Therapeutic Potential of Axl Blockade in BCR-ABL Negative Myeloproliferative Neoplasms (MPN)

Abstract Axl, a member of the TAM family of receptor tyrosine kinases, mediates survival and therapy resistance of different cancer cells. The Axl ligand growth-arrest specific gene 6 (Gas6) was discovered to promote proliferation of leukemia cells in acute and chronic myeloid leukemia and Axl was identified as a potential therapeutic target in these diseases. Based on these data we investigated the role of Axl in BCR-ABL negative myeloproliferative neoplasms (MPN) and the therapeutic potential of Axl blockade in this group of diseases. We studied the effects of Axl blockade using the small molecule Axl inhibitor BGB324 and performing a lentivirus shRNA mediated knockdown of Axl in human SET-2 and murine BaF3-Jak2V617F MPN cell lines. Pharmacologic Axl blockade resulted in a significant dose dependent decrease in viability of MPN cell lines as measured by WST-1 cell viability assay. Annexin+ staining revealed an increased rate of apoptotic cells upon BGB324 treatment for SET-2 (increase by 15% at 1µM, p<0.001) and BaF3-Jak2V617F cells (increase by 54% at 2µM, p<0.05). Moreover, Western Blot analysis showed higher levels of cleaved caspase 3 in BGB324 treated SET-2 cells and decreased levels of anti-apoptotic bcl-2 in BGB324 treated BaF3-Jak2V617F cells. Additionally, BrdU incorporation assays showed a dose dependent decrease in proliferating cells upon treatment with BGB324 in MPN cell lines (p<0.05). Genetic knockdown of Axl in SET-2 cells decreased cell viability by 75% (p<0.01), increased apoptosis levels as measured by Annexin+ staining by 61% (p<0.05) and decreased proliferation as measured by BrdU incorporation by 35% (p<0.001) compared to control-transduced cells. Furthermore, Western Blot analysis revealed that genetic knockdown of Axl resulted in decreased phosphorylation of Stat3 and Stat5 compared to control-transduced cells. Combined Axl and Jak2 blockade, using BGB324 and the Jak2-inhibitor ruxolitinib, showed additive effects on reducing cell viability in SET-2 and BaF3-Jak2V617F cells (p<0.01 and p<0.001, respectively). Western Blot analysis identified inhibition of Stat5 by BGB324 single treatment in SET-2 cells whereas additive effects of combined Axl and Jak2 blockade resulted from additional inhibition of Stat3. In BaF3-Jak2V617F cells, BGB324 single treatment resulted in downstream inhibition of Akt signaling whereas additive effects of combined Axl and Jak2 blockade were exerted via additional inhibition of Stat5, Stat3 and Erk. The finding that BGB324 inhibits growth of MPN cells was further corroborated in vivo. A xenograft tumor model with SET-2 cells was set up in vivo. SET-2 tumor bearing mice treated with BGB324 50mg/kg showed a slower tumor growth (n=8, p<0.01), with a 60% reduction of tumor weight compared to vehicle treated mice (n=8/8, p<0.01). As a second in vivo model, a systemic model of Jak2V617F driven disease was used. After intravenous injection of BaF3-Jak2V617F cells, mice were treated with 50mg/kg BGB324 or vehicle starting the day after inoculation. BGB324 treated mice had a longer overall survival compared to vehicle treated mice (n=10/11, p*<0.05). Furthermore, to evaluate the potential of BGB324 in primary MPN cells, peripheral blood mononuclear cells (PBMC) were isolated from MPN patients and healthy donors. Western Blot analysis showed higher levels of Axl expression by PBMC from MPN patients compared to PBMC from healthy donors. Moreover, colony-forming assays with PBMC were performed in the presence of different concentrations of BGB324. Here, a higher reduction in the number of colony forming units (BFU-E and CFU-GEMM) was observed in samples from MPN patients compared to healthy donors upon treatment with 1µM (77% vs. 5%, respectively; p<0.001) or 2µM (100% vs. 60%, respectively; p<0.01) of BGB324 (n=5/5). In conclusion, these data indicate therapeutic potential of Axl blockade in BCR-ABL negative MPN as monotherapy and in combination with Jak2-inhibition, supporting the need for clinical investigation. Disclosures von Amsberg: Novartis: Honoraria, Speakers Bureau; Ipson: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Sanofi: Honoraria, Speakers Bureau; Astellas: Honoraria, Speakers Bureau; MSD: Honoraria, Speakers Bureau. Loges:BerGenBio: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Abnormal Expression of TRAF Adapter Proteins in Waldenstrom’s Macroglobulinemia.

