scholarly journals Activation of the STAT1-BCL-2/MCL-1 Axis in Leukemic Cells Carrying a SPAG9-JAK2 Fusion

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4326-4326
Author(s):  
Azusa Mayumi ◽  
Toshihiro Tomii ◽  
Takuyo Kanayama ◽  
Takashi Mikami ◽  
Kuniaki Tanaka ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Fusion Protein ◽  
Western Blot ◽  
Gene Expression Analysis ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Jak Inhibitor ◽  
Tyrosine Residues

Abstract [Background and aim of this study] Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a distinct subtype of B-ALL with poor prognosis. JAK2 related fusion genes have been identified in this subtype, especially in adolescent and young adults (AYA). Previously, we identified SPAG9-JAK2 fusion gene in 14-year-old boy with Ph-like ALL (Kawamura M, et al. Genes Chromosomes Cancer. 2015). In this study, we performed functional analysis of the SPAG9-JAK2 fusion protein, and evaluated the efficacy of treatment with a JAK inhibitor against cells carrying the fusion. In addition, we assessed therapeutic options other than JAK2 inhibition following comprehensive molecular analysis. [Materials and Methods] Full length of SPAG9-JAK2 cDNA was cloned into retroviral construct with Tet-On system. Ba/F3 cells, which are IL-3 dependent murine pro B-ALL cells, were transduced with retroviral vector to establish Ba/F3 cells expressing SPAG9-JAK2 (Ba/F3-SPAG9-JAK2) under doxycycline (DOX) dependent manner. Ba/F3-SPAG9-JAK2 were analyzed whether IL-3 independent growth was achieved. Aberrant activation of JAK-STAT pathway achieved by SPAG9-JAK2 was evaluated by western blot. To clarify whether the tyrosine residues of JAK2 in this fusion protein were critical for IL-3-independent proliferation, Ba/F3 cells expressing SPAG9-JAK2 mutants (SPAG9-JAK2 mut) in which both tyrosine residues of JAK2 were replaced with phenylalanine was established. Gene expression analysis using Mouse Genome 430 2.0 Array was performed for comprehensive analysis of gene expression profile related to SPAG9-JAK2. Sensitivity of Ba/F3-SPAG9-JAK2 to ruxolitinib (a JAK inhibitor) was tested in cytotoxic assay and in xenograft model established using Ba/F3-SPAG9-JAK2 cells. [Results and discussions] The expression of SPAG9-JAK2 in Ba/F3 cells under DOX dependent manner was confirmed by western blot. Ba/F3-SPAG9-JAK2 proliferated without IL-3 in contrast to Ba/F3 cells (p<0.01), suggesting SPAG9-JAK2 had the proliferation activity. Western blot revealed that constitutive phosphorylation of tyrosine residue of SPAG9-JAK2, STAT3/STAT5, suggesting constitutive activation of JAK2-STAT3/STAT5 pathway. SPAG9-JAK2 mut abolished IL-3 independence (p<0.01), but had no influence on STAT3/STAT5 phosphorylation levels detected by western blot. Gene expression analysis revealed that Stat1 was significantly up-regulated in Ba/F3-SPAG9-JAK2 cells compared with mock Ba/F3 cells (fold change 8.04 with p < 0.01) [Fig. 1], confirmed by western blot. STAT1 was also phosphorylated in Ba/F3-SPAG9-JAK2 but not SPAG9-JAK2 mut cells detected by western blot [Fig. 2], suggesting that STAT1 is a key mediator for SPAG9-JAK2-mediated cell proliferation. Consistently, STAT1 induced expression of the anti-apoptotic proteins, BCL-2 and MCL-1, as did SPAG9-JAK2, but not SPAG9-JAK2 mut confirmed by western blot [Fig. 3]. Ruxolitinib abrogated Ba/F3-SPAG9-JAK2-mediated proliferation in vitro (p<0.01), with an 50% inhibitory concentration (IC50) value of 65.9 ± 9.8 nM, causing decrease of JAK2, STAT1/STAT3/STAT5 phospholyration in western blot and apoptosis in annexin V/PI staining. Ruxolitinib prolonged survival time of xenotransplanted mice (p = 0.0213), however, the proliferation of leukemic cells in mouse bone marrow was not suppressed by ruxolitinib. Ba/F3-SPAG9-JAK2 cells showed a dose-dependent response for venetoclax (a BCL-2 inhibitor) with IC50 2.57 ± 1.11 µM and AZD5991 (an MCL-1 inhibitor) with IC50 6.76 ± 3.31 µM. Treatment of Ba/F3-SPAG9-JAK2 cells with a combination of ruxolitinib and venetoclax or AZD5991 resulted in a significant reduction in the IC50 of ruxolitinib (p<0.01) [Fig. 4, 5], with a combined index (CI) value of 0.61 or 0.92, indicating a moderately or weak synergistic effect in vitro. [Conclusion] SPAG9-JAK2 promotes cell proliferation and that tyrosine phosphorylation of the JAK2 kinase domain is critical for IL-3-independent cell growth. Ruxolitinib shows sufficient cytotoxic effects against Ba/F3-SPAG9-JAK2 cells in vitro, but is only partially effective in vivo. Activation of the JAK2-STAT1-BCL-2/MCL-1 axis contributes to aberrant growth promotion by SPAG9-JAK2. BCL-2 or MCL-1 inhibitors in combination with ruxolitinib shows efficacy against Ph-like ALL carrying the SPAG9-JAK2 fusion in vitro. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Leukemic Cell Expressing a Novel Kinase Fusion Protein NCOR1-LYN Exhibits High Sensitivity to Dasatinib and Rapamycin

