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Biologic activity of the novel small molecule STAT3 inhibitor LLL12 against canine osteosarcoma cell lines.

Couto JI, Bear MD, Lin J, Pennel M, Kulp SK, Kisseberth WC, London CA - BMC Vet. Res. (2012)

Bottom Line: The present study sought to characterize the biologic activity of a novel allosteric STAT3 inhibitor, LLL12, in canine OS cell lines.Lastly, LLL12 exhibited synergistic anti-proliferative activity with the chemotherapeutic doxorubicin in the OS lines.LLL12 exhibits biologic activity against canine OS cell lines through inhibition of STAT3 related cellular functions supporting its potential use as a novel therapy for OS.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210, USA.

ABSTRACT

Background: STAT3 [1] has been shown to be dysregulated in nearly every major cancer, including osteosarcoma (OS). Constitutive activation of STAT3, via aberrant phosphorylation, leads to proliferation, cell survival and resistance to apoptosis. The present study sought to characterize the biologic activity of a novel allosteric STAT3 inhibitor, LLL12, in canine OS cell lines.

Results: We evaluated the effects of LLL12 treatment on 4 canine OS cell lines and found that LLL12 inhibited proliferation, induced apoptosis, reduced STAT3 phosphorylation, and decreased the expression of several transcriptional targets of STAT3 in these cells. Lastly, LLL12 exhibited synergistic anti-proliferative activity with the chemotherapeutic doxorubicin in the OS lines.

Conclusion: LLL12 exhibits biologic activity against canine OS cell lines through inhibition of STAT3 related cellular functions supporting its potential use as a novel therapy for OS.

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Related in: MedlinePlus

Evaluation of canine OS cell lines for apoptosis following LLL12 treatment. Canine OS cell lines treated with vehicle or LLL12 for 24 h. Apoptosis was assessed by measuring activated caspases 3 and 7 (A) using the Sensolyte® Homogeneous AMC Caspase-3/7 Assay kit. Experiments were performed in quadruplicate and repeated three times. The same canine OS cell lines were treated under identical conditions as above and stained with annexin V-FITC/PI and analyzed by flow cytometry (B). Normal canine osteoblasts were treated under identical conditions and stained as above (C). There was a significant increasing trend in caspase activity for all lines except OSA 8 (p<0.01).
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Figure 2: Evaluation of canine OS cell lines for apoptosis following LLL12 treatment. Canine OS cell lines treated with vehicle or LLL12 for 24 h. Apoptosis was assessed by measuring activated caspases 3 and 7 (A) using the Sensolyte® Homogeneous AMC Caspase-3/7 Assay kit. Experiments were performed in quadruplicate and repeated three times. The same canine OS cell lines were treated under identical conditions as above and stained with annexin V-FITC/PI and analyzed by flow cytometry (B). Normal canine osteoblasts were treated under identical conditions and stained as above (C). There was a significant increasing trend in caspase activity for all lines except OSA 8 (p<0.01).

Mentions: To determine if LLL12 growth inhibition was mediated via apoptosis, canine OS cell lines were treated with DMSO or LLL12 for 24 hours, and caspase 3/7 activity was measured. In all cell lines, caspase 3/7 activity was increased at 24 hours post treatment with LLL12 at concentrations of 0.4-0.8 μM (Figure2A). OS cells were also stained with Annexin V-FITC/PI and analyzed by flow cytometry to assess the percentage of early and late apoptotic cells in the population. After a 24 hour exposure to 0.5 μM LLL12 there was an increase in the proportion of early apoptotic (Annexin V positive, up to 22-fold increase) and late apoptotic (Annexin V/PI positive, up to 13-fold increase) cells. This correlated with data generated from the caspase assay (Figure2B). Normal canine osteoblasts were treated and analyzed by flow cytometry as described above, and were far less sensitive to the apoptosis inducing effects of LLL12 (Figure2C).


