Anti-AML activity of a novel beta-catenin antagonist BC2059.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 10605-10605
Author(s):  
Kapil N. Bhalla ◽  
Warren Fiskus ◽  
Sunil Sharma ◽  
Stephen Horrigan ◽  
Uma Mudunuru ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cyclin D1 ◽  
Progenitor Cells ◽  
Survivin Expression ◽  
Beta Catenin ◽  
Tail Vein ◽  
Nsg Mice ◽  
Induced Apoptosis

10605 Background: The canonical WNT-β-catenin pathway is essential for self-renewal, growth and survivalof AML stem and progenitor cells. Deregulated WNT signaling inhibits degradation of β-catenin, causing increased nuclear translocation and interaction of β-catenin with the TCF/LEF transcription factor, which up regulates cyclin D1, Myc and survivin expression in AML progenitor cells. BC2059 (β-Cat Pharmaceuticals) is a potent, small molecule, anthraquinone oxime-analog, which inhibits WNT-β catenin pathway by promoting the degradation and attenuation of β-catenin levels. Methods: We determined the in vitro anti-AML activity of BC2059 (BC) (250 to 1000 nM) against cultured and primary human AML blast progenitors, as well as evaluated the in vivo anti-AML efficacy of BC in NOD-SCID and NOD-SCID-IL2γ receptor deficient (NSG) mice. Results: BC induced cell cycle G1 phase accumulation and apoptosis (40%) of the cultured OCI-AML3, HL-60 and HEL92.1.7 (HEL) AML cells. BC dose-dependently also induced apoptosis of primary AML versus normal progenitors. Treatment with BC resulted in proteasomal degradation and decline in the nuclear levels of β-catenin, which led to decreased activity of the LEF1/TCF4 transcription factor highlighted by reduced TOP-FLASH luciferase activity in the AML cells. This was associated with reduced protein levels of cyclin D1, MYC and survivin. Co-treatment with BC and the histone deacetylase inhibitor panobinostat (PS) (10 to 20 nM) synergistically induced apoptosis of cultured and primary AML blasts. Following tail vein infusion and establishment of AML by OCI-AML3 or HEL cells in NOD-SCID mice, treatment with BC (5, 10 or 15 mg/kg b.i.w, IV) for three weeks demonstrated improved survival, as compared to the control mice (p <0. 001). Survival was further improved upon co-treatment with BC and PS (5 mg/kg IP, MWF). BC treatment (5 or 10 mg/kg IV) also dramatically improved survival of NSG mice with established human AML following tail-vein injection of primary AML blasts expressing FLT3 ITD. Mice did not experience any toxicity or weight loss. Conclusions: These findings highlight the notable pre-clinical in vitro and in vivo activity and warrant further development and in vivo testing of BC against human AML.

Treatment with β-Catenin Antagonist BC2059 Exerts Single Agent Efficacy and Exerts Improved Activity with Tyrosine Kinase Inhibitor (TKI) or Pan-Histone Deacetylase (HDAC) Inhibitor Against Human CML and Myeloproliferative Neoplasm (MPN) Progenitor Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 65-65
Author(s):  
Warren Fiskus ◽  
Jacqueline Smith ◽  
Uma Mudunuru ◽  
Stacey Hembruff ◽  
Ruben Reyes ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cyclin D1 ◽  
Progenitor Cells ◽  
Hdac Inhibitor ◽  
Single Agent ◽  
Tail Vein ◽  
Equity Ownership ◽  
Induced Apoptosis ◽  
20 Nm