Blood ◽

10.1182/blood.v108.11.4640.4640 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4640-4640

Author(s):

Xavier Leleu ◽

Lian Xu ◽

Zachary R. Hunter ◽

Anne-Sophie Moreau ◽

Xiaoying Jia ◽

...

Keyword(s):

Western Blot ◽

Cell Lines ◽

Blot Analysis ◽

Western Blot Analysis ◽

Low Grade ◽

Adapter Proteins ◽

Rt Pcr ◽

Growth And Survival ◽

Healthy Donors ◽

Sirna Knockdown

Abstract Background: Waldenström’s Macroglobulinemia (WM) is an incurable low-grade lymphoplasmacytic lymphoma with as yet unknown genetic basis for its pathogenesis. Several TNF family members (CD40L, APRIL and BAFF/BLYS) are known to regulate WM growth and survival. TRAFs are a novel family of adapter proteins that facilitate pro-apoptotic (TACI) or pro-survival/differentiation (CD40, BAFFR, BCMA) receptor signaling mediated by TNF family ligands. Therefore, understanding the TRAF system in WM may yield important clues about WM growth and survival. Methods: WM cell lines (BCWM.1 and WSU-WM), IgM secreting low-grade lymphoma cell lines (MEK1, RL, Namalwa), and primary bone marrow CD19+ selected lymphoplasmacytic cells (LPC) from 20 WM patients and 6 healthy donors were evaluated for TRAF (TRAF 2, 3, 5, 6) expression using semi quantitative RT-PCR and/or western blot analysis. Results: The TNF familiy receptors CD40, BAFFR, BCMA, and TACI were expressed in all cell lines tested as well as in CD19+ selected LPC from WM patients and healthy donors. Moreover, TRAF 2, 3, 5, 6 were expressed in all cell lines by both RT-PCR and western blot analysis. In contrast, we observed loss or abnormally low expression of both TRAF 2 and 5 in 6/20 (30%) patients, whilst TRAF 3 was absent or abnormally low in 3/30 (15%) patients. TRAF 6 was expressed in all patients. Among healthy donors, we observed expression of all TRAF adapter proteins. Conclusion: Up to one third of WM patients demonstrate loss of TRAF 2 and 5 adapter proteins which facilitate signaling through the pro-apoptotic receptor TACI. Ongoing studies including gene sequencing and siRNA knockdown models are delineating a role for TRAF loss in the pathogenesis of WM.

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Inhibition Of H3K27-Methylome As a Novel Therapeutic Strategy In Multiple Myeloma

Blood ◽

10.1182/blood.v122.21.3162.3162 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3162-3162

Author(s):

Maria Gkotzamanidou ◽

Masood A Shammas ◽

Jun Qi ◽

Mehmet Kemal Samur ◽

Teru Hideshima ◽

...

Keyword(s):