Biologic activity of the novel small molecule STAT3 inhibitor LLL12 against canine osteosarcoma cell lines.

Couto JI, Bear MD, Lin J, Pennel M, Kulp SK, Kisseberth WC, London CA - BMC Vet. Res. (2012)

Evaluation of canine OS cell lines for apoptosis following LLL12 treatment. Canine OS cell lines treated with vehicle or LLL12 for 24 h. Apoptosis was assessed by measuring activated caspases 3 and 7 (A) using the Sensolyte® Homogeneous AMC Caspase-3/7 Assay kit. Experiments were performed in quadruplicate and repeated three times. The same canine OS cell lines were treated under identical conditions as above and stained with annexin V-FITC/PI and analyzed by flow cytometry (B). Normal canine osteoblasts were treated under identical conditions and stained as above (C). There was a significant increasing trend in caspase activity for all lines except OSA 8 (p<0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585923&req=5

Figure 2: Evaluation of canine OS cell lines for apoptosis following LLL12 treatment. Canine OS cell lines treated with vehicle or LLL12 for 24 h. Apoptosis was assessed by measuring activated caspases 3 and 7 (A) using the Sensolyte® Homogeneous AMC Caspase-3/7 Assay kit. Experiments were performed in quadruplicate and repeated three times. The same canine OS cell lines were treated under identical conditions as above and stained with annexin V-FITC/PI and analyzed by flow cytometry (B). Normal canine osteoblasts were treated under identical conditions and stained as above (C). There was a significant increasing trend in caspase activity for all lines except OSA 8 (p<0.01).
Mentions: To determine if LLL12 growth inhibition was mediated via apoptosis, canine OS cell lines were treated with DMSO or LLL12 for 24 hours, and caspase 3/7 activity was measured. In all cell lines, caspase 3/7 activity was increased at 24 hours post treatment with LLL12 at concentrations of 0.4-0.8 μM (Figure2A). OS cells were also stained with Annexin V-FITC/PI and analyzed by flow cytometry to assess the percentage of early and late apoptotic cells in the population. After a 24 hour exposure to 0.5 μM LLL12 there was an increase in the proportion of early apoptotic (Annexin V positive, up to 22-fold increase) and late apoptotic (Annexin V/PI positive, up to 13-fold increase) cells. This correlated with data generated from the caspase assay (Figure2B). Normal canine osteoblasts were treated and analyzed by flow cytometry as described above, and were far less sensitive to the apoptosis inducing effects of LLL12 (Figure2C).

Bottom Line: The present study sought to characterize the biologic activity of a novel allosteric STAT3 inhibitor, LLL12, in canine OS cell lines.Lastly, LLL12 exhibited synergistic anti-proliferative activity with the chemotherapeutic doxorubicin in the OS lines.LLL12 exhibits biologic activity against canine OS cell lines through inhibition of STAT3 related cellular functions supporting its potential use as a novel therapy for OS.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210, USA.

ABSTRACT

Background: STAT3 [1] has been shown to be dysregulated in nearly every major cancer, including osteosarcoma (OS). Constitutive activation of STAT3, via aberrant phosphorylation, leads to proliferation, cell survival and resistance to apoptosis. The present study sought to characterize the biologic activity of a novel allosteric STAT3 inhibitor, LLL12, in canine OS cell lines.

Results: We evaluated the effects of LLL12 treatment on 4 canine OS cell lines and found that LLL12 inhibited proliferation, induced apoptosis, reduced STAT3 phosphorylation, and decreased the expression of several transcriptional targets of STAT3 in these cells. Lastly, LLL12 exhibited synergistic anti-proliferative activity with the chemotherapeutic doxorubicin in the OS lines.

Conclusion: LLL12 exhibits biologic activity against canine OS cell lines through inhibition of STAT3 related cellular functions supporting its potential use as a novel therapy for OS.

Show MeSH
Related in: MedlinePlus