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1557-1557 ◽  
Author(s):  
Toshihiro Tomii ◽  
Toshihiko Imamura ◽  
Mio Yano ◽  
Kenichi Sakamoto ◽  
Itaru Kato ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fusion Protein ◽  
Western Blot ◽  
Retroviral Vector ◽  
Dependent Manner ◽  
Cytotoxic Assay ◽  
Targeting Therapy ◽  
Tyrosine Residues ◽  
Proliferation Activity ◽  
Constitutive Phosphorylation

Abstract [Background and aim of this study] Kinase fusion protein is putative therapeutic target of hematological malignancies. Previously, we identified NCOR1-LYN fusion gene in which NCOR1 exon 34 was fused in-frame to LYN exon 7 in a pediatric patient with Ph-like acute lymphoblastic leukemia (ALL) treated according to Japan Childhood Leukemia Study Group ALL-02 protocol (Yano M, et al. Br J Haematol 2015). Because rearrangement of LYN is the recurrent genetic abnormality in high risk B-ALL, targeting therapy for LYN rearranged ALL could be attractive. Thus, in this study, we performed functional analysis of NCOR1-LYN fusion protein to get insight of biological property of this fusion protein to establish targeting therapy for LYN rearranged ALL. [Materials and Methods] Full length of NCOR1-LYN cDNA was cloned into pRetroX-Tight-Pur retroviral vector. Then, Ba/F3 cells, which are IL-3 dependent murine pro B-ALL cells, were transduced with this retroviral vector to establish Ba/F3 cells expressing NCOR1-LYN under doxycycline (DOX) dependent manner. Once Ba/F3 cells expressing NCOR1-LYN were established, cells were analyzed whether IL-3 independent growth was achieved. To determine whether the tyrosine residues of LYN (Y397 and Y508) in this fusion protein were critical for IL-3-independent proliferation, Ba/F3 cells expressing NCOR1-LYN mutants in which both tyrosine residues of LYN were replaced with phenylalanine (Y397F and Y508F) was established. In cytotoxic assay, proliferation of Ba/F3 cells expressing NCOR1-LYN was assessed under the media with tyrosine kinase inhibitors (TKI), such as imatinib and dasatinib. To determine changes of gene expression pattern in Ba/F3 cells expressing NCOR1-LYN, gene expression analysis was performed using Mouse Genome 430 2.0 Array. [Results and discussions] The expression of NCOR1-LYN in Ba/F3 cells under DOX dependent manner was confirmed by western blot. Ba/F3 cells expressing NCOR1-LYN could proliferate without IL-3 in contrast to Ba/F3 cells not expressing NCOR1-LYN couldn't, suggesting that NCOR1-LYN could induce IL-3 independent proliferation of Ba/F3 cells. Western blot analysis also revealed that constitutive tyrosine phosphorylation of NCOR1-LYN fusion protein was present, intriguing that constitutive phosphorylation of tyrosine residues in LYN was associated with IL-3 independent proliferation activity of NCOR1-LYN. Consistent with our hypothesis, Ba/F3 cells expressing NCOR1-LYN mutant (Y397F and Y508F), which didn't show constitutive phosphorylation of NCOR1-LYN, could not proliferate without IL-3. Considering that TKIs could block constitutive phosphorylation of LYN, TKIs might block the IL-3 independent proliferation of Ba/F3 cells expressing NCOR1-LYN. Cytotoxic assay revealed that 1nM of dasatinib suppressed the proliferation of Ba/F3 cells expressing NCOR1-LYN completely (p<0.01), although imatinib didn't show any effect. Annexin V assay also determined that 96% of Ba/F3 cells treated with dasatinib (10nM, 48hrs) were Annexin positive. However, dasatinib didn't show any effect on Ba/F3 cells not expressing NCOR1-LYN. These findings suggest that dasatinib abolishes the proliferation activity of NCOR1-LYN selectively. To determine other signaling pathways to be targeted in Ba/F3 cells expressing NCOR1-LYN, Gene Set Enrichment Analysis (GSEA) was performed. GSEA revealed that multiple signaling pathways including mTOR pathway, MYC, E2A and EZH2 related pathways. Because mTOR pathway was activated in Ba/F3 cells expressing NCOR1-LYN (NOM p-value<0.01), mTOR inhibitor might suppress the proliferation activity of NCOR1-LYN. Consistent with our hypothesis, cytotoxic assay revealed that rapamycin (20nM) could suppress the proliferation of Ba/F3 cells expressing NCOR1-LYN efficiently compared to Ba/F3 cells not expressing NCOR1-LYN. [Conclusion] Our in vitro study clearly demonstrated that dasatinib and rapamycin could be effective for the patient with B-ALL harboring LYN rearrangement. Disclosures No relevant conflicts of interest to declare.