Abstract Abstract 65 The canonical WNT-β-catenin pathway is essential to the cellular processes of self-renewal, growth and survival. Deregulated WNT-β-catenin in transformed hematopoietic progenitor cells inhibits the multi-protein degradation complex formed by axin, adenomatous polyposis coli (APC) and glycogen synthase kinase 3β (GSK3β). This results in the preservation, nuclear translocation and interaction of β-catenin with the T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factor, which regulates the expression of genes such as cyclin D1, Myc, survivin and Axin. BC2059 (β-Cat Pharmaceuticals) is a potent small molecule, anthraquinone oxime-analog inhibitor of the WNT-β catenin pathway. Treatment with BC2059 mediates the degradation of β-catenin. In the present studies, we determined the activity of BC2059 in human cultured and primary CML and advanced MPN versus normal progenitor cells. Exposure to 50 to 100 nM of BC2059 induced cell cycle G1 phase accumulation and apoptosis (40 to 80%) of the cultured MPN cells HEL92.1.7 (HEL) and UKE1 cells expressing the mutant JAK2V617F, as well as of the CML K562 and LAMA-84 cells expressing BCR-ABL. BC2059 treatment also induced apoptosis of CD34+ primary MPN cells derived from the peripheral blood of patients with advanced MPN expressing mutant JAK2, as well as of primary CD34+ CML progenitor cells. In contrast, as compared to the untreated controls, BC2059 treatment did not induce apoptosis of normal CD34+ progenitor cells. Exposure to BC2059 resulted in marked down regulation of β-catenin protein levels and the activity of the LEF1/TCF4 transcription factor, which was accompanied with reduced levels of cyclin D1, MYC, survivin and up regulation of Axin 2 levels, as detected by immunoblot analyses of the cell lysates of BC2059-treated CML and MPN cells. We also determined the in vivo anti-MPN activity of BC2059. Following the tail vein infusion of HEL cells and establishment of MPN, NOD-SCID mice were treated with 15 or 20 mg/Kg of BC2059 administered b.i.w for three weeks via the tail vein. As compared to the control, BC2059-treated mice demonstrated significantly improved survival (p <0. 001). Next, we examined the effects of co-treatment with BC2059 (20 to 50 nM) and JAK2-targeted TKI TG101209 (TG) (200–1000 nM) or BCR-ABL-targeted TKI nilotinib (10–20 nM) against MPN or CML cells, respectively. As compared to treatment with each agent alone co-treatment with BC2059 and TG synergistically induced apoptosis of HEL and primary CD34+ MPN cells. Additionally, co-treatment with BC2059 and nilotinib induced synergistic apoptosis of K562 and primary CML progenitor cells. Further, combined treatment with BC2059 and the HDAC inhibitor panobinostat (10 to 20 nM) also induced significantly more apoptosis of HEL and K562 (p < 0.01), as well as of the primary CD34+ MPN and primary CML progenitor cells. In normal CD34+ progenitor cells, the BC2059-based combinations were remarkably less toxic (p < 0.01). These findings demonstrate that BC2059 exerts notable in vitro and in vivo activity against human MPN and CML versus normal CD34+ progenitor cells. Additionally, BC2059 may exert superior activity in combination with JAK2 or BCR-ABL-targeted TKI, or with pan-HDAC inhibitor against human MPN or CML progenitor cells. Disclosures: Reyes: Millennium, Sanofi Aventis: Consultancy. Horrigan:BetaCat Pharmaceuticals: Employment, Equity Ownership. Sharma:Beta Cat Pharmaceuticals: Equity Ownership.

Pre-Clinical Efficacy of Combined Therapy with Novel β-Catenin Antagonist BC2059 and Histone Deacetylase Inhibitor Against Cultured and Primary AML Blast Progenitor Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 287-287
Author(s):  
Kapil Bhalla ◽  
Warren Fiskus ◽  
Stephen K. Horrigan ◽  
Sunil Abhyankar ◽  
Joseph McGuirk ◽  
...  
Keyword(s):  
Cyclin D1 ◽  
Progenitor Cells ◽  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitor ◽  
Nuclear Accumulation ◽  
Hematopoietic Stem ◽  
Tail Vein ◽  
Deacetylase Inhibitor ◽  
Induced Apoptosis