Multiple Myeloma ◽

Western Blot ◽

Cell Lines ◽

Blot Analysis ◽

Western Blot Analysis ◽

Inhibitory Effect ◽

Epigenetic Changes ◽

Healthy Donors ◽

Equity Ownership ◽

Induced Apoptosis

Abstract Multiple myeloma (MM) remains incurable, despite recent therapeutic advances; newer insights into the pathogenic mechanisms that cause this disease and additional therapies are urgently needed. Recent studies of the epigenome and in particular the methylome, have shown that myeloma is characterized by widespread epigenetic changes. Epigenetic changes may precede genetic mutations and genomic instability; Ubiquitously Transcribed Tetratricopeptide Repeat Protein X (UTX), a histone H3K27 demethylase may represent such an example. Previous studies have shown inactivating somatic mutations in UTX (KDM6A) in 10% of MM cases. Aberrant methylation of core histone tails and deregulation of the corresponding enzymes such as UTX and JMJD3 have been implicated in leukemia as well as other types of cancers, but their role in MM remains unknown. We evaluated the activity of the selective Jumonji H3K27 demethylase (UTX/JMJD3) inhibitor, GSK-J4, in MM. In a panel of 15 human MM cell lines (HMCLs) including cell lines resistant to bortezomib and dexamethasone, GSK-J4 induced significant survival and proliferation inhibition, as measured by luminescence-based viability assay (CTG), MTT (inhibition>68% in 48 hours) and 3H thymidine uptake, with the exception of OPM2 that was resistant up to 5 uM concentration. GSK-J4 induced apoptosis as measured by flow cytometry upon staining with Annexin-V/Propidium Iodide. The compound did not induce cytotoxicity in PBMCs from healthy donors and normal human skin fibroblasts. The inhibitory effect of GSK-J4 was observed also in CD138+ primary plasma cells from newly diagnosed MM patients (n=5) compared to PBMCs from healthy donors (n=9) (p<0.001). The compound also resulted in significant inhibition of MM cell colony formation in soft agar after 2 weeks in compared to controls. Moreover, interaction between MM cells and bone marrow stromal cells from MM patients or HS-5 stromal cell line did not overcome the inhibitory effect of GSK-J4 in all the HMCLs (KMS-12BM, LP-1, MM1S, OPM2, RPMI-8226, H929 and INA6) tested. To further investigate the mechanism of GSK-J4-induced apoptosis, we evaluate the activation of caspases 3/7, 8 and 9, and observed significant activation of caspase 3/7 and 9, indicating the involvement of intrinsic apoptotic pathway into the GSK-J4 function, as also confirmed by western blot analysis of Bcl-xl, p53, and Bax. We evaluated the enzymatic activity of UTX/JMJD3 in HMCLs by using a fluometric-based assay. The sensitivity of the HMCLs to demethylase inhibitor was directly related with UTX/JMJD3 activity (p<0.05). As the methylation of H3K27 mark plays a major role in the maintenance of active and silent states of gene expression in important cellular processes, we next mapped H3K27me3 and H3K27me2 chromatin modifications by genome-wide chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) analysis in RPMI8226 and KMS-12BM cells before and after GSK-J4 treatment and in UTX knockdown cells. Transcription factors of OSKM complex: Oct4, Sox2, and Nanog were found to be targets of aberrant demethylation after treatment with GSK-J4, indicating possible involvement of H3K27 demethylases in pathogenesis of MM through the regulation of “stemness” genes. Western blot analysis showed that the inhibitor reduced the expression of these genes. In conclusion, we demonstrate a significant beneficial impact of inhibition of H3K27 demethylation in MM. These results provide new insights into the mechanism of altering methylome as a potent epigenetic intervention in treatment of MM. Disclosures: Hideshima: Acetylon Pharmaceuticals: Consultancy. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.

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Single Agent and Combinatorial Efficacy of First-in-Class Small Molecule ONC201 in Acute Leukemia and Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.2759.2759 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2759-2759 ◽

Cited By ~ 1

Author(s):

Varun V Prabhu ◽

Amriti Lulla ◽

Christina L Kline ◽

Peter J Van den Heuvel ◽

Mala K. Talekar ◽

...

Keyword(s):