Effect of Auraptene on angiogenesis in Xenograft model of breast cancer

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Mohammad Reza Shiran ◽  
Elham Mahmoudian ◽  
Abolghasem Ajami ◽  
Seyed Mostafa Hosseini ◽  
Ayjamal Khojasteh ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Animal Model ◽  
Protein Expression ◽  
Western Blot ◽  
Xenograft Model ◽  
Dependent Manner ◽  
Concentration Dependent Manner

Abstract Objectives Angiogenesis is the most important challenge in breast cancer treatment. Recently, scientists become interesting in rare natural products and intensive researches was performed to identify their pharmacological profile. Auraptene shows helpful effects such as cancer chemo-preventive, anti-inflammatory, anti-oxidant, immuno-modulatory. In this regard, we investigated the anti-angiogenesis effect of Auraptene in in-vitro and in-vivo model of breast cancer. Methods In this study, 4T, MDA-MB-231 and HUVEC cell lines were used. The proliferation study was done by MTT assay. For tube formation assay, 250 matrigel, 1 × 104 HUVEC treated with Auraptene, 20 ng/mL EGF, 20 ng/mL bFGF and 20 ng/mL VEGF were used. Gene expression of important gene related to angiogenesis in animal model of breast cancer was investigated by Real-time PCR. Protein expression of VCAM-1 and TNFR-1 gene related to angiogenesis in animal model of breast cancer was investigated by western-blot. Results Auraptene treatment led to reduction in cell viability of MDA-MB-231 in a concentration-dependent manner. Also, we observed change in the number of tubes or branches formed by cells incubated with 40 and 80 μM Auraptene. Auraptene effect the gene expression of important gene related to angiogenesis (VEGF, VEGFR2, COX2, IFNɣ). Moreover, the western blot data exhibited that Auraptene effect the protein expression of VCAM-1 and TNFR-1. Conclusions Overall, this study shows that Auraptene significantly suppressed angiogenesis via down-regulation of VEGF, VEGFR2, VCAM-1, TNFR-1, COX-2 and up-regulation of IFNγ.

scholarly journals A Novel PAX5-KIDINS220 Fusion Protein Activates Multiple Signals Associated with Therapeutic Resistance

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3928-3928
Author(s):  
Takuyo Kanayama ◽  
Toshihiko Imamura ◽  
Kenichi Sakamoto ◽  
Fumihiko Hayakawa ◽  
Akihiko Tanizawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fusion Protein ◽  
Expression Analysis ◽  
Stromal Cell ◽  
Gene Expression Analysis ◽  
Dominant Negative ◽  
Leukemic Cells ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Proliferation Ability