Abstract Abstract 287 The canonical WNT-β-catenin pathway is essential for self-renewal, growth and survival of AML stem and early blast progenitor cells (BPCs). Deregulated WNT signaling in AML inhibits polyubiquitylation and proteasomal degradation of β-catenin by the multi-protein complex. The inactivation of the degradation complex for β-catenin results in the preservation, nuclear translocation and activity of β-catenin in AML BPCs. This is especially notable in AML BPCs expressing FLT3-ITD, where increased activity of PI3K/AKT phosphorylates and inactivates GSK3β, thereby inhibiting phospho-degradation, increasing stability and nuclear accumulation of β-catenin. Activated FLT3 kinase has also been shown to directly phosphorylate β-catenin and promote its stabilization and nuclear localization. As a co-activator, in the nucleus β-catenin interacts with the bipartite T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factor, which increases the expression of pro-growth and pro-survival genes, including cyclin D1, Myc and survivin. Aberrant expression of LEF1 in hematopoietic stem cells has been shown to induce AML. β-catenin is also required for the HOXA9 and MEIS1 or MLL-AF9-mediated transformation of hematopoietic stem/progenitor cells. Thus, reactivation of β-catenin is required for maintenance of leukemic transformation making it an attractive drug target in AML. Here, we determined the in vitro and in vivo anti-AML activity of BC2059 (β-Cat Pharmaceuticals), a potent, small molecule, anthraquinone oxime-analog, against cultured (HL-60, OCI-AML3 and MV4-11 cells) and primary AML BPCs with or without the expression of FT3-ITD. First, as compared to the normal human CD34+ cells, FLT3-ITD expressing AML BPCs showed increased levels of β-catenin, mostly in the nucleus, as determined by confocal immunofluorescence microscopy. Exposure to 100 nM of BC2059 (BC) induced β-catenin degradation through the proteasome, as well as attenuated the nuclear and cytoplasmic levels of β-catenin in the cultured and primary AML BPC. Chromatin immunoprecipitation with anti-β-catenin antibody demonstrated that treatment with BC2059 (BC) reduced the binding of β-catenin to the WNT response elements (WRE) in the promoter DNA of its target genes, including Myc, cyclin D1 and survivin. Estimation of the intracellular luciferase levels in AML cells transfected with the TOP/FLASH versus FOP/FLASH construct showed that treatment with BC2059 significantly reduced only the TOP-FLASH luciferase activity, indicating that BC inhibits the expression of genes with promoters containing WRE elements. This was associated with reduced mRNA and protein levels of cyclin D1, MYC and survivin. Treatment with BC dose-dependently induced apoptosis of cultured and primary AML BPCs (up to 70%), including apoptosis of the CD34+CD38-Lin- AML BPCs. In contrast, BC induced apoptosis in < 10% of normal CD34+ progenitor cells. We have previously reported that treatment with the histone deacetylase inhibitor panobinostat (PS) attenuates p-FLT3, p-AKT and p-GSK3β levels in AML BPCs expressing FLT3-ITD. Consistent with this, here, we determined that co-treatment with BC and PS (10 to 20 nM) synergistically induced apoptosis of cultured and primary AML BPCs. Against primary AML BPCs expressing FLT3-ITD, co-treatment with the FLT3 kinase inhibitor AC220 (100 nM) further augmented BC mediated depletion of the cytoplasmic and nuclear levels of β-catenin and significantly enhanced BC-induced apoptosis (p < 0.01). This was associated with induction of BIM and p27 with depletion of MCL-1 levels. Following tail vein infusion and establishment of AML by OCI-AML3 cells in NOD-SCID mice, treatment with BC (5 or 10 mg/kg b.i.w, IV) for three weeks demonstrated improved survival of the mice compared to the vehicle control treated mice (p <0. 001). Survival was further improved upon co-treatment with BC and PS (5 mg/kg IP, MWF). BC treatment (5 or 10 mg/kg IV) also dramatically improved survival of the NOD/SCID/IL2Rγ-depleted mice with established human AML following tail-vein injection of primary AML BPCs expressing FLT3 ITD. Mice did not experience any toxicity or weight loss. These findings support a compelling rationale for further development and in vivo testing of the BC-based combination with PS and FLT3 antagonist against human AML BPCs. Disclosures: Horrigan: BetaCat Pharmaceuticals: Employment. Sharma:BetaCat Pharmaceuticals: Equity Ownership.

scholarly journals Combined Targeting of β-Catenin and FLT-3 Is Synergistically Lethal Against Cultured and Primary AML Blast Progenitor Cells Expressing FLT3 Internal Tandem Duplication (ITD)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 618-618
Author(s):  
Saikat Saha ◽  
Warren Fiskus ◽  
Sunil Sharma ◽  
Bhavin Shah ◽  
Anna T Rogojina ◽  
...  
Keyword(s):  
Cyclin D1 ◽  
Progenitor Cells ◽  
Nuclear Localization ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Adaptor Protein ◽  
Beta Catenin ◽  
Induced Apoptosis ◽  
Nuclear Levels

