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In JoVE (1)
Other Publications (7)
- European Journal of Haematology
- European Journal of Cancer (Oxford, England : 1990)
- Neoplasia (New York, N.Y.)
- Clinical Cancer Research : an Official Journal of the American Association for Cancer Research
- Clinical Cancer Research : an Official Journal of the American Association for Cancer Research
- Molecular Medicine (Cambridge, Mass.)
- Molecular and Cellular Biology
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Articles by Rajesh Kumar Gandhirajan in JoVE
JoVE General
Visualisering af Vascular Ca 2 + Signaling Triggered ved Paracrine Afledte ROS
1Department of Biochemistry, Temple University, 2Department of Anesthesiology and Pain Medicine, University of Washington
En effektiv metode til at få indblik i at visualisere paracrine-afledte ROS induktion af endothel Ca2 + signalering er beskrevet. Denne metode benytter sig af at måle paracrine afledt ROS udløst Ca2 + mobilisering i vaskulære endotelceller i en co-kultur model.
Other articles by Rajesh Kumar Gandhirajan on PubMed
Small Molecule Inhibitors of WNT Signaling Effectively Induce Apoptosis in Acute Myeloid Leukemia Cells
In a significant proportion of acute myeloid leukemia (AML) cases the canonical WNT pathway is upregulated and targeting the WNT/LEF1 signaling cascade in AML may be a promising approach to develop new treatments for this entity. Recently two compounds (CGP049090 and PFK115-584) have been identified, which specifically inhibit complexation of beta-catenin (CTNNB1) and lymphoid enhancer-binding factor 1 (LEF1) leading to transcriptional inactivation of LEF1 in colon carcinoma cell lines. To evaluate the effect of WNT inhibition utilizing theses compounds with regard to their effectivity in AML we treated the AML cell lines Kasumi-1 and HL-60, primary AML blasts and healthy peripheral blood mononuclear cells (PBMCs) with varying concentrations of both substances. Treatment with both compounds for 24 h resulted in a significant killing of AML cell lines and primary AML blasts with 50% effective concentration doses (EC(50)) within the submicromolar range. PBMCs were not significantly affected as indicated by EC(50)-values 100-fold higher than for AML cells. Cell kill was mediated by apoptosis as indicated by induction of caspases 3 and 7 and cleavage of poly(ADP-ribose) polymerase (PARP) upon treatment. Furthermore, we could show that both compounds substantially decrease expression of CTNNB1/LEF1 target genes c-myc, cyclin D1 and survivin, proofing the specificity of the substances. This was shown in both, AML cell lines and most of the tested primary samples. Our data demonstrate that targeting this pathway seems to be an innovative approach in the treatment of AML.
Targeting the WNT/beta-catenin/TCF/LEF1 Axis in Solid and Haematological Cancers: Multiplicity of Therapeutic Options
Among aberrantly regulated signalling pathways in cancer the WNT/beta-catenin pathway plays an outstanding role, since it was shown to be critically involved in a wide range of neoplasias. While the underlying mechanisms vary, overexpression of WNTs was found to mediate active signalling in some of these diseases. Other cancers show a mutation in pathway members further downstream, such as APC, Axin or beta-catenin, leading to aberrant signalling activation. Another mechanism initiating activation of WNT/beta-catenin signalling is the silencing of expression of negative WNT/beta-catenin regulators, such as DKK and WIF1, by, for example, promoter hypermethylation. All these mechanisms result in a common consequence, the activation of TCF/LEF1 transcription factors and subsequent target gene expression. Several target genes are known to be key players in tumourigenesis, such as c-myc, cyclin D1 or survivin. The variety of possible underlying mechanisms leading to beta-catenin/TCF/LEF1 activation offers multiple options to target the aberrantly activated pathway in order to prevent target gene expression and/or their gene products to exert their tumourigenic function. Here, we summarise the physiological role of WNT/beta-catenin signalling and the consequences of its aberrant activation during tumourigenesis. Furthermore, we discuss the possible strategies to target this pathway and their potential importance in cancer treatment.
Small Molecule Inhibitors of Wnt/beta-catenin/lef-1 Signaling Induces Apoptosis in Chronic Lymphocytic Leukemia Cells in Vitro and in Vivo
Lymphoid enhancer factor-1 (lef-1) is overexpressed in B-cell chronic lymphocytic leukemia (CLL) when compared with normal B cells and transcribes several genes implicated in the pathogenesis of CLL. We therefore hypothesize that antagonism of lef-1 might lead to killing of CLL cells. We used two small molecule inhibitors of Wnt/beta-catenin/lef-1 signaling (CGP049090 and PKF115-584) to test our hypothesis.
The Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Inhibitors Vatalanib and Pazopanib Potently Induce Apoptosis in Chronic Lymphocytic Leukemia Cells in Vitro and in Vivo
There is evidence that vascular endothelial growth factor (VEGF) is a critical microenvironmental factor that exerts angiogenesis-independent effects on the survival of chronic lymphocytic leukemia (CLL) cells. Vatalanib and pazopanib are potent orally available VEGF receptor tyrosine kinase inhibitors. We investigated the efficacy and selectivity of both compounds in CLL cells, simulated potential combination with conventional cytostatics, and tested the effect of both substances on CLL-like tumor xenografts.
Nitric Oxide-donating Acetylsalicylic Acid Induces Apoptosis in Chronic Lymphocytic Leukemia Cells and Shows Strong Antitumor Efficacy in Vivo
Nitric oxide-donating acetylsalicylic acid (NO-ASA) has been shown to possess an antineoplastic effect in Wnt-/β-catenin-active cancers. As chronic lymphocytic leukemia (CLL) cells exhibit aberrantly active Wnt signaling, we investigated the effect of the para-isomer of NO-ASA on CLL cell survival in vitro and in a CLL-like xenograft mouse model.
Bone Marrow Stromal Cell-derived Vascular Endothelial Growth Factor (VEGF) Rather Than Chronic Lymphocytic Leukemia (CLL) Cell-derived VEGF is Essential for the Apoptotic Resistance of Cultured CLL Cells
Chronic lymphocytic leukemia (CLL) cells feature a pronounced apoptotic resistance. The vascular endothelial growth factor (VEGF) possesses a role in this apoptotic block, although underlying functional mechanisms and the involvement of the microenvironment are unclear. In this study, the VEGF status in CLL was assessed by enzyme-linked immunosorbent assay and immunofluorescence. VEGF receptor 2 (VEGFR2) phosphorylation was determined flow cytometrically and by immunofluorescence. For co-culture, CLL cells were cultivated on a monolayer of the bone marrow-derived stromal cell (BMSC) line HS5. Secreted VEGF was neutralized using the monoclonal antibody mAb293 (R&D Systems, Minneapolis, MN, USA). To block protein secretion, we used Brefeldin A. VEGF was downregulated in BMSCs by small interfering RNA (siRNA), and we assessed survival by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. CLL cells express and secrete VEGF and possess phosphorylated VEGFR2. This positive VEGF status is not sufficient to prevent spontaneous apoptosis in vitro. Coculture with BMSCs, which secrete vast amounts of VEGF, maintains in vitro CLL cell survival. Blockage of secreted VEGF using the monoclonal antibody mAb293 significantly reduced the survival support for cocultured CLL cells. Both general blockage of protein secretion by Brefeldin A in BMSCs, but not in CLL cells, and siRNA-mediated downregulation of VEGF in BMSCs, significantly reduced the coculture-mediated survival support for CLL cells. It can be concluded that BMSC-derived proteins and VEGF, in particular, but not CLL cell-derived VEGF, is essentially involved in the coculture-mediated survival support for CLL cells. Hence, therapeutic targeting of VEGF signaling might be a promising approach to overcome the apoptotic resistance CLL cells feature within their natural microenvironment.
Requirement of FADD, NEMO, and BAX/BAK for Aberrant Mitochondrial Function in Tumor Necrosis Factor Alpha-induced Necrosis
Necroptosis represents a form of alternative programmed cell death that is dependent on the kinase RIP1. RIP1-dependent necroptotic death manifests as increased reactive oxygen species (ROS) production in mitochondria and is accompanied by loss of ATP biogenesis and eventual dissipation of mitochondrial membrane potential. Here, we show that tumor necrosis factor alpha (TNF-α)-induced necroptosis requires the adaptor proteins FADD and NEMO. FADD was found to mediate formation of the TNF-α-induced pronecrotic RIP1-RIP3 kinase complex, whereas the IκB Kinase (IKK) subunit NEMO appears to function downstream of RIP1-RIP3. Interestingly, loss of RelA potentiated TNF-α-dependent necroptosis, indicating that NEMO regulates necroptosis independently of NF-κB. Using both pharmacologic and genetic approaches, we demonstrate that the overexpression of antioxidants alleviates ROS elevation and necroptosis. Finally, elimination of BAX and BAK or overexpression of Bcl-x(L) protects cells from necroptosis at a later step. These findings provide evidence that mitochondria play an amplifying role in inflammation-induced necroptosis.
