Characterization of STAT3 activation and expression in canine and human osteosarcoma - PubMed (original) (raw)
Characterization of STAT3 activation and expression in canine and human osteosarcoma
Stacey L Fossey et al. BMC Cancer. 2009.
Abstract
Background: Dysregulation of signal transducer and activator of transcription 3 (STAT3) has been implicated as a key participant in tumor cell survival, proliferation, and metastasis and is often correlated with a more malignant tumor phenotype. STAT3 phosphorylation has been demonstrated in a subset of human osteosarcoma (OSA) tissues and cell lines. OSA in the canine population is known to exhibit a similar clinical behavior and molecular biology when compared to its human counterpart, and is often used as a model for preclinical testing of novel therapeutics. The purpose of this study was to investigate the potential role of STAT3 in canine and human OSA, and to evaluate the biologic activity of a novel small molecule STAT3 inhibitor.
Methods: To examine STAT3 and Src expression in OSA, we performed Western blotting and RT-PCR. OSA cells were treated with either STAT3 siRNA or small molecule Src (SU6656) or STAT3 (LLL3) inhibitors and cell proliferation (CyQUANT), caspase 3/7 activity (ELISA), apoptosis (Western blotting for PARP cleavage) and/or viability (Wst-1) were determined. Additionally, STAT3 DNA binding after treatment was determined using EMSA. Expression of STAT3 targets after treatment was demonstrated with Western blotting, RT-PCR, or gel zymography.
Results: Our data demonstrate that constitutive activation of STAT3 is present in a subset of canine OSA tumors and human and canine cell lines, but not normal canine osteoblasts. In both canine and human OSA cell lines, downregulation of STAT3 activity through inhibition of upstream Src family kinases using SU6656, inhibition of STAT3 DNA binding and transcriptional activities using LLL3, or modulation of STAT3 expression using siRNA, all resulted in decreased cell proliferation and viability, ultimately inducing caspase-3/7 mediated apoptosis in treated cells. Furthermore, inhibition of either Src or STAT3 activity downregulated the expression of survivin, VEGF, and MMP2, all known transcriptional targets of STAT3.
Conclusion: These data suggest that STAT3 activation contributes to the survival and proliferation of human and canine OSA cells, thereby providing a potentially promising target for therapeutic intervention. Future investigational trials of LLL3 in dogs with spontaneous OSA will help to more accurately define the role of STAT3 in the clinical setting.
Figures
Figure 1
Activation of Src and STAT3 in OSA cell lines and tissues. A) OSA cells and normal canine osteoblasts were serum starved then left untreated or stimulated with rhHGF (50 ng/ml). Protein lysates were generated and separated by SDS PAGE and Western blotting for pSTAT3 (Y705), pSrc (Y416), total Src, and total STAT3 was performed. B) OSA cell lines were serum starved for 2 hours while in suspension culture (S), serum starved for 2 hours while remaining adherent to tissue culture flasks (A), or left in 1% serum while adherent in flasks for 2 hours prior to collection (C). Protein lysates were generated and separated by SDS-PAGE and Western blotting for pSTAT3 (Y705), pSrc (Y416), total Src, and total STAT3 was performed. C) Fresh frozen canine OSA tumor tissues and control normal muscle tissue were processed for protein lysates. Protein was separated by SDS-PAGE and Western blotting for pSTAT3 (Y705), STAT3, VEGF, and β-actin was performed. D) RNA was collected from canine OSA cell lines and RT-PCR was performed for SFK members Src, Fyn, Yes, and Lyn as well as GAPDH as a control.