Western Blot ◽

Cell Viability ◽

Cell Lines ◽

Blot Analysis ◽

Western Blot Analysis ◽

Single Agent ◽

Employment Equity ◽

Equity Ownership ◽

Dose Dependent Decrease ◽

Dose Dependent

Abstract ONC201 is the founding member of the imipridone class of anti-cancer small molecules that possess a unique core chemical structure. ONC201 is currently being evaluated in several Phase I/II clinical trials for advanced cancers. In the current study, we evaluated the single agent and combinatorial efficacy of ONC201 in preclinical models of acute leukemia and multiple myeloma (MM). In acute leukemia, we evaluated ONC201 anti-cancer effects in acute myeloid leukemia (AML) (Kasumi-1, HL60) and acute lymphoblastic leukemia (ALL) (Reh, Jurkat and MOLT-4) cell lines. We observed a time- and dose-dependent decrease in cell viability for every cell line in the panel (EC50 1-5 µM). Vincristine-resistant cells HL60/VCR were also sensitive to single agent ONC201 with EC50 values on par with corresponding vincristine-sensitive parental cells. Dose- and time-dependent induction of apoptosis was noted in Western blot analysis of caspase-3 cleavage in AML cell lines treated with 2.5 µM or 5 µM of ONC201 for 48 hr. Western Blot analysis further demonstrated inhibition of Akt and Foxo3a phosphorylation in Kasumi-1 cells, in line with the previously reported late-stage signaling effects of ONC201 in solid tumor cells (Allen et al, 2013). Sub-G1 analysis indicated that ONC201 induces apoptosis in ALL cells and a pan-caspase inhibitor reduced ONC201-mediated apoptosis. Western blot analysis revealed ONC201-mediated apoptosis involves PARP cleavage and caspase-9 activation in ALL cells. Anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xl were downregulated while the pro-apoptotic Bcl-2 family member Bim is upregulated in response to ONC201 treatment in ALL cells. ONC201 also downregulates the inhibitor of apoptosis (IAP) family proteins cIAP1 and cIAP2 in ALL cells. We observed inhibition of Akt phosphorylation upon ONC201 treatment of ALL cells. Fresh AML patient cells were also found to be sensitive to ONC201 in cell viability and caspase 3/7 activity assays at 5µM. We observed that independent clones of cancer cells with acquired resistance to ONC201 were more sensitive to cytarabine compared to parental ONC201-sensitive cancer cells. In addition, ONC201 demonstrated synergistic reduction in cell viability in combination with cytarabine in AML cell lines. Determination of combination indices (CI) revealed synergy at several concentrations (CI 0.336-0.75 in CMK cells). Also, ONC201 combined additively with midostaurin in CMK cells and vincristine in HL60/VCR cells. Thus, ONC201 is a promising combinatorial partner for AML therapies based on these preclinical sensitization results. In accordance with ONC201-mediated activation of the integrated stress response that B cells are highly sensitive to (Kline et al and Ishizawa et al, 2016), MM was identified as one of the most ONC201-sensitive tumor types in the Genomics of Drug Sensitivity in Cancer collection of cell lines. Three human MM cell lines were used for validation (KMS18, MM.1S and RPMI-8226), which revealed a time- and dose-dependent decrease in cell viability (EC50 1-2.5 µM). Bortezomib-resistant cells MM.1S 33X were sensitive to ONC201 as a single agent with EC50 values comparable to bortezomib-sensitive parental cells. We observed an average of 10-fold induction of ONC201-mediated apoptosis using Sub-G1 analyses in MM cells at 5 µM, 48 hrs post-treatment. Rescue of ONC201-mediated apoptosis was demonstrated using the pan-caspase inhibitor (Z-VAD-FMK). In addition, Western blot analysis in MM cells indicated a dose-dependent decrease in the anti-apoptotic protein XIAP which is a key mediator of apoptosis inhibition and is reported to be highly up-regulated in MM cells. Furthermore, ONC201 demonstrated synergistic reduction in cell viability at various concentrations in combination with either ixazomib or dexamethasone, which are used in the clinical treatment of MM, in RPMI8226 cells (CI 0.228-0.75). Also, ONC201 combined additively with bortezomib in RPMI8226 and MM.1S 33X cells. In summary, these preclinical studies support the ongoing ONC201 single agent trials in acute leukemias and MM. Our findings suggest that ONC201 may be an important therapeutic option for patients with hematological malignancies who have developed resistance to approved therapies. Additionally, our results point to specific standard-of-care therapies that may be combined with ONC201 to exert durable responses without adding to the burden of toxicity. Disclosures Prabhu: Oncoceutics: Employment. Tarapore:Oncoceutics: Employment, Equity Ownership. Oster:Oncoceutics: Employment, Equity Ownership. Allen:Oncoceutics: Employment, Equity Ownership. El-Deiry:Oncoceutics: Equity Ownership.

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Paeonol Induces Protective Autophagy in Retinal Photoreceptor Cells

Frontiers in Pharmacology ◽

10.3389/fphar.2021.667959 ◽

2021 ◽

Vol 12 ◽

Author(s):

Daowei Zhang ◽

Jiawen Wu ◽

Jihong Wu ◽

Shenghai Zhang

Keyword(s):

Oxidative Stress ◽

Western Blot ◽

Cell Viability ◽

Blot Analysis ◽

Western Blot Analysis ◽

Critical Role ◽

Cell Counting ◽

Terminal Deoxynucleotidyl Transferase ◽

Retinal Photoreceptor

Background: Retinal photoreceptor (RP) cells are widely involved in retina-related diseases, and oxidative stress plays a critical role in retinal secondary damage. Herein, we investigated the effectiveness and potential mechanisms of autophagy of paeonol (Pae) in terms of oxidation resistance.Methods: The animal model was induced by light damage (LD) in vivo, whereas the in vitro model was established by H2O2 stimulation. The effectiveness of Pae was evaluated by hematoxylin and eosin, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, immunofluorescence, transmission electron microscopy, electroretinogram, and Western blot analysis in vivo, and the underlying mechanisms of Pae were assessed by Cell Counting Kit-8 assay, reactive oxygen species (ROS) assay, and Western blot analysis in 661W cells. We mainly evaluated the effects of Pae on apoptosis and autophagy.Results: Increased apoptosis of the LD-induced and decreased autophagy of RPs were mitigated by Pae treatment. Pea, which increased the expression of mitochondrial functional protein cytochrome c, reversed the decreased cell viability and autophagy induced by oxidative stress in 661W cells. Experiments showed that autophagy was downregulated in PINK1/Parkin dependent and the BNIP3L/Nix dependent pathways under H2O2 stimulation and was upregulated by Pae treatment. Pae increased the cell viability and reduced ROS levels through autophagy.Conclusion: Pretreatment with Pae preserved RP cells by enhancing autophagy, which protected retinal function.

