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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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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.
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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.

Mentions: STAT3 is known to regulate the transcription of MMP2, a matrix metalloproteinase known to be important in tumor cell migration and metastasis. Treatment of the canine OSA8 and OSA32 cell lines with the STAT3 inhibitor LLL3 resulted in a significant reduction in MMP2 mRNA expression (Fig. 7A). Treatment of canine and human OSA cell lines with SU6656 or LLL3 for 72 hours resulted in a corresponding dose and time dependent downregulation of both the proenzyme and active form of MMP2 as assessed by gel zymography (Fig. 7B). Changes in MMP2 band intensity following drug treatment were quantified via densitometry using NIH Image J (Fig. 7C).


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)

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

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

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.
Mentions: STAT3 is known to regulate the transcription of MMP2, a matrix metalloproteinase known to be important in tumor cell migration and metastasis. Treatment of the canine OSA8 and OSA32 cell lines with the STAT3 inhibitor LLL3 resulted in a significant reduction in MMP2 mRNA expression (Fig. 7A). Treatment of canine and human OSA cell lines with SU6656 or LLL3 for 72 hours resulted in a corresponding dose and time dependent downregulation of both the proenzyme and active form of MMP2 as assessed by gel zymography (Fig. 7B). Changes in MMP2 band intensity following drug treatment were quantified via densitometry using NIH Image J (Fig. 7C).

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