BACKGROUND
the signal transducers and activators of transcription  are a family of cell signaling proteins that play critical roles in inflammation, proliferation and differentiation
<cit> .
the stat family is comprised of  <dig> isoforms with a variety of unique but also overlapping functions.
stat proteins play critical roles in responding to extracellular signals from growth factors and cytokines, as well as regulating gene transcription in the nucleus.
stat <dig> in particular has been shown to be dysregulated in many cancers including osteosarcoma  and is frequently associated with malignant transformation and resistance to apoptosis in other tumor types
<cit> .
in the normal cell, activation of cell surface receptors induces phosphorylation of specific tyrosine residues on stat <dig>  either through activation of receptor tyrosine kinase’s  or janus kinases , depending on the nature of the signaling stimulus.
the phosphorylated stat <dig>  molecules then homodimerize via their sh- <dig> domains and subsequently translocate into the nucleus where binding to promoter elements of target genes acts to regulate their transcription
<cit> .
while stat <dig> activation is transient in normal cells due to a host of endogenous protein regulators , neoplastic cells often display constitutive stat <dig> activation, which contributes to increased angiogenesis, metastasis and chemotherapy resistance
<cit> .
although originally discovered as a protein involved in the pathway transducing a signal in response to interferon
<cit> , stat <dig> was not linked to cancer until it was shown to be essential for v-src mediated cellular transformation
<cit> .
the importance of stat <dig> in tumor progression and survival is supported by the fact that overexpression of pstat <dig> has been linked to poor prognosis in several cancers and as such, has been proposed as a relevant target for therapeutic intervention
<cit> .
our work and that of others has demonstrated that both human and canine os cell lines and tumors constitutively express pstat <dig> and as such, stat <dig> represents a potential therapeutic target for this disease
<cit> .
the identification of novel therapeutic targets for os is critical given that approximately 40% of children and over 90% of dogs will die from os
<cit> .
to this end, several small molecule stat <dig> inhibitors have been developed and some have shown promising activity both in vitro and in mouse xenograft models
<cit> .
however, most of these inhibitors have suffered from issues such as poor solubility that preclude their clinical development.
using structure based design, we have developed lll <dig> as a non-peptide small molecule inhibitor of stat <dig> that possesses good solubility and predictable oral bioavailability
<cit> .
lll <dig> binds to the phosphorylated tyrosine on stat <dig> monomers, blocking dimerization and subsequent translocation into the nucleus, abrogating its function as a transcription factor.
the purpose of this study was to characterize the biologic activity of this new stat <dig> inhibitor, lll <dig>  in canine os cells and evaluate its ability to inhibit stat <dig> and its downstream targets.
methods
cell lines and reagents
canine os cell lines osa  <dig> and osa  <dig> were provided by jaime modiano , the canine d <dig> os cell line was purchased from american type cell culture collection , and the canine abrams os cell line was provided by doug thamm .
osa  <dig>  osa  <dig> and d <dig> were maintained in rpmi- <dig> supplemented with 10% fbs, non-essential amino acids, sodium pyruvate, penicillin, streptomycin, l-glutamine, and hepes -1-piperazineethanesulfonic acid) at 35°c, supplemented with 5% co <dig>  the abrams cell line was cultured in dmem medium with 10% fbs and l-glutamine.
normal canine osteoblasts  were cultured in canine osteoblast medium .
lll <dig> was synthesized and purified as described previously
<cit> .
the following antibodies were used for western blotting experiments: pstat <dig> , total stat <dig> , survivin  and β-actin .
cell proliferation
os cells  were seeded in triplicate in 96-well plates overnight in 10% fbs supplemented medium and incubated with dmso or increasing concentrations of lll <dig>  doxorubicin, or both for  <dig> hours.
the medium was removed and the plates were frozen at −80°c overnight before processing with the cyquant® cell proliferation assay kit  according to the manufacturer’s instructions.
cell proliferation was calculated as a percentage of the dmso-treated control wells and ic <dig> values derived after plotting proliferation values on a logarithmic curve.
each experiment was repeated  <dig> times.
detection of apoptosis
os cells  were seeded in triplicate in 96-well plates overnight in 10% fbs supplemented medium and incubated with medium only, dmso or lll <dig> at increasing concentrations for  <dig> hours.
caspase 3/ <dig> activity was determined using the sensolyte® homogeneous amc caspase 3/ <dig> assay kit  according to manufacturer’s instructions.
to further assess apoptosis, 2× <dig> cells were plated in a t <dig> plate and allowed to grow overnight before being treated with dmso or lll <dig>  for  <dig> hours.
the cells were then harvested and incubated with fitc conjugated annexin v and propidium iodide dye  following the manufacturer’s protocol  before evaluation by flow cytometry .
cellquest software  was used to analyze the samples for early and late apoptosis.
western blotting
os cells or canine osteoblasts  in 1% fbs medium were treated with dsmo or  <dig>  μm lll <dig> for  <dig> hours.
normal canine osteoblasts were serum starved for  <dig> hours prior to identical treatment.
protein lysates were prepared and quantified, separated by sds-page, and western blotting was performed using previously described methods
<cit> .
the membranes were incubated overnight with anti-pstat <dig>  or anti-survivin  antibodies, then incubated with appropriate horseradish peroxidase linked secondary antibodies, washed, and exposed to substrate .
blots were stripped, washed, and reprobed for total stat <dig>  or β-actin , respectively.
rt-pcr and qrt-pcr
total rna was extracted from canine os cells in 10% fbs supplemented medium following  <dig> hours of treatment with dmso or  <dig>  μm lll <dig> using rneasy mini kits  according to the manufacturer’s instructions.