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Caspase-Dependent Anti-Tumor Effects of ONC201/TIC10 on Acute Myeloid Leukemia (AML) and Multiple Myeloma (MM)

Blood ◽

10.1182/blood.v124.21.5224.5224 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5224-5224

Author(s):

Amriti R. Lulla ◽

Christina Leah B Kline ◽

Liz J. Hernandez-Borrero ◽

Varun Vijay Prabhu ◽

Jessica M Wagner ◽

...

Keyword(s):

Western Blot ◽

Tumor Cells ◽

Cell Lines ◽

Blot Analysis ◽

Western Blot Analysis ◽

Caspase 3 ◽

Therapeutic Potential ◽

Fold Induction ◽

Dose Dependent Decrease ◽

Resistant Cells

Abstract PI3K/Akt and Ras/MAPK pathways are attractive therapeutic targets in almost all tumor types, including AML and MM. Apo2L/TRAIL has been deemed a promising therapeutic given its selectivity towards cancer cells although its clinical development has been hampered by various limitations including short half-life and general shortcoming of protein-based therapeutics. ONC201/TIC10 (Oncoceutics, Inc.) is a first-in-class small molecule inducer of TRAIL expression. ONC201/TIC10 has previously been shown to up-regulate TRAIL and its death inducing receptor DR5 in HCT116 colon cancer cells, in part through the inhibition of Foxo3a phosphorylation mediated by dual inhibition of Akt and ERK (Allen JE et al, Sci Transl Med., 2013). Currently, ONC201/TIC10 is set to enter clinical trials for patients with advanced malignancies after the IND was approved by the FDA in March, 2014. We thus investigated the therapeutic potential of ONC201/TIC10 in AML and MM given a major unmet need when conventional therapy fails. We explored the possibility that ONC201/TIC10 induces apoptosis in MM and AML in part through dual inhibition of the PI3K/Akt and Ras/MAPK pathways. We tested a panel of four human MM cell lines (KMS18, MM.1S, MM.1S 33X and RPMI-8226) and three human AML cell lines (Kasumi-1, HL60, HL60/VCR). The Cell-titer Glo assay demonstrated a time and dose-dependent decrease in viability in the entire panel of MM and AML cells. EC50 values ranged from 1-2.5 µM for the MM and 2-5µM for the AML cell lines, respectively. Bortezomib-resistant cells MM.1S 33X and vincristine- resistant cells HL60/VCR were also significantly sensitive to ONC201/ TIC10 as a single agent with EC50s on par with the corresponding parental cell lines. Given the previously reported pro-apoptotic effects of ONC201/TIC10 against solid tumor cells, we assessed apoptosis by performing Sub-G1 analyses and assessing caspase-3 cleavage as two widely used methods to analyze apoptotic cell death. We observed an average of 10-fold induction of ONC201/TIC10–mediated apoptosis in MM cells at 5 mM at 48 hrs post-treatment. Rescue of ONC201/TIC10-mediated apoptosis was demonstrated using the pan-caspase inhibitor (Z-VAD-FMK). In addition, western blot analysis in MM cells indicated a dose-dependent decrease in the anti-apoptotic protein XIAP which is a key mediator of apoptosis inhibition and is reported to be highly up-regulated in MM cells. Dose and time dependent induction of apoptosis was noted in western blot analysis of caspase-3 cleavage in AML cell lines treated with 2.5 µM or 5 µM of ONC201/TIC10 for 48 hrs prior to analysis. Western Blot analysis further demonstrated inhibition of Akt and Foxo3a phosphorylation in Kasumi-1 cells, in line with the previously proposed mechanism of ONC201/TIC10 against solid tumor cells. To further investigate the therapeutic potential of ONC201/TIC10 in the context of AML, fresh AML cells were treated with ONC201/TIC10. The primary cells were also found to be sensitive to ONC201/TIC10 (60% decrease in cell viability 72 hrs post 5mM ONC201/TIC10 treatment). Similarly, caspase 3/7 activity was significantly increased as assessed by the Caspase Glo 3/7 assay (~5 fold induction in activity 72 hrs post 5mM ONC201/TIC10 treatment). To explore further the therapeutic potential of ONC201/TIC10, we performed combinatorial experiments with bortezomib and vincristine using the MM.1S 33X MM cells and the HL60/VCR AML cell lines. ONC201/ TIC10 showed an additive effect with both these compounds against the MM and AML lines. Our work demonstrates activity of ONC201/TIC10 against AML and MM cell lines including fresh AML tumor cells. The efficacy data with resistant cells is in par with the applicability of TIC10 in patients with refractory/relapsed hematological malignancies. The long-term goal of this project is to provide a rationale for a phase 1b trial of ONC201/TIC10 for refractory/relapsed MM and AML in combination with existing therapies. Figure 1: Efficacy of ONC201/TIC10 in AML and MM cells Figure 1:. Efficacy of ONC201/TIC10 in AML and MM cells Disclosures Allen: Oncoceutics, Inc.: Employment, Equity Ownership, Patents & Royalties. El-Deiry:Oncoceutics, Inc.: Equity Ownership, Patents & Royalties.