Abstract Background: It is well known that PAX5 related fusion proteins are mainly associated with leukemogenesis of B cell precursor acute lymphoblastic leukemia (B-ALL) through dominant negative effect against normal PAX5, resulting in impairment of B cell differentiation. However, different biological property of PAX5 fusion protein which is related to B-ALL has not been fully investigated. Here, we performed functional analysis of novel fusion protein, PAX5-KIDINS220 (P-K220) which was identified in pediatric Ph-like ALL patient. Methods: Full length of P-K220 fusion gene was cloned into pRetroX-Tight-Pur retroviral vector. We also established the PAX5-N construct that contained only PAX5 region (1-306 aa) of P-K220 so that we could analyze the importance of KIDINS220 in P-K220. Then, Ba/F3 cells, which are IL-3 dependent murine pro B-ALL cells, were transduced with this retroviral vector to establish Ba/F3 cells expressing P-K220 or PAX5-N under doxycycline (DOX) dependent manner. P-K220 was also cloned into pAcGFP1-C1 vector and pMSCVneo vector to perform localization assay and luciferase reporter assay. Gene expression analysis was performed using Mouse Genome 430 2.0 Array. In cytotoxic assay using co-culture system with murine mesenchymal stromal cell, MS-5, Ba/F3 cells that were pre-stained with Carboxyfluorescein diacetate succinimidyl ester (CFSE) were seeded on MS-5 stromal cell, then 10 nM vincristine (VCR) was applied. 72 hours later, flow cytometric analysis was performed to determine the fraction and proliferation ability of viable Ba/F3 cells. The proliferation ability was estimated by CFSE fluorescence that attenuates depending on cell proliferation. Results and discussions: P-K220 and PAX5-N were successfully expressed in Ba/F3 cells, confirmed by western blotting. Localization assay revealed that GFP tagged P-K220 was localized in a nucleus of HEK293-T cells under confocal microscopy, suggesting that P-K220 acted as a transcriptional factor. Luciferase reporter assay revealed that P-K220 protein inhibited PAX5 transcriptional activity in dominant negative fashion. Although P-K220 was identified in Ph-like ALL patient and activated JAK2-STAT5 pathway through reduction of Socs5 expression, Ba/F3 cells expressing P-K220 protein did not acquire IL-3 independency. P-K220 attenuated proliferation of Ba/F3 cells (p=0.02), which was in contrast to PAX5-JAK2. To reveal which pathways were affected by P-K220, gene expression analysis was performed. Gene set enrichment analysis revealed that multiple pathways related to chemotaxis, migration, such as IL-15 pathway, were activated in P-K220 expressing Ba/F3 cells. RQ-PCR and western blotting confirmed P-K220 induced expression of IL-15 in RNA (p=0.018) and protein level, suggesting that P-K220 might be associated with infiltration of leukemic cells into extramedullary site. Next, we examined whether P-K220 was associated with sensitivity of chemotherapeutic agents, such as vincristine (VCR), cytarabine, prednisolone, methotrexate, and 6-mercaptoprine. P-K220 expressing Ba/F3 cells showed higher VCR IC50 (0.4 nM vs 2.5 nM) and decreased Annexin V positive fraction under the condition of 10 nM VCR (39.4 % vs 23.8 %, p=0.04). In co-culture analysis with MS-5 stromal cell under the 10 nM VCR, the fraction of viable Ba/F3 cells that adhered to MS-5 was significantly increased with expression of P-K220 than PAX5-N or Dox (-) (11.6% vs 7.3% vs 6.8%, p<0.001), and proliferation ability was also significantly increased with expression of P-K220, that was estimated by mean CFSE fluorescence (479.6 vs 990.0 vs 909.7, p<0.001), suggesting P-K220 was associated with VCR resistance. Gene expression analysis and RQ-PCR revealed that P-K220 induced expression of Abcc5 which was reported to be associated with VCR resistance (p=0.003). Conclusion: P-K220 did not induce high proliferation of Ba/F3 cells even though identified in Ph-like ALL patient. However, P-K220 might be associated with treatment resistance through the infiltration of leukemic cells into extramedullary sites partly via activation of IL-15 pathway and resistance to VCR via induction of Abcc5. Disclosures No relevant conflicts of interest to declare.

