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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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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].
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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].

Mentions: Given the correlation between dysregulated STAT3 activation and tumor cell survival, we wanted to determine whether inhibition of Src and STAT3 activity in OSA cell lines affected their capacity to proliferate and survive. Human and canine OSA cell lines were cultured with SU6656 or LLL3 at increasing concentrations for 1, 3, or 5 days. Cell proliferation was assessed using the CyQUANT assay and apoptosis was determined using the SensoLyte Homogeneous AMC Caspase 3/7 Assay kit. Although there was some variation in response among cell lines, SU6656 and LLL3 caused a dose and time dependent loss in cell proliferation in all human and canine OSA cell lines (Fig. 5A and 5B). Significant increases in apoptosis as evidenced by caspase 3,7 activity were observed in many OSA cell lines even at 24 hours post treatment (Fig. 5A and 5B). The induction of apoptosis was further confirmed by demonstration of PARP cleavage in OSA cell lines treated with LLL3 or SU6656 for 24 and 48 hours (Fig. 6A and 6B). At both time points, there was a dose and time dependent corresponding decrease in survivin expression in the canine (Fig. 6A) and human OSA cell lines (Fig. 6B).


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)

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

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

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].
Mentions: Given the correlation between dysregulated STAT3 activation and tumor cell survival, we wanted to determine whether inhibition of Src and STAT3 activity in OSA cell lines affected their capacity to proliferate and survive. Human and canine OSA cell lines were cultured with SU6656 or LLL3 at increasing concentrations for 1, 3, or 5 days. Cell proliferation was assessed using the CyQUANT assay and apoptosis was determined using the SensoLyte Homogeneous AMC Caspase 3/7 Assay kit. Although there was some variation in response among cell lines, SU6656 and LLL3 caused a dose and time dependent loss in cell proliferation in all human and canine OSA cell lines (Fig. 5A and 5B). Significant increases in apoptosis as evidenced by caspase 3,7 activity were observed in many OSA cell lines even at 24 hours post treatment (Fig. 5A and 5B). The induction of apoptosis was further confirmed by demonstration of PARP cleavage in OSA cell lines treated with LLL3 or SU6656 for 24 and 48 hours (Fig. 6A and 6B). At both time points, there was a dose and time dependent corresponding decrease in survivin expression in the canine (Fig. 6A) and human OSA cell lines (Fig. 6B).

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