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RUNX2 Plays An Oncogenic Role in Esophageal Carcinoma by Activating the PI3K/AKT and ERK Signaling Pathways

Cellular Physiology and Biochemistry ◽

10.1159/000492872 ◽

2018 ◽

Vol 49 (1) ◽

pp. 217-225 ◽

Cited By ~ 11

Author(s):

Huibin Lu ◽

Tian Jiang ◽

Kewei Ren ◽

Zongming Li Li ◽

Jianzhuang Ren ◽

...

Keyword(s):

Western Blot ◽

Esophageal Carcinoma ◽

Cell Viability ◽

Signaling Pathways ◽

Blot Analysis ◽

Western Blot Analysis ◽

Tumor Formation ◽

Erk Signaling ◽

Migration And Invasion

Background/Aims: Esophageal carcinoma is a frequently occurring cancer at upper gastrointestinal tract. We aimed to evaluate the roles and possible mechanism of Runt Related Transcription Factor 2 (RUNX2) in the development of esophageal cancer. Methods: The expression of RUNX2 in esophageal carcinoma tissues and cells was investigated by qRT-PCR. Effects of RUNX2 on cell viability, apoptosis, migration and invasion were assessed using MTT assay, flow cytometry assay/western blot analysis, and Transwell assays, respectively. Afterwards, effects of RUNX2 on of the activation of the PI3K/AKT and ERK pathways were explored by Western blot analysis. In addition, a PI3K/AKT pathway inhibitor LY294002 and an ERK inhibitor U0126 were applied to further verify the regulatory relationship between RUNX2 and the PI3K/AKT and ERK signaling pathways. Besides, the RUNX2 function on tumor formation in vivo was investigated by tumor xenograft experiment. Results: The result showed that RUNX2 was highly expressed in esophageal carcinoma tissues and cells. Knockdown of RUNX2 significantly inhibited TE-1 and EC-109 cell viability, repressed TE-1 cell migration and invasion, and increased TE-1 cell apoptosis. RUNX2 overexpression showed the opposite effects on HET-1A cells. Moreover, RUNX2-mediated TE-1 cell viability, migration and invasion were associated with the activation of the PI3K/AKT and ERK pathways. Besides, knockdown of RUNX2 markedly suppressed tumor formation in vivo. Conclusion: Our results indicate that RUNX2 may play an oncogenic role in esophageal carcinoma by activating the PI3K/ AKT and ERK pathways. RUNX2 may serve as a potent target for the treatment of esophageal carcinoma.

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Neogenin-1 Promotes Cell Proliferation, Motility, and Adhesion by Up-Regulation of Zinc Finger E-Box Binding Homeobox 1 Via Activating the Rac1/PI3K/AKT Pathway in Gastric Cancer Cells

Cellular Physiology and Biochemistry ◽

10.1159/000492255 ◽

2018 ◽

Vol 48 (4) ◽

pp. 1457-1467 ◽

Cited By ~ 6

Author(s):

Hengyi Qu ◽

Huabo Sun ◽

Xueping Wang

Keyword(s):