Irisin recovers osteoarthritic chondrocytes: a muscle-cartilage cross-talk boosted by physical activity

2020 ◽  
Author(s):  
Gianluca Vadalà ◽  
Giuseppina Di Giacomo ◽  
Luca Ambrosio ◽  
Francesca Cannata ◽  
Claudia Cicione ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Western Blot ◽  
P38 Mapk ◽  
Collagen Gene ◽  
Type Ii ◽  
Protein Levels ◽  
Collagen Gene Expression ◽  
Osteoarthritic Chondrocytes

Abstract Background Physical exercise favors weight loss and ameliorates both articular pain and function in patients suffering from osteoarthritis (OA). Irisin, a myokine released by skeletal muscles upon muscle contraction, has demonstrated to yield anabolic effects on different cell types. The study aimed to investigate the effect of irisin on human osteoarthritic chondrocytes (hOAC) in vitro . The hypothesis of this study was that irisin would improve hOAC metabolism and proliferation. Methods hOAC were isolated from osteochondral tissues of 5 patients undergoing total knee joint replacement. Cells were cultured in growing media and then exposed to either phosphate-buffered saline (control group) or human recombinant irisin (experimental group). Cell proliferation (Picogreen assay), glycosaminoglycan content (dimethylmethylene blue), type II/X collagen gene expression (Real-Time polymerase chain reaction) and quantification (Western blot and densitometric analysis), p38/ERK MAPK and Akt involvement (Western blot and densitometric analysis) were evaluated in both groups. Results Irisin increased hOAC proliferation ( p < 0.001) and both type II collagen gene expression ( p < 0.001) and protein levels ( p < 0.01), while decreased type X collagen gene expression ( p < 0.05) and protein levels ( p < 0.001). These effects seemed to be mediated by the inactivation of the p38 MAPK and PI3K-Akt intracellular pathways, as irisin reduced phosphorylated p38 (p-p38), ( p < 0.01) and phosphorylated Akt (p-Akt) ( p < 0.001) protein levels. Conclusion Irisin stimulated cell proliferation and anabolism in hOAC through p38 MAPK and PI3K-Akt inactivation in vitro , demonstrating for the first time the existence of a cross-talk between muscle and cartilage.

The tyrosine kinase src plays a crucial role in leukemic cell proliferation in AML

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 10531-10531
Author(s):  
G. Schuch ◽  
E. Schäfer ◽  
K. Eggert ◽  
S. Loges ◽  
M. Görn ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Western Blot ◽  
Cell Lines ◽  
Crucial Role ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Clinical Samples ◽  
Dependent Manner ◽  
Rt Pcr

10531 Background: Src-family tyrosine kinases are known to be involved in signal transduction pathways of growth factors and cytokines in hematopoetic cells. While the role of other src family members has been studied widely, only few data exist about c-src in leukemia. The actual study was performed to analyze src mutations in leukemic cells and to determine the role of pp60src in leukemic cell proliferation. Methods: AML cell lines and primary samples were analyzed for expression and activation of src by RT-PCR and Western blot analyses. Mutational analyses were performed by sequencing of C-terminal cDNA from 60 AML samples. The effects of src inhibition were studied by src-specific inhibitors PP1 and PP2 or by siRNA transfection. Effetcs of src inhibition were monitored in proliferation assays and analyzes of signalling through Erk1/2 and apoptosis by annexin V staining and DNA laddering. Results: In all 60 patients analyzed expression of c-src mRNA was detected by RT-PCR. Western blot analyses confirmed strong expression of src on the protein level and revealed a robust activation of the protein as determined by tyrosine phosphorylation. Incubation of leukemic cells with PP1 and PP2 caused significant inhibition of proliferation in a dose dependent manner. Similar results were observed after transfection with specific siRNAs. Src inhibition blocked phosphorylation of pp60src, Erk1/2 and induced apoptosis in leukemic cells. Mutational analyses as performed by SSCP/heteroduplex and bi-directionally sequencing revealed wildtype sequence in cell lines and 60 clinical samples. Conclusions: In summary, pp60src is highly expressed and activated in cell lines and clinical samples of human AML. Moreover, phosphorylation of src is essential for leukemic cell proliferation. Underlying mutations in the coding sequence of c-src causing constitutive activation could be excluded. These data suggest that pp60src plays a crucial role in AML and src inhibition by targeted therapy might offer a useful new approach in the treatment of AML. No significant financial relationships to disclose.

