N-terminal MLL portion of MLL fusions down-regulate RUNX1 and CBFβ protein expression via the MLL CXXC domain and flanking regions. To understand the impact of RUNX1/CBFβ down-regulation on hematopoietic stem and progenitor cells (HSPCs), we generated RUNX1+/–/CBFβ+/– mice as a hypomorph model. The percentage of bone marrow (BM) LSK cells from RUNX1+/–/CBFβ+/– mice was significantly increased compared with that from WT mice. Using BM cells from these mice, we performed in vitro CFU assay and in vivo bone marrow transplantation (BMT) assay. BM cells from RUNX1+/–/CBFβ+/– mice provided more colonies in CFU assay compared with those from WT mice. To determine whether restoration of RUNX1 could repress the MLL mediated leukemogenesis, we retrovirally overexpressed WT RUNX1 in BM cells from Mll-Af9 knock-in mice. Using transduced BM cells, we performed in vitro CFU assay and in vivo BMT assay. RUNX1 overexpressed Mll-Af9 (Mll-Af9/RUNX1) cells underwent terminal differentiation after 2 times replating, while control vector transduced Mll-Af9 (Mll-Af9/Control) cells could still be replated more than 4 times. All the recipient mice transplanted with Mll-Af9/Control cells developed AML. In contrast, all the recipient mice transplanted with Mll-Af9/RUNX1 never develop AML. Furthermore, when we treated MLL leukemia cell lines with DOT1L inhibitor (EPZ-5676), RUNX1 protein levels in these MLL leukemia cell lines were significantly increased 48 hours after the treatment in comparing with controls treated with DMSO. However, there was no significant mRNA expression level change of RUNX1within 48 hours. Future studies are needed to fully understand the mechanism of whether this increasing RUNX1 protein level by DOT1L inhibitor is through blocking CXXC domain and flanking regions mediated degradation. In conclusion, MLL aberrations down-regulate RUNX1/CBFβ via their CXXC domain and flanking regions. Down-regulation of RUNX1/CBFβ plays critical role for MLL mediated leukemia development. Targeting RUNX1/CBFβ levels allows us to test novel therapies for MLL leukemias. Disclosures Mulloy: Celgene: Research Funding; Seattle Genetics: Research Funding; Amgen: Research Funding; NovImmune: Research Funding.

scholarly journals Development of Novel Chalcone Analogs as Potential Multi-Targeted Therapies for Castration-Resistant Prostate Cancer

2021 ◽  
Author(s):  
Ola Hussein ◽  
Feras Alali ◽  
Ala‐Eddin Al Mustafa ◽  
Ashraf Khalil
Keyword(s):  
Prostate Cancer ◽  
Cell Lines ◽  
In Vivo Studies ◽  
Treatment Modalities ◽  
Castration Resistant Prostate Cancer ◽  
Castration Resistant ◽  
Biological Studies ◽  
In Silico Admet

Prostate cancer (PCa) is the second most frequently diagnosed malignancy, as well as a leading cause of cancer-related mortality in men globally. Despite the initial response to hormonal targeted therapy, the majority of patients ultimately progress to a lethal form of the disease, castration-resistant prostate cancer (CRPC). Therefore, the objective of this study was to discover and develop novel treatment modalities for CRPC. Chalcones are among the highly attractive scaffolds being investigated for their antitumor activities. A library of 26 chalcone analogs were designed, synthesized and evaluated as potential therapies for CRPC. The design was guided by in-silico ADMET prediction in which analogs with favorable drug-likeness properties were prioritized. The new compounds were synthesized, purified and characterized by extensive structural elucidation studies. The compounds in vitro cytotoxicity was evaluated against two androgen receptor (AR)-negative prostate cancer cell lines (PC3 and DU145). Among the tested compounds, pyridine containing analogs (13, 15 and 16) showed potent antiproliferative activities with IC50 values ranging between 4.32-6.47 µM against PC3 and DU145 cell lines. Detailed biological studies of the lead molecule 16 revealed that it can significantly induce apoptosis through upregulation of Bax and downregulation of Bcl-2. In addition, compound 16 potently inhibited colony formation and reduced cell migration of AR-negative PCa cell lines (PC3 and DU145). The molecular pathway analysis showed that the anticancer activity of compound 16 is associated with blocking of ERK1/2 and Akt activities. Furthermore, compound 16 inhibited angiogenesis in the chick chorioallantoic membrane (CAM) model as compared to control. Structure-activity relationship study revealed that the cytotoxicity could dramatically improve via changing the methoxylation pattern by more than 2-folds (IC50 << 2.5 μM). These results indicate that pyridine-based chalcones could serve as promising lead molecules for the treatment of CRPC; thus, further in vitro and in vivo studies are warranted.