Abstract β-catenin acts as a co-activator for the T-cell factor (TCF) 4/lymphoid enhancer factor (LEF) 1 bipartite transcription factor at the promoters of the WNT-β-catenin target genes, including cyclin D1, c-Myc and survivin. The canonical WNT-β-catenin pathway is documented to be essential for self-renewal, growth and survival of the AML stem and blast progenitor cells (BPCs), which has also been correlated with a poor prognosis in AML. In AML stem/BPCs expressing mutant FLT3-ITD, increased PI3K/AKT activity causes phosphorylation and inactivation of GSK3β, thereby preventing degradation, promoting stabilization and nuclear localization of β-catenin. Additionally, FLT3 can also directly mediate the tyrosine phosphorylation of β-catenin, thereby stabilizing and promoting the nuclear localization and binding of β-catenin to TCF4. TBL1 (transducin beta-like) is an adaptor protein, which binds to nuclear β-catenin and promotes its co-factor activity with TCF4/LEF1 in mediating transcription of the target genes, including c-Myc, cyclin D1 and survivin. Therefore, we hypothesized that targeted disruption of TBL1-β-catenin binding or depletion of TBL1 would abrogate the pro-growth and oncogenic signaling of β-catenin in AML BPCs, especially those expressing FLT3-ITD. Here, we demonstrate that treatment with 20 to 100 nM of BC2059 (β-Cat Pharmaceuticals), a small molecule, anthraquinone oxime-analog, disrupts the binding of β-catenin to TBL1 (by anti-TBL1 pull down and immunofluorescence analyses) and promotes proteasomal degradation of β-catenin, thereby attenuating the nuclear levels of β-catenin in the cultured (OCI-AML3, MOLM13 and MV4-11), as well as in primary (p) AML BPCs. Concomitantly, BC2059 treatment inhibited the mRNA and protein expression of c-Myc, cyclin D1 and survivin, while de-repressing p21 and Axin2. BC2059 also dose dependently inhibited growth and induced apoptosis of cultured and CD34+ pAML BPCs expressing FLT3-ITD (40 to 60%), but not of normal CD34+ bone marrow progenitor cells (p < 0.01). Transient knockdown of TBL1 or beta catenin (60 to 70%) by lentivirus-transduced shRNA caused loss of viability in MOLM13 cells, which was significantly enhanced by treatment with BC2059 (p < 0.01). BC2059 also induced apoptosis of MOLM13-TKIR cells that were isolated in vitro to exhibit resistance to FLT3 antagonists (approximately 50-fold). Notably, BC2059 treatment (10 mg/kg, t.i.w., by IV injection) also exerted potent in vivo anti-AML activity and significantly improved the survival of immune depleted mice engrafted with cultured and patient-derived pAML BPCs (p < 0.001). Since compared to the control OCI-AML3 cells, BC2059 demonstrated significantly greater lethality against the OCI-AML3 cells ectopically overexpressing FLT3-ITD (approximately 8-fold), we hypothesized that co-treatment with a FLT3 antagonist would further reduce the nuclear levels of β-catenin and enhance the lethal activity of FLT3-antagonist against AML BPCs expressing FLT3-ITD. Indeed, co-treatment with BC2059 (50 nM) and the FLT3-antagonist quizartinib or ponatinib (100 to 200 nM), versus each agent alone, caused more reduction in the nuclear levels and binding of β-catenin to TBL1 (by confocal immunofluorescence analysis). This was associated with greater decline in the expression of c-Myc, cyclin D1 and survivin, but increase in the levels of p21 and BIM. Compared to each agent alone, co-treatment with BC2059 and quizartinib or ponatinib also synergistically induced apoptosis of the FLT3-ITD expressing cultured (MOLM13 and MV4-11) and pAML BPCs (combination indices of < 1.0, by isobologram analyses) but not of normal CD34+ progenitor cells. Treatment with BC2059 (25 to 100 nM) also significantly increased the apoptosis observed by the shRNA mediated incomplete knockdown of TBL1 or β-catenin (approximately 70%) in MOLM13 cells (p < 0.01). Collectively, our findings support that targeted inhibition of the levels and binding of β-catenin to TBL by BC2059 and FLT3-antagonist is a promising approach to exert lethal activity against AML BPCs expressing FLT3-ITD. Further pre-clinical development of this combination therapy against FLT3-ITD expressing AML is progressing. Disclosures No relevant conflicts of interest to declare.