Gastric Cancer ◽

Cell Proliferation ◽

Western Blot ◽

Cell Viability ◽

Cell Lines ◽

Zinc Finger ◽

Blot Analysis ◽

Western Blot Analysis ◽

Akt Pathway ◽

E Box

Background/Aims: Neogenin-1 (Neo1) has been reported to be involved in diverse physiology and pathology functions, including cell proliferation, differentiation and migration. The present study aimed to explore the functional role of neogenin-1 (Neo1) in gastric cancer (GC), together with underlying mechanisms. Methods: Neo1 expression was analyzed by qRT-PCR and Western blot analysis in both human GC cell lines and normal gastric epithelial cell line. Neo1 was respectively overexpressed or silenced by transfection with pcDNA3.1 or siRNA, and then the cells were incubated with or without different concentrations of cisplatin, transforming growth factor (TGF)-β1, and/or inhibitors of Rac-1 and PI3K. Thereafter, cell viability, invasion, and adhesion were measured by CCK-8, wound healing and adhesion assays, respectively. The expression levels of key factors involved in epithelial mesenchymal transition (EMT) and the PI3K/AKT pathway were analyzed by Western blot analysis. Results: The results showed that the Neo1 level was significantly increased in GC cell lines, with the highest level in SGC-7901 cells. Overexpression of Neo1 significantly reduced the GC cell sensitivity to cisplatin and increased the cell viability, motility and adhesion ability, and while silencing of Neo1 showed contrary results. Moreover, overexpression of Neo1 dramatically downregulated the E-Cadherin level and upregulated the levels of N-Cadherin and Vimentin. In addition, the data revealed that Neo1 positively regulated the expression of Zinc finger E-box-binding homeobox 1 (ZEB1) by activating the Rac1/PI3K/AKT pathway. Conclusions: Neo1 could promote cell proliferation, motility, and adhesion by up-regulation of ZEB1 via activating the Rac1/PI3K/AKT pathway in GC cells.

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Sulforaphane and PEITC Augment Activity of Conventional and Novel Anti-Myeloma Drugs

Blood ◽

10.1182/blood.v112.11.2648.2648 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2648-2648

Author(s):

Jana Jakubikova ◽

Jan Sedlak ◽

Steffen Klippel ◽

Merav Leiba ◽

Yu-Tzu Tai ◽

...

Keyword(s):

Western Blot ◽

Cell Lines ◽

Stress Responses ◽

Blot Analysis ◽

Western Blot Analysis ◽

Cytotoxic Effect ◽

Therapeutic Potential ◽

Cruciferous Vegetables ◽

M Cell ◽

Mapk Activation

Abstract Isothiocyanates (ITCs) represent a family of phytochemicals found in cruciferous vegetables. Several epidemiological studies indicated that high intake of diet-derived ITCs may provide chemopreventive effect associated with a reduced risk of renal, prostate, pancreatic and colorectal carcinoma. ITCs have also been reported to enhance the chemosensitivity of diverse types of tumor cells. We therefore evaluated the cytotoxic effect of ITC, sulforaphane (SFN) and phenylethyl isothiocyanate (PEITC) on a panel of human MM cell lines, including cells resistant to doxorubicin (RPMI-Dox40), mitoxantrone (RPMI-MR20), melphalan (RPMI-LR5), and dexamethasone (MM.1R, OPM1 and OPM2); as well as cells with low sensitivity to thalidomide derivatives (RPMI 8226-S) and sensitive cell lines (MM.1S). We evaluated the anti-MM activity of these compounds using both MTT and flow cytometric assays. Our results suggest that all tested MM cell lines are susceptible to the cytotoxic effect of both ITCs at concentrations in the same order of magnitude as those achieved in vivo by dietary consumption of cruciferous vegetables. PEITC (IC50 of 3.5–8.2 μM) was more potent than SFN (IC50 of 5–15 mM) at 48 h. ITCs induce apoptotic death of MM cells, evidenced by Annexin V-FITC staining, increased sub-G1 population measured by flow cytometry, and cleavage of PARP and caspase-3 by western blot analysis, ITCs also induced G2/M cell cycle arrest and depletion of mitochondrial potential in JC-1 probed MM cell lines. Multiplex analysis of phoshorylation signaling pathways, using the Luminex system and confirmed by conventional western blot analysis, revealed that PEITC at 2hrs triggers MAPK activation (MEK1 (4-fold), ERK1/2 (4.4-fold), JNK (25.4-fold) and p38MAPK (6-fold)) at 2h, which likely are stress responses of cells to PEITC treatment. SFN induced less pronounced but a more sustained MAPK activation (up to 24 h) than PEITC. Concentration-dependent increases in phosphorylation of GSK3α/β, c-jun, p70S6 kinase, and p90RSK were also observed at early time-points after ITCs-treatment. In contrast, decreased phoshorylation of Akt was observed in MM cells treated with SFN at 2 h and PEITC at 24 h. Chou-Talalay analysis of the effects of combinations of ITCs with anti-MM drugs (dexamethasone, melphalan and bortezomib) revealed all combinations to have synergistic MM cytotoxicity. Importantly, ITCs treatment, both alone and in combination with the aforementioned agents, also significantly suppressed proliferation of CFSE-labeled MM cell lines co-cultured with the human bone stromal cell line HS-5. These results indicate that SFN and PEITC suppress survival and proliferation of MM cells, both alone and in combination, and suggest their therapeutic potential in MM.