Preclinical Rationale for the Use of the Multikinase Inhibitor Sorafenib in the Treatment of Human Lymphomas

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2605-2605
Author(s):  
Carmelo Carlo-Stella ◽  
Cristiana Lavazza ◽  
Arianna Giacomini ◽  
Loredana Cleris ◽  
Daniela Sia ◽  
...  
Keyword(s):  
Growth Factor ◽  
Tumor Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Scid Mice ◽  
Multikinase Inhibitor ◽  
Preclinical Data

Abstract Introduction: The multikinase inhibitor Sorafenib (Nexavar, Bayer) exerts a remarkable activity against a variety of nonhematological tumors by blocking tumor cell proliferation and angiogenesis through the inhibition of the RAF/MEK/ERK pathway, as well as the receptor tyrosine kinases vascular endothelial growth factor receptors (VEGFRs), platelet-derived growth factor receptor (PDGFR), c-KIT, Flt3, and RET. Several lines of evidence suggest that sorafenib might have a relevant clinical impact in the therapy of malignant lymphomas by overcoming the cytoprotective effects of ERK, Mcl-1, and Bcl-XL. However, preclinical data establishing a rationale for the clinical use of sorafenib in lymphomas are still lacking. The present studies aimed to investigate the activity and the mechanism(s) of action of sorafenib in human lymphomas. Methods: The effects of sorafenib were evaluated in vitro using a panel of six human cell lines of different phenotypes, including JVM-2 (B-Chronic Lymphocytic Leukemia), Granta-519 (Mantle Cell Lymphoma), DOHH2 (Follicular Lymphoma), SU-DHL-4V (Diffuse Large B-Cell Lymphoma), HD-MY-Z (Hodgkin Lymphoma), and KMS-11 (Multiple Myeloma) cell lines. Additionally, the antitumor efficacy and mechanism of action of sorafenib were investigated in vivo by means of five lymphoma xenograft models in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Results: In vitro, the response of cell lines to sorafenib (1–10 μM for 24–48 hours) was evaluated by detecting apoptotic cell death with the annexin-V/propidium iodide double staining assay, and viable cell countings with the Trypan blue dye exclusion test. All 6 cell lines responded to sorafenib with values of 50% inhibitory concentrations ranging from 1 to 7.5 μM. In contrast, normal CD34+ cells remain insensitive to the drug up to 15 μM. Despite significant rates of sorafenib-induced apoptosis were seen in all cell lines, activation of caspase-3 analyzed by fluorescent-activated cell sorter was only detected in DOHH-2 and JVM-2 cell lines. The phosphorylation status of mitogen-activated protein kinase (MAPK) was investigated using the human phospho-MAPK Array kit (R&D systems), analyzed with the open source imaging software ImageJ (http://rsb.info.nih.gov/ij/), and then validated by Western blotting. Sorafenib induced a significant reduction of pAkt1, pAkt2, and pAkt3 in SU-DHL-4V, Granta-519, and JVM-2 cell lines, whereas p38 phosphorylation levels were significantly reduced in all but one cell line (KMS-11). Reduced levels of pMEK, pERK1 and pERK2 were detected in SU-DHL-4V, KMS-11, Granta-519, and HD-MY-Z cell lines. Down-regulation of MCL-1 was seen in HD-MY-Z, JVM-2, and DOHH-2 cell lines. In vivo, the activity of sorafenib was evaluated in NOD/SCID mice bearing subcutaneous tumor nodules. Animals with tumors averaging from 140 to 160 mg were randomly grouped to receive sorafenib (90 mg/kg body weight, IP, once daily for 15 days) or control vehicle. Sorafenib significantly (P ≤0.001) reduced the growth of subcutaneous HD-MY-Z, KMS-11, Granta-519, SU-DHL-4V, and JVM-2 nodules, with values of tumor growth inhibition of 70%, 52%, 40%, 37%, and 24%, respectively. In control mice, TUNEL staining of tumor sections showed large areas of viable cells without significant necrosis, whereas a 2- to 5-fold increase of necrotic areas was detected in sorafenib-treated mice bearing the different lymphoma xenografts. Analysis of tumor vasculature by means of in vivo biotinylation of endothelial cells with sulfo-NHS-LC-biotin showed a 30% to 60% reduction of vessel density in sorafenib-treated mice bearing the different lymphoma xenografts. Conclusions: Sorafenib efficiently targets a variety of human lymphomas representative of different phenotypes by inhibiting tumor angiogenesis and directly affecting tumor cell survival. Our preclinical data establish a rationale for exploring the clinical activity of sorafenib in human lymphomas.

Sign in / Sign up

Export Citation Format

Share Document