Selective elimination of leukemic CD34+ progenitor cells by cytotoxic T lymphocytes specific for WT1

Blood ◽  
2000 ◽  
Vol 95 (7) ◽  
pp. 2198-2203 ◽  
Author(s):  
Liquan Gao ◽  
Ilaria Bellantuono ◽  
Annika Elsässer ◽  
Stephen B. Marley ◽  
Myrtle Y. Gordon ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Chronic Myeloid Leukemia ◽  
T Lymphocytes ◽  
Progenitor Cells ◽  
Cytotoxic T Lymphocytes ◽  
Colony Formation ◽  
Myeloid Leukemia ◽  
Leukemia Cell

Abstract Hematologic malignancies such as acute and chronic myeloid leukemia are characterized by the malignant transformation of immature CD34+ progenitor cells. Transformation is associated with elevated expression of the Wilm's tumor gene encoded transcription factor (WT1). Here we demonstrate that WT1 can serve as a target for cytotoxic T lymphocytes (CTL) with exquisite specificity for leukemic progenitor cells. HLA-A0201– restricted CTL specific for WT1 kill leukemia cell lines and inhibit colony formation by transformed CD34+ progenitor cells isolated from patients with chronic myeloid leukemia (CML), whereas colony formation by normal CD34+ progenitor cells is unaffected. Thus, the tissue-specific transcription factor WT1 is an ideal target for CTL-mediated purging of leukemic progenitor cells in vitro and for antigen-specific therapy of leukemia and other WT1-expressing malignancies in vivo.

Pre-Clinical Efficacy of Combined Therapy with LSD1 Antagonist SP-2509 and Pan-Histone Deacetylase Inhibitor Against AML Blast Progenitor Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 868-868 ◽  
Author(s):  
Warren Fiskus ◽  
Sunil Sharma ◽  
Sunil Abhyankar ◽  
Joseph McGuirk ◽  
David J. Bearss ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Histone Deacetylases ◽  
Combined Therapy ◽  
Colony Growth ◽  
Clinical Activity ◽  
Gene Promoters ◽  
Chromatin Mark ◽  
Tail Vein ◽  
Nsg Mice