after rna extraction, samples were treated with dnase i using rq <dig> rnase-free dnase .
cdna was generated from  <dig> μg of total rna using superscript iii reverse transcriptase kit  according to the manufacturer’s instructions.
for each pcr reaction, 1/ <dig> of the resultant cdna was used in a total volume of  <dig> μl.
primers designed and utilized for canine survivin, cyclin d <dig>  bcl- <dig>  vegfa, mcl- <dig> and  <dig> s are listed in table
<dig>  as are the annealing temperatures for each reaction.
standard pcr was performed with all primer sets and amplicon length verified through agarose gel electrophoresis and visualization of products using the alpha imager system .
to quantitatively measure the effect of lll <dig> treatment on stat <dig> downstream targets, total rna was collected as described above.
real-time quantitative pcr was performed using applied biosystem’s stepone plus real-time pcr system .
canine survivin, cyclin d <dig>  bcl- <dig>  vegfa, mcl- <dig> and  <dig> s mrna were detected using fast sybr green pcr master mix  according to the manufacturer’s protocol.
all reactions were performed in triplicate and included non-template controls for each gene.
relative expression was calculated using the comparative threshold cycle method
<cit> .
experiments were repeated  <dig> times using samples in triplicate.
drug combination analysis
experiments were performed in 96-well plates.
os cells were seeded at a density of  <dig> × <dig> cells per well in rpmi medium containing 10% fcs.
stock solutions of lll <dig> and doxorubicin were generated and serial dilutions  for each compound were prepared, with the concentration range from .0625x to 4x the ic <dig> value of each drug.
to assess potential synergistic interactions, the treatment regimen involved simultaneous treatment of cells with lll <dig> and doxorubicin for  <dig> hours, in addition to controls consisting of cells treated with the individual compounds alone for  <dig> hours.
all treatments were performed in triplicate wells.
following drug treatment, the number of viable cells in each well was determined using cyquant® as described previously.
drug interactions were analyzed using compusyn  <dig> .
<dig> , which is based on the median effect model of chou and talalay
<cit> .
statistical analysis
all the values reported are mean ± sd.
delta cts from qrt-pcr were compared using two sample t-tests and holm’s method
<cit>  was used to control type-i error across tests of multiple genes.
the jonckhere-terpstra  test
<cit>  was used to test for a monotone trend in cell proliferation and caspase activity with dose of drug.
if the jt test was insignificant, we performed the mack-wolfe test
<cit>  for a non-monotone, or umbrella, dose–response.
all analyses were performed using sas version  <dig>  .
the mack-wolfe test was performed using the mwuspu and mwuspk sas macros developed by juneau
<cit> .
RESULTS
lll <dig> inhibits the proliferation of canine os cell lines
canine os cell lines were treated with increasing concentrations of lll <dig>  for  <dig> hours and effects on cell proliferation were assessed.
lll <dig> significantly reduced cell proliferation at concentrations as low as  <dig>  μm with the calculated ic <dig> concentrations in the nanomolar range  for all cell lines .
normal canine osteoblasts were comparatively resistant to the anti-proliferative effects of lll <dig>  with an approximately 7-fold higher calculated ic <dig> of  <dig>  μm .
lll <dig> promotes apoptosis of canine os lines
to determine if lll <dig> growth inhibition was mediated via apoptosis, canine os cell lines were treated with dmso or lll <dig> for  <dig> hours, and caspase 3/ <dig> activity was measured.
in all cell lines, caspase 3/ <dig> activity was increased at  <dig> hours post treatment with lll <dig> at concentrations of  <dig> - <dig>  μm .
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  <dig> hour exposure to  <dig>  μm lll <dig> there was an increase in the proportion of early apoptotic  and late apoptotic  cells.
this correlated with data generated from the caspase assay .
normal canine osteoblasts were treated and analyzed by flow cytometry as described above, and were far less sensitive to the apoptosis inducing effects of lll <dig> .
lll <dig> treatment decreases pstat <dig> and survivin expression in canine os lines
canine os cells and normal canine osteoblasts were treated with dmso,  <dig>  μm lll <dig> or  <dig>  μm lll <dig> for  <dig>   <dig> or  <dig> hours to determine the time and dose dependence of its effect on stat <dig> phosphorylation and survivin expression.
western blot analysis revealed pstat <dig> was completely downregulated following treatment with  <dig>  μm lll <dig> for only  <dig> hours, with a concomitant downregulation of survivin expression .
as expected, these results were time- and dose-dependent.
importantly, normal canine osteoblasts treated identically to their os counterparts had significantly lower pstat <dig> expression and demonstrated no change in survivin expression  following  <dig>  μm lll <dig> treatment at  <dig> hours.
lll <dig> treatment decreases stat3-mediated gene transcription
to assess the effects of lll <dig> on transcriptional targets of stat <dig> the expression of cyclin d <dig>  bcl- <dig>  mcl- <dig> and survivin was assessed using quantitative rt-pcr.
standard pcr was run with all primer sets and amplicon length verified prior to quantitative analysis.
expression of the stat <dig> regulated genes evaluated was significantly downregulated in all  <dig> os cell lines after  <dig> hours of treatment with  <dig>  μm lll <dig> when compared to dmso treated cells  supporting the notion that inhibition of pstat <dig> by lll <dig> affects its transcriptional activity.
lll <dig> enhances the antiproliferative effects of doxorubicin in canine os cells
to assess whether inhibition of pstat <dig> would enhance the biologic activity of chemotherapy in os cell