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Characterization of STAT3 expression, signaling and inhibition in feline oral squamous cell carcinoma.

Brown ME, Bear MD, Rosol TJ, Premanandan C, Kisseberth WC, London CA - BMC Vet. Res. (2015)

Bottom Line: Exposure of SCCF2 cells to LLL12 resulted in decreased expression of pSTAT3 and total STAT3, apoptosis as assessed by caspase 3/7 activation, inhibition of colony formation and reduced expression of the STAT3 transcriptional target survivin.The OSCC cell lines with low basal STAT3 phosphorylation did not exhibit these effects, suggesting that STAT3 inhibition was responsible for the observed results.However, given the up-regulation of several STAT3 transcriptional targets following treatment, further investigation of STAT3 and its related signaling pathways in OSCC is warranted.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA.

ABSTRACT

Background: Signal transducer and activator of transcription 3 (STAT3) plays a critical role in tumor development by regulating signaling pathways involved in cell proliferation, survival, metastasis and angiogenesis. STAT3 is activated in many cancers, including head and neck squamous cell carcinoma (HNSCC) in people. Feline oral squamous cell carcinoma (OSCC) is similar to advanced or recurrent HNSCC as it is poorly responsive to traditional therapies and carries a poor long-term prognosis. The purpose of this study was to characterize expression and activation of STAT3 in feline OSCC cell lines and tumor samples and to investigate the biologic activity of a novel, allosteric STAT3 inhibitor, LLL12, in feline OSCC cell lines.

Results: We evaluated 3 feline OSCC cell lines and one of these (SCCF2) exhibited high levels of constitutive STAT3 phosphorylation and high sensitivity to LLL12 treatment. Exposure of SCCF2 cells to LLL12 resulted in decreased expression of pSTAT3 and total STAT3, apoptosis as assessed by caspase 3/7 activation, inhibition of colony formation and reduced expression of the STAT3 transcriptional target survivin. In contrast, the STAT3 transcriptional targets VEGF and MCL-1 increased after LLL12 treatment. This was, in part, likely due to LLL12 mediated upregulation of HIF-1α, which is known to drive VEGF and MCL-1 expression. The OSCC cell lines with low basal STAT3 phosphorylation did not exhibit these effects, suggesting that STAT3 inhibition was responsible for the observed results. Lastly, immunohistochemistry for pSTAT3 was performed using a feline OSCC tissue microarray, demonstrating expression in 48 % of samples tested.

Conclusions: These data demonstrate that LLL12 has biologic activity against a feline OSCC cell line expressing pSTAT3 and that STAT3 represents a target for therapeutic intervention in this disease. However, given the up-regulation of several STAT3 transcriptional targets following treatment, further investigation of STAT3 and its related signaling pathways in OSCC is warranted.

No MeSH data available.


Related in: MedlinePlus

Impact of LLL12 on colony formation in feline OSCC cell lines. a Feline OSCC cells were seeded at 1,000 cells per well in 6 –well plates for 24 h, followed by treatment with DMSO, 0.02, 0.2 or 2 μM LLL12 until formation of visible colonies. Cells were then fixed and stained with crystal violet and colonies greater than 50 cells were counted. After counting colonies, plating efficiency and survival fraction were calculated. Plating efficiency was defined as the number of colonies formed divided by the number of cells seeded in DMSO treated groups. Survival fraction was defined as the number of colonies formed divided by the number of cells seeded in LLL12 treated groups, normalized to the plating efficiency (*p < 0.0001). b Photographs of representative 6-well plates
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Fig5: Impact of LLL12 on colony formation in feline OSCC cell lines. a Feline OSCC cells were seeded at 1,000 cells per well in 6 –well plates for 24 h, followed by treatment with DMSO, 0.02, 0.2 or 2 μM LLL12 until formation of visible colonies. Cells were then fixed and stained with crystal violet and colonies greater than 50 cells were counted. After counting colonies, plating efficiency and survival fraction were calculated. Plating efficiency was defined as the number of colonies formed divided by the number of cells seeded in DMSO treated groups. Survival fraction was defined as the number of colonies formed divided by the number of cells seeded in LLL12 treated groups, normalized to the plating efficiency (*p < 0.0001). b Photographs of representative 6-well plates

Mentions: To further assess the effects of LLL12 on feline OSCC cell growth, SCCF2 and SCCF3 cells were seeded in 6-well plates and treated with DMSO or LLL12 and evaluated daily. Once colony formation was observed in DMSO treated wells, the plates were collected and colonies were counted following staining with crystal violet. As shown in Fig. 5, colony formation was significantly inhibited in both SCCF2 and SCCF3 at concentrations above 0.02 and 0.2 μM, respectively. Photographs of representative 6-well plates are shown in Figure 5b.Fig. 5


Characterization of STAT3 expression, signaling and inhibition in feline oral squamous cell carcinoma.