Abstract Abstract 868 LSD1 (KDM1A) is an FAD-dependent histone demethylase, with homology to amine oxidases. LSD1 demethylates di- and mono-methylated lysine (K) 4 on histone H3, reducing the permissive H3K4Me3 chromatin mark for gene expression. LSD1 forms a complex with the histone deacetylases (HDAC) 1 and 2 and with the co-repressor CoREST, which stimulates the activity of LSD1 toward nucleosomes. While high LSD1 expression may be an effector of blocked differentiation and confers poor prognosis in AML, LSD1 inhibition induces the expression of myeloid–differentiation associated genes and attenuates growth of AML blast progenitor cells (BPCs). Recently, LSD1 was shown to sustain the in vivo leukemogenic potential of MLL-AF9 expressing leukemia stem cells. Also, co-treatment with the LSD1 inhibitor tranylcypromine (TCP) and all-trans retinoic acid (ATRA) was shown to diminish the engraftment of primary AML BPCs in vivo in NOD-SCID-γIL-2 receptor deficient (NSG) mice. Previous studies have shown that HDAC inhibitors attenuate the levels of LSD1 through Sp1 inhibition. SP-2509 is a potent and selective FAD-binding pocket, non-MAOA and MAOB, inhibitor with an IC50 of 13 nM for LSD1. In the present studies, we determined the chromatin effects and anti-AML efficacy of SP-2509 alone and in combination with the pan-HDAC inhibitor panobinostat (PS) (Novartis Pharmaceuticals) in cultured (HL-60, OCI-AML3, MV4-11, MOLM13, THP1 and SKM1 cells) and primary human AML BPCs. Treatment with SP-2509 (250 to 1000 nM) dose-dependently increased the levels of H3K4Me2 & Me3 chromatin mark, and chromatin immunoprecipitation followed by QPCR analyses showed an increase in the H3K4Me3 mark on the gene promoters of KLF4, HMOX1, p57 and p21 in AML BPCs. SP-2509 treatment attenuated the binding of LSD1 with CoREST, accompanied with increased levels of p16, p21 and p27 in AML BPCs. Consistent with this, treatment with SP-2509 inhibited the suspension and colony growth of AML BPCs regardless of whether they expressed MLL fusion oncoproteins. Knockdown of LSD1 by shRNA also inhibited the suspension and colony growth of AML blast progenitor cells. SP-2509 also induced C/EBPα expression and features of morphologic differentiation in the cultured and primary AML BPCs. Following tail vein infusion and establishment of AML by OCI-AML3 or MOLM13 cells in NOD/SCID mice, treatment with SP-2509 (25 mg/kg b.i.w. via IP injection) for three weeks demonstrated improved survival of the mice compared to the vehicle control treated mice (p <0. 001). We have previously reported that treatment with PS depleted polycomb repressive complex proteins EZH2, SUZ12 and BMI1 but also reduced LSD1 expression in AML cells. Co-treatment with PS enhanced SP-2509-induced chromatin effects and differentiation of AML cells. Also, PS and SP-2509 synergistically induced apoptosis of the cultured AML OCI-AML3, MOLM13 and MV4-11cells (combination indices, CI <1.0). Additionally, co-treatment with SP-2509 sensitized AML cells to ATRA-induced differentiation. Notably, co-treatment with SP-2509 and PS also induced significantly greater loss of viability of primary AML BPCs but not of normal CD34+ cells. SP-2509 treatment (15 mg/kg b.i.w. IP) also dramatically improved survival of NSG mice with established human AML following tail-vein injection of primary AML blasts. Survival was further significantly improved upon co-treatment with SP-2509 and PS (5 mg/kg IP, MWF) (p < 0.001). Mice did not experience any toxicity or weight loss. Taken together, these findings demonstrate promising pre-clinical activity of combined therapy with SP-2509 and PS, warranting further in vivo development and testing of SP-2509 against human AML. Disclosures: Sharma: Salarius Pharmaceuticals: Equity Ownership.

scholarly journals Isoliquiritigenin inhibits the proliferation, migration and metastasis of Hep3B cells via suppressing cyclin D1 and PI3K/AKT pathway

2020 ◽  
Vol 40 (1) ◽  
Author(s):  
Yun Huang ◽  
Chen Liu ◽  
Wu-Cha Zeng ◽  
Guo-Yan Xu ◽  
Jian-Min Wu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Inhibitory Effect ◽  
Cycle Arrest ◽  
Novel Drugs ◽  
Epithelial Marker ◽  
Hep3b Cells ◽  
Induced Apoptosis

Abstract The overall survival rate of patients with hepatocellular carcinoma (HCC) has remained unchanged over the last several decades. Therefore, novel drugs and therapies are required for HCC treatment. Isoliquiritigenin (ISL), a natural flavonoid predominantly isolated from the traditional Chinese medicine Glycyrrhizae Radix (Licorice), has a high anticancer potential and broad application value in various cancers. Here, we aimed to investigate the anticancer role of ISL in the HCC cell line Hep3B. Functional analysis revealed that ISL inhibited the proliferation of Hep3B cells by causing G1/S cell cycle arrest in vitro. Meanwhile, the inhibitory effect of ISL on proliferation was also observed in vivo. Further analysis revealed that ISL could suppress the migration and metastasis of Hep3B cells in vitro and in vivo. Mechanistic analysis revealed that ISL inhibited cyclin D1 and up-regulated the proteins P21, P27 that negatively regulate the cell cycle. Furthermore, ISL induced apoptosis while inhibiting cell cycle transition. In addition, phosphatidylinositol 3′-kinase/protein kinase B (PI3K/AKT) signal pathway was suppressed by ISL treatment, and the epithelial marker E-cadherin was up-regulated when the mesenchymal markers Vimentin and N-cadherin were down-regulated. In brief, our findings suggest that ISL could be a promising agent for preventing HCC tumorigenesis and metastasis.