Figure 2
STAT3 siRNA induces downregulation of STAT3 and its downstream targets with subsequent loss of viability and apoptosis of canine OSA cells. A) Canine OSA cell lines OSA8 and OSA16 were transfected with Lipofectamine 2000 alone, 50 pMol scrambled siRNA, or 50 pMol STAT3 siRNA and collected 48 hours post transfection. Protein lysates were generated and separated via SDS-PAGE. Western blotting for STAT3, VEGF, survivin and β-actin was performed. B) OSA8 and OSA16 were transfected with Lipofectamine 2000, scrambled siRNA, or STAT3 siRNA at 0 and 48 hours. Cell viability was assessed at 0, 72, or 96 hours post transfection using the Wst-1 assay. Values are reported as percentage of control wells. C) OSA8 and OSA16 were transfected with Lipofectamine 2000, scrambled siRNA, or STAT3 siRNA and generation of active caspase-3/7 was assessed 48 hours post transfection using the SensoLyte® Homogeneous AMC Caspase-3/7 Assay kit. D) OSA8 cells were left untreated or transfected with STAT3 siRNA or scrambled siRNA and evaluated by digital photography 72 hours post transfection. *p < 0.05
Figure 3
SU6656 inhibits phosphorylation of Src and STAT3 in OSA lines. Canine and human OSA cell lines were serum starved then left untreated or incubated with SU6656 for 2 hours. Cells were collected and protein separated by SDS-PAGE, followed by Western blotting for pSrc (Y416), pSTAT3 (Y705), total Src, and total STAT3.
Figure 4
Downregulation of Src or STAT3 leads to decreased STAT3 DNA binding. The human OSA cell line SJSA and canine OSA cell line OSA8 were incubated with media, DMSO, or drug (A) SU6656, B) LLL3) for 72 hours. Cells were harvested and nuclear protein isolated. Nuclear protein was added to binding reactions with labeled species specific DNA probes for the STAT3 recognition sequences located in the promoter for survivin in the presence or absence of unlabelled competitor probe. Additionally, anti-STAT3 antibody was added to nuclear protein from cells treated with media alone to demonstrate specificity of the binding reaction. Reactions were separated on an acrylamide gel, transferred to a nylon membrane, and the DNA was crosslinked. The membranes were processed using the LightShift Chemiluminescent EMSA kit.
Figure 5
Inhibition of Src or STAT3 leads to loss of OSA cell proliferation and apoptosis via the caspase 3,7 pathway. A) Canine or B) human OSA cell lines were treated with DMSO, SU6656, or LLL3 for 1, 3, or 5 days. Proliferation was analyzed using the CyQUANT cell proliferation assay kit. Apoptosis was assessed by measuring active caspase-3/7 using the SensoLyte® Homogeneous AMC Caspase-3/7 Assay kit. Proliferation values are listed as a percentage of DMSO control. *p < 0.05
Figure 6
Inhibition of Src or STAT3 leads to PARP cleavage and downregulates survivin expression in OSA cell lines. A) Canine or B) human OSA cell lines were treated for 24 or 48 hours with DMSO, SU6656, or LLL3. Cells were collected and protein lysates were separated via SDS-PAGE. Western blotting for survivin, PARP and β-actin was performed. The anti-PARP antibody utilized in our laboratory has been applied to experiments with canine cell lines and recognizes a 113 kDa intact PARP protein and a 23 kDa cleaved PARP fragment [53].
Figure 7
Downregulation of Src or STAT3 leads to loss of MMP2 expression in OSA cells. A) Canine OSA cell lines OSA8 and OSA 32 were treated with DMSO or LLL3 (40 uM), for 72 hours. RNA was collected and RT-PCR was performed for MMP2 and GAPDH. B) Canine and human OSA cell lines were treated with DMSO, SU6656, or LLL3 for 72 hours. Media was collected and MMP2 was assessed via gel zymography. C) Gel zymography images were evaluated by densitometry using NIH Image J.
Figure 8
VEGF expression is reduced after Src or STAT3 inhibition. A) Canine OSA cell line OSA8 and human OSA cell line U2OS were treated with LLL3 for 72 hours. RNA was collected and RT-PCR was performed for VEGF and GAPDH. B) Canine and human OSA cell lines were treated with DMSO, SU6656, or LLL3 for 72 hours. Cells were collected and protein lysates were separated via SDS-PAGE. Western blotting for VEGF and β-actin was performed.
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