Brown ME, Bear MD, Rosol TJ, Premanandan C, Kisseberth WC, London CA - BMC Vet. Res. (2015)

Impact of LLL12 on colony formation in feline OSCC cell lines. a Feline OSCC cells were seeded at 1,000 cells per well in 6 –well plates for 24 h, followed by treatment with DMSO, 0.02, 0.2 or 2 μM LLL12 until formation of visible colonies. Cells were then fixed and stained with crystal violet and colonies greater than 50 cells were counted. After counting colonies, plating efficiency and survival fraction were calculated. Plating efficiency was defined as the number of colonies formed divided by the number of cells seeded in DMSO treated groups. Survival fraction was defined as the number of colonies formed divided by the number of cells seeded in LLL12 treated groups, normalized to the plating efficiency (*p < 0.0001). b Photographs of representative 6-well plates
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4536595&req=5

Fig5: Impact of LLL12 on colony formation in feline OSCC cell lines. a Feline OSCC cells were seeded at 1,000 cells per well in 6 –well plates for 24 h, followed by treatment with DMSO, 0.02, 0.2 or 2 μM LLL12 until formation of visible colonies. Cells were then fixed and stained with crystal violet and colonies greater than 50 cells were counted. After counting colonies, plating efficiency and survival fraction were calculated. Plating efficiency was defined as the number of colonies formed divided by the number of cells seeded in DMSO treated groups. Survival fraction was defined as the number of colonies formed divided by the number of cells seeded in LLL12 treated groups, normalized to the plating efficiency (*p < 0.0001). b Photographs of representative 6-well plates
Mentions: To further assess the effects of LLL12 on feline OSCC cell growth, SCCF2 and SCCF3 cells were seeded in 6-well plates and treated with DMSO or LLL12 and evaluated daily. Once colony formation was observed in DMSO treated wells, the plates were collected and colonies were counted following staining with crystal violet. As shown in Fig. 5, colony formation was significantly inhibited in both SCCF2 and SCCF3 at concentrations above 0.02 and 0.2 μM, respectively. Photographs of representative 6-well plates are shown in Figure 5b.Fig. 5

Bottom Line: Exposure of SCCF2 cells to LLL12 resulted in decreased expression of pSTAT3 and total STAT3, apoptosis as assessed by caspase 3/7 activation, inhibition of colony formation and reduced expression of the STAT3 transcriptional target survivin.The OSCC cell lines with low basal STAT3 phosphorylation did not exhibit these effects, suggesting that STAT3 inhibition was responsible for the observed results.However, given the up-regulation of several STAT3 transcriptional targets following treatment, further investigation of STAT3 and its related signaling pathways in OSCC is warranted.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA.

ABSTRACT

Background: Signal transducer and activator of transcription 3 (STAT3) plays a critical role in tumor development by regulating signaling pathways involved in cell proliferation, survival, metastasis and angiogenesis. STAT3 is activated in many cancers, including head and neck squamous cell carcinoma (HNSCC) in people. Feline oral squamous cell carcinoma (OSCC) is similar to advanced or recurrent HNSCC as it is poorly responsive to traditional therapies and carries a poor long-term prognosis. The purpose of this study was to characterize expression and activation of STAT3 in feline OSCC cell lines and tumor samples and to investigate the biologic activity of a novel, allosteric STAT3 inhibitor, LLL12, in feline OSCC cell lines.

Results: We evaluated 3 feline OSCC cell lines and one of these (SCCF2) exhibited high levels of constitutive STAT3 phosphorylation and high sensitivity to LLL12 treatment. Exposure of SCCF2 cells to LLL12 resulted in decreased expression of pSTAT3 and total STAT3, apoptosis as assessed by caspase 3/7 activation, inhibition of colony formation and reduced expression of the STAT3 transcriptional target survivin. In contrast, the STAT3 transcriptional targets VEGF and MCL-1 increased after LLL12 treatment. This was, in part, likely due to LLL12 mediated upregulation of HIF-1α, which is known to drive VEGF and MCL-1 expression. The OSCC cell lines with low basal STAT3 phosphorylation did not exhibit these effects, suggesting that STAT3 inhibition was responsible for the observed results. Lastly, immunohistochemistry for pSTAT3 was performed using a feline OSCC tissue microarray, demonstrating expression in 48 % of samples tested.

Conclusions: These data demonstrate that LLL12 has biologic activity against a feline OSCC cell line expressing pSTAT3 and that STAT3 represents a target for therapeutic intervention in this disease. However, given the up-regulation of several STAT3 transcriptional targets following treatment, further investigation of STAT3 and its related signaling pathways in OSCC is warranted.

No MeSH data available.


Related in: MedlinePlus