scholarly journals EZN-2208 treatment suppresses chronic lymphocytic leukaemia by interfering with environmental protection and increases response to fludarabine

Open Biology ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 190262
Author(s):  
Roberta Valsecchi ◽  
Nadia Coltella ◽  
Daniela Magliulo ◽  
Lucia Bongiovanni ◽  
Lydia Scarfò ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Chronic Lymphocytic Leukaemia ◽  
Environmental Protection ◽  
Lymphocytic Leukaemia ◽  
Therapeutic Approaches ◽  
Leukaemic Cells ◽  
Induced Apoptosis ◽  
Early Phases

The transcription factor HIF-1α is overexpressed in chronic lymphocytic leukaemia (CLL), where it promotes leukaemia progression by favouring the interaction of leukaemic cells with protective tissue microenvironments. Here, we tested the hypothesis that a pharmacological compound previously shown to inhibit HIF-1α may act as a chemosensitizer by interrupting protective microenvironmental interactions and exposing CLL cells to fludarabine-induced cytotoxicity. We found that the camptothecin-11 analogue EZN-2208 sensitizes CLL cells to fludarabine-induced apoptosis in cytoprotective in vitro cultures; in vivo EZN-2208 improves fludarabine responses, especially in early phases of leukaemia expansion, and exerts significant anti-leukaemia activity, thus suggesting that this or similar compounds may be considered as effective CLL therapeutic approaches.

Survivin Is Negatively Regulated by Early Growth Response (Egr)-1 Transcription Factor in Acute Myeloid Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1943-1943 ◽  
Author(s):  
Ingo Tamm ◽  
Karin Schmelz ◽  
Bernd Dörken ◽  
Mandy Wagner
Keyword(s):  
Transcription Factor ◽  
Cell Lines ◽  
Growth Response ◽  
Survivin Expression ◽  
Early Growth ◽  
Survivin Promoter ◽  
Over Expression ◽  
Early Growth Response ◽  
Induced Apoptosis

Abstract Survivin, a member of the inhibitor of apoptosis protein family, is involved in both, inhibition of apoptosis and regulation of cell division. It is expressed in embryonic and fetal tissues as well as in the majority of human leukemias, but is undetectable in normal differentiated adult tissue in vivo. The molecular mechanisms involved in the cancer-specific re-expression of survivin are unclear. In this study, we describe a novel mechanism for over-expression of survivin in AML. Using electrophoretic mobility shift assays (EMSA), we show that the early growth response (Egr)-1 transcription factor binds to the sequence 5′ GAGGGGGCG 3′ within the human survivin promoter after induction by phorbol 12-myristate-13-acetate (PMA) in vitro. Furthermore, chromatin immunoprecipitation (ChIP) analysis confirmed the specific binding of Egr-1 to the proximal survivin promoter in PMA treated entire leukemia cells. To further analyze the functionality of the Egr-1 site within the survivin promoter in entire cells, transient transfections of p53 wildtype and mutated cell lines with wildtype Egr-1 expression vector were performed. In these overexpression experiments, mRNA and protein levels of survivin but not of control protein were downregulated after exogenous expression of wildtype Egr-1. Using reporter-gene assays, basal survivin promoter activity was decreased significantly by wildtype Egr-1 transfection, whereas mutant Egr-1 did not change activity of the survivin promoter, implying that Egr-1 specifically blocks survivin expression at the transcriptional level. In addition, Egr-1 over-expression sensitized cells to TRAIL-induced apoptosis. To investigate the expression pattern of Egr-1 in different AML cell lines and control samples from healthy donors, we analyzed the expression of Egr-1 by RT-PCR. Fitting with the above shown data, Egr-1 was expressed at significantly lower levels in AML cell lines expressing high survivin levels than in healthy control samples with low survivin levels. Taken together, we show that the transcription factor Egr-1 binds to the human survivin promoter in vitro and in entire cells, downregulates survivin expression independently of p53 and sensitizes cells to TRAIL-induced apoptosis. Since Survivin is often overexpressed in AML, downregulating Survivin expression by activating Egr-1 may be an interesting therapeutic option.