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764 Characterization of RVU-27065 a novel small-molecule STING agonist suitable for systemic administration

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.764 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A799-A799

Author(s):

Maciej Rogacki ◽

Stefan Chmielewski ◽

Magdalena Zawadzka ◽

Aniela Gołas ◽

Aleksandra Poczkaj ◽

...

Keyword(s):

T Cell ◽

Tumor Growth ◽

Western Blot ◽

Cell Viability ◽

Small Molecule ◽

Blot Analysis ◽

Targeted Delivery ◽

Western Blot Analysis ◽

Dual Reporter

BackgroundSTimulator of INterferon Genes (STING) is a key signaling protein involved in activation of the immune system in response to self-DNA. In recent years, STING signaling has been demonstrated to play a major role in activating the antitumor immune response and therefore is considered an attractive drug target in immuno-oncology. The first wave of STING agonists, cyclic-dinucleotide analogues of the internal ligand cGAMP, were developed for local, intratumoral administration. Herein we present the most recent profiling results of our frontrunner molecule RVU-27065, a potent and selective systemic STING agonist with a favorable drug profile.MethodsBinding to recombinant STING protein was examined using Fluorescence Thermal Shift and Fluorescence Polarisation. Primary activity screen was performed in THP-1 Dual reporter cells. Selectivity was confirmed in THP-1 reporter cells with knocked out STING or expressing STING variants. T cell viability and proliferation was assessed by flow cytometry using activated human T cells. PBMCs were isolated by density gradient from whole blood of healthy donors. Downstream STING pathway activation in cells treated with RVU-27065 was confirmed using Western blot analysis. BALB/c mice were inoculated with EMT6 tumor cells and the compound was administered intravenously followed by regular monitoring of tumor growth. Cured animals were rechallenged by repeated inoculation of EMT6 cells.ResultsRVU-27065 binds and strongly thermostabilizes recombinant STING proteins of all tested species. Binding to the protein results in activation of downstream signalling pathway, confirmed by western blot analysis. The agonist is characterized by selectivity and excellent potency in THP-1 dual reporter cells as well as in human PBMCs and dendritic cells. Short term incubation of RVU-27065 has no impact on T cell viability, activation or proliferation. Furthermore, STING activation with RVU-27065 leads to repolarization of immunosuppressive M2 macrophages into pro-inflammatory M1-like phenotype. In vivo efficacy of RVU-27065 was confirmed, leading to significant tumor growth inhibition and complete tumor regressions in an EMT6 mouse breast cancer syngeneic tumor model.ConclusionsRVU-27065 is a novel representative of a 3rd generation of Ryvu STING agonists – small-molecule, non-macrocyclic molecules built around a unique chemotype. The compound is characterized by high in vitro potency which translates to efficacy in vivo in preclinical animal models. Drug-like properties, excellent selectivity and a good safety profile make RVU-27065 an attractive candidate for further development for standalone as well as targeted delivery, which holds high potential for improved immunotherapy in cancer patients.

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Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

Journal of Neurosurgery ◽

10.3171/2010.7.gks10972 ◽

2010 ◽

Vol 113 (Special_Supplement) ◽

pp. 228-235 ◽

Cited By ~ 4

Author(s):

Qiang Jia ◽

Yanhe Li ◽

Desheng Xu ◽

Zhenjiang Li ◽

Zhiyuan Zhang ◽

...

Keyword(s):

Western Blot ◽

Gamma Knife ◽

Blot Analysis ◽

Western Blot Analysis ◽

Glioma Cells ◽

C6 Glioma ◽

C6 Glioma Cells ◽

C6 Cells

Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

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