scholarly journals RNAi-mediated TCF-3 gene silencing inhibits proliferation of Eca-109 esophageal cancer cells by inducing apoptosis

2017 ◽  
Vol 37 (6) ◽  
Author(s):  
Jie Ma ◽  
Xian-Bin Wang ◽  
Rui Li ◽  
Shu-Hong Xuan ◽  
Fang Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Esophageal Cancer ◽  
Cell Growth ◽  
Gene Silencing ◽  
Colony Formation ◽  
Specific Gene ◽  
Dependent Manner

Esophageal cancer (EC) remains an important health problem in China. In the present study, through the use of siRNA, specific gene knockdown of transcription factor 3 gene (TCF-3) was achieved in vitro and the effect of TCF-3 gene on human EC Eca-109 cell proliferation and apoptosis. Eca-109 cells were treated using negative control (NC) of siRNA against TCF-3 (siTCF-3) and siTCF-3 group. Colony formation assay was used to detect the colony formation ability in Eca-109 cells. MTT assay was used to measure the cell growth and viability, whereas BrDU assay was used to evaluate cell proliferation, and flow cytometry (FCM) to assess cell apoptosis. Reverse-transcription quantitative PCR (RT-qPCR) was applied to measure TCF-3 gene expression. Protein expressions of TCF-3, apoptosis-related proteins, Bcl-2, Bax, and caspase-3 were determined using Western blotting. Transfection of siTCF-3 successfully down-regulated TCF-3 gene expression. In addition, siTCF-3, reduced Eca-109 cell viability and proliferation, in a time-dependent manner, and inhibited progression of cell cycle from G0/G1 to S-stage. When treated with siTCF-3, the Eca-109 cells exhibited increased apoptosis, with up-regulated cleaved caspase and Bax expressions, whereas Bcl-2 expression was down-regulated. The present study shows that TCF-3 gene silencing inhibits Eca-109 cell growth and proliferation, suppresses cell cycle progression, and promotes apoptosis, which might serve as a new objective for EC treatment.

Resveratrol Exerts Antiproliferative Effect and Induces Apoptosis in Waldenstrom’s Macroglobulinemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1383-1383
Author(s):  
Aldo Roccaro ◽  
Xavier Leleu ◽  
Anne-Sophie Moreau ◽  
Antonio Sacco ◽  
Lian Xu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Western Blot ◽  
Cell Lines ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Downstream Target ◽  
Dose Dependent

Abstract Background: Resveratrol is a polyphenolic natural product, synthesized by a wide variety of plant species including grapes. It has gained considerable attention because of its anti-cancer properties, as demonstrated in solid and haematological malignancies. We therefore examined Resveratrol for its anti-tumor activity in Waldenstrom’s Macroglobulinemia (WM). Methods: We examined the effect of increasing concentrations of resveratrol (5–80 μM) on WM cell lines (BCWM.1), IgM secreting low-grade lymphoma cell lines (WM-WSU, MEC-1, RL), peripheral blood mononuclear cells (PBMCs) isolated from healthy donors, primary CD19+ WM cells and bone marrow stromal cells (BMSCs) isolated from bone marrow of patients with WM. [3H]-thymidine uptake and calcein-AM assay were used to evaluate the effect of resveratrol on proliferation and cytotoxicity respectively. Apoptosis and cell cycle analysis were investigated at 24h by flow cytometry using Annexin V-propidium iodide (PI) staining and PI-staining respectively. Apoptotic and cell signaling pathways targeted by resveratrol were investigated by Western Blot at 24 h and 6 h respectively. Since BMCSc confer growth and resistance to conventional treatments, we also tested the effect of resveratrol on WM cells co-cultured with BMSCs. Gene expression analysis has been performed on BCWM.1 cultured in presence or absence of resveratrol. Results: Resveratrol induced significant cytotoxicity and inhibition of DNA synthesis at 24 and 48 h on BCWM.1 with an IC50 of 10–20μM. Similar data was obtained with primary CD19+ WM cells. In contrast, resveratrol did not trigger significant reduction of proliferation of PBMCs. Resveratrol induced apoptosis in BCWM.1, as demonstrated by flow cytometry. Dose-dependent apoptosis at 24h with induction of JNK followed by caspases 3, 8, 9 and PARP cleavage was also observed. Resveratrol induced reduction of Mcl-1 and increase of p53, p63 and p73, as shown by gene expression analysis and western blot, providing an alternative mechanism of cell growth arrest in absence or mutation of p53. In parallel, resveratrol induced down-regulation of cyclin-D1, -D2, -E1, cdk-2, -4, -6 and up-regulation of p21Cip1 and p27Kip1, demonstrated in terms of transcript by gene expression analysis and protein levels by western blotting. We next observed that resveratrol inhibited ERK and Akt phosphorylation in BCWM.1 in a dose-dependent manner, as well as Akt activity, as shown by the in vitro Akt kinase assay. Phosphorylation of GSK3α/β and ribosomal protein-S6, downstream target proteins of Akt, were also markedly inhibited. Resveratrol also down-regulated Wnt signaling pathway with a reduction of nuclear β-catenin levels and a decrease of myc and survivin, both downstream target proteins of β-catenin. Lastly, adherence to BMSCs did not confer protection to WM cells against resveratrol-induced cytotoxicity Furthermore, resveratrol demonstrated synergistic cytotoxicity when combined with dexamethasone, fludarabine and bortezomib. Conclusion: These in vitro data demonstrated that resveratrol has significant antitumor activity in WM, providing the framework for clinical trials in WM patients.