scholarly journals Inhibition of Cyclin D1 Expression in Human Glioblastoma Cells is Associated with Increased Temozolomide Chemosensitivity

2018 ◽  
Vol 51 (6) ◽  
pp. 2496-2508 ◽  
Author(s):  
Danfeng Zhang ◽  
Dawei Dai ◽  
Mengxia Zhou ◽  
Zhenxing Li ◽  
Chunhui Wang ◽  
...  
Keyword(s):  
Malignant Glioma ◽  
Cyclin D1 ◽  
Cell Lines ◽  
Effective Means ◽  
Malignant Gliomas ◽  
Glioma Cells ◽  
Normal Brain ◽  
Induced Apoptosis

Background/Aims: Cyclin D1 (CCND1) is frequently overexpressed in malignant gliomas. We have previously shown ectopic overexpression of CCND1 in human malignant gliomas cell lines. Methods: Quantitative reverse transcriptase PCR (qRT-PCR) and Western Blot (WB) was performed to investigate the expression of CCND1 in glioma tissues and cell lines. The biological function of CCND1 was also investigated through knockdown and overexpression of BCYRN1 in vitro. Results: Here we reported that CCND1 expression was positively associated with the pathological grade and proliferative activity of astrocytomas, as the lowest expression was found in normal brain tissue (N = 3) whereas the highest expression was in high-grade glioma tissue (N = 25). Additionally, we found that the expression level of CCND1 was associated with IC50 values in malignant glioma cell lines. Forced inhibition of CCND1 increased temozolomide efficacy in U251 and SHG-44 cells. After CCND1 overexpression, the temozolomide efficacy decreased in U251 and SHG-44 cells. Colony survival assay and apoptosis analysis confirmed that CCND1 inhibition renders cells more sensitive to temozolomide treatment and temozolomide-induced apoptosis in U251 and SHG-44 cells. Inhibition of P-gp (MDR1) by Tariquidar overcomes the effects of CCND1 overexpression on inhibiting temozolomide-induced apoptosis. Inhibition of CCND1 inhibited cell growth in vitro and in vivo significantly more effectively after temozolomide treatments than single temozolomide treatments. Finally, inhibition of CCND1 in glioma cells reduced tumor volume in a murine model. Conclusion: Taken together, these data indicate that CCND1 overexpression upregulate P-gp and induces chemoresistance in human malignant gliomas cells and that inhibition of CCND1 may be an effective means of overcoming CCND1 associated chemoresistance in human malignant glioma cells.

scholarly journals Transcription factor Tlx1 marks a subset of lymphoid tissue organizer-like mesenchymal progenitor cells in the neonatal spleen

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Yuta Ueno ◽  
Keiko Fujisaki ◽  
Shoko Hosoda ◽  
Yusuke Amemiya ◽  
Shogo Okazaki ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Lymphoid Tissue ◽  
Stromal Cell ◽  
Lineage Tracing ◽  
Mesenchymal Progenitor Cells ◽  
Reticular Cells

AbstractThe spleen is comprised of spatially distinct compartments whose functions, such as immune responses and removal of aged red blood cells, are tightly controlled by the non-hematopoietic stromal cells that provide regionally-restricted signals to properly activate hematopoietic cells residing in each area. However, information regarding the ontogeny and relationships of the different stromal cell types remains limited. Here we have used in vivo lineage tracing analysis and in vitro mesenchymal stromal cell assays and found that Tlx1, a transcription factor essential for embryonic spleen organogenesis, marks neonatal stromal cells that are selectively localized in the spleen and retain mesenchymal progenitor potential to differentiate into mature follicular dendritic cells, fibroblastic reticular cells and marginal reticular cells. Furthermore, by establishing a novel three-dimensional cell culture system that enables maintenance of Tlx1-expressing cells in vitro, we discovered that signals from the lymphotoxin β receptor and TNF receptor promote differentiation of these cells to express MAdCAM-1, CCL19 and CXCL13, representative functional molecules expressed by different subsets of mature stromal cells in the spleen. Taken together, these findings indicate that mesenchymal progenitor cells expressing Tlx1 are a subset of lymphoid tissue organizer-like cells selectively found in the neonatal spleen.

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