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Characterization of STAT3 activation and expression in canine and human osteosarcoma.

Fossey SL, Liao AT, McCleese JK, Bear MD, Lin J, Li PK, Kisseberth WC, London CA - BMC Cancer (2009)

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA. fossey.1@osu.edu

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.

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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
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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

Mentions: We designed a small interfering RNA specifically for canine STAT3 to determine the effect of STAT3 downregulation in canine OSA cells. Expression of STAT3 48 hours post transfection was significantly reduced in cells treated with 50 pMol siRNA (Fig. 2A) as evidenced by Western blotting. Additionally, expression of the downstream targets of STAT3, VEGF and survivin, was reduced in both OSA cell lines tested. Downregulation of STAT3 and survivin correlated with a significant loss in viability after 72 and 96 hours and induction of caspase-3/7 activity after 48 hours in both cell lines treated with STAT3 siRNA when compared to those treated with transfection reagent Lipofectamine 2000 alone or scrambled control siRNA (Fig. 2B and 2C). Furthermore, a marked reduction in cellularity occurred at 72 hours post transfection with Stat3 siRNA when compared to cells receiving media alone or scrambled siRNA (Fig. 2D).


Characterization of STAT3 activation and expression in canine and human osteosarcoma.

Fossey SL, Liao AT, McCleese JK, Bear MD, Lin J, Li PK, Kisseberth WC, London CA - BMC Cancer (2009)

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
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2666757&req=5

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
Mentions: We designed a small interfering RNA specifically for canine STAT3 to determine the effect of STAT3 downregulation in canine OSA cells. Expression of STAT3 48 hours post transfection was significantly reduced in cells treated with 50 pMol siRNA (Fig. 2A) as evidenced by Western blotting. Additionally, expression of the downstream targets of STAT3, VEGF and survivin, was reduced in both OSA cell lines tested. Downregulation of STAT3 and survivin correlated with a significant loss in viability after 72 and 96 hours and induction of caspase-3/7 activity after 48 hours in both cell lines treated with STAT3 siRNA when compared to those treated with transfection reagent Lipofectamine 2000 alone or scrambled control siRNA (Fig. 2B and 2C). Furthermore, a marked reduction in cellularity occurred at 72 hours post transfection with Stat3 siRNA when compared to cells receiving media alone or scrambled siRNA (Fig. 2D).

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA. fossey.1@osu.edu

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.

Show MeSH
Related in: MedlinePlus