scholarly journals Leukemic Cell with a Novel Kinase Fusion Gene SPAG9-JAK2 Exhibits High Sensitivity to Ruxolitinib

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5044-5044
Author(s):  
Azusa Mayumi ◽  
Toshihiro Tomii ◽  
Toshihiko Imamura ◽  
Machiko Kawamura ◽  
Hajime Hosoi
Keyword(s):  
Gene Expression ◽  
Western Blot ◽  
Signaling Pathway ◽  
Gene Expression Analysis ◽  
Fusion Gene ◽  
Dependent Manner ◽  
Cytotoxic Assay ◽  
Proliferation Activity ◽  
Constitutive Phosphorylation ◽  
Stat Pathway

[Background and aim of this study] Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a distinct subtype of B-ALL with poor prognosis. JAK2 related fusion genes with diverse partner genes have been identified in this subtype, especially in adolescent and young adults (AYA). Previously, we identified SPAG9-JAK2 fusion gene in 14-year-old boy with Ph-like ALL (Kawamura M, et al. Genes Chromosomes Cancer. 2015). JAK2 fusion protein is putative therapeutic target of Ph-like ALL with JAK2 rearrangement, so it should be clarified whether JAK inhibitor is effective for the leukemic cells with JAK2 fusion genes. In this study, we performed functional analysis of SPAG9-JAK2 to establish molecular targeting therapy for the patients with SPAG9-JAK2. [Materials and Methods] Full length of SPAG9-JAK2 cDNA was cloned into retroviral construct with Tet-On system. Then, Ba/F3 cells, which are IL-3 dependent murine pro B-ALL cells, were transduced with retroviral vector to establish Ba/F3 cells expressing SPAG9-JAK2 (Ba/F3-SPAG9-JAK2) under doxycycline (DOX) dependent manner. Once Ba/F3-SPAG9-JAK2 was established, cells were analyzed whether IL-3 independent growth was achieved. To determine whether aberrant activation of JAK-STAT pathway achieved by SPAG9-JAK2, activation of JAK-STAT pathway was evaluated by western blot. In addition, gene expression analysis using Mouse Genome 430 2.0 Array was performed for comprehensive analysis of gene expression profile related to SPAG9-JAK2. Finally, in cytotoxic assay, proliferation of Ba/F3-SPAG9-JAK2 was assessed under the media with various concentrations of ruxolitinib (JAK inhibitor, RUX). [Results and discussions] The expression of SPAG9-JAK2 in Ba/F3 cells under DOX dependent manner was confirmed by western blot. Ba/F3-SPAG9-JAK2 could proliferate without IL-3 in contrast to Ba/F3 cells (p<0.0001), suggesting that SPAG9-JAK2 had the IL-3 independent proliferation activity of Ba/F3 cells. Western blot revealed that constitutive phosphorylation of tyrosine residue of SPAG9-JAK2, Stat5, and Stat3, suggesting that constitutive activation of JAK2-STAT pathway contributes to IL-3 independent proliferation activity. Gene expression analysis revealed that, in Ba/F3-SPAG9-JAK2, expression of 187 genes among 45,037 probes was increased (log 2 ratio, ≥2.0) and the expression of 371 genes was decreased (log 2 ratio, ≤2.0), compared with those in Ba/F3. Pathway analysis revealed that the expression level of several genes related to type II interferon signaling pathway, p53 signaling pathway, Il4 receptor signaling pathway and G1 to S cell cycle control in Ba/F3-SPAG9-JAK2. In detail, Stat1, Stat2, Ptpn6, Irf1 and Cxcr4 were significantly up-regulated and Cdkn2a and Cdkn2b were significantly down-regulated in Ba/F3-SPAG9-JAK2. These findings were in accordance with activation of JAK-STAT pathway and aberrant growth promotion of Ba/F3-SPAG9-JAK2. Considering that JAK inhibitors could block constitutive phosphorylation of SPAG9-JAK2, RUX might block the IL-3 independent proliferation of Ba/F3-SPAG9-JAK2. Cytotoxic assay revealed that 100nM of RUX suppressed the proliferation of Ba/F3-SPAG9-JAK2 completely (p<0.00001), although 100nM RUX didn't show any effect on Ba/F3. Annexin V assay also determined that 100 nM RUX induced more apoptosis in Ba/F3-SPAG9-JAK2 than Ba/F3 (p<0.01). These findings suggest that RUX abolishes the proliferation activity of SPAG9-JAK2 selectively and is possibly effective for ALL with SPAG9-JAK2. [Conclusion] Although in vivo study is required, our in vitro study clearly demonstrated that RUX could be effective for the B-ALL patients withSPAG9-JAK2. Further studies are on-going to determine other therapeutic target of B-ALL with SPAG9-JAK2. Figure Disclosures No relevant conflicts of interest to declare.

Effects of N-carbamylglutamate and L-arginine on gonadotrophin-releasing hormone (GnRH) gene expression and secretion in GT1-7 cells

2018 ◽  
Vol 30 (5) ◽  
pp. 759 ◽  
Author(s):  
Y. Liu ◽  
J. H. Bai ◽  
X. L. Xu ◽  
Z. L. Chen ◽  
L. J. Spicer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Mrna Abundance ◽  
Metastasis Suppressor ◽  
Dependent Manner ◽  
Releasing Hormone ◽  
Gnrh Secretion ◽  
Gonadotrophin Releasing Hormone ◽  
High Concentrations

Recent studies have shown that N-carbamylglutamate (NCG) and arginine (ARG) supplementation improves reproductive performance in livestock. The objectives of the present study were to evaluate the effects of NCG and ARG on GT1-7 cell gonadotrophin-releasing hormone (GnRH) secretion, gene expression and cell proliferation. GT1-7 cells were treated in vitro with different concentrations of NCG (0–1.0 mM) or ARG (0–4.0 mM) in serum-free medium for 12 or 24 h. For GnRH secretion and cell proliferation, GT1-7 cells were more sensitive to NCG than ARG. NCG treatment after 12 h increased cell numbers and inhibited GnRH secretion in a dose-dependent manner (P < 0.05), although there was no significant effect of NCG on these parameters after 24 h culture. ARG treatment decreased GnRH secretion after 24 h (P < 0.05), whereas it had no effect after 12 h. GT1-7 cells express GnRH, Kiss-1 metastasis-suppressor (Kiss1), G-protein coupled receptor 54 (GPR54), neuronal nitric oxide synthase (nNOS) and estrogen receptor α (ERα) genes. High concentrations of NCG (1.0 mM) and ARG (4.0 mM) inhibited (P < 0.05) GnRH and nNOS mRNA abundance in GT1-7 cells. ARG treatment decreased Kiss1 and increased ERα mRNA abundance. Thus, high concentrations of NCG (1.0 mM) and ARG (4.0 mM) may act both directly and indirectly to regulate GnRH neuron function by downregulating genes related to GnRH synthesis and secretion to slow GnRH production while stimulating GT1-7 cell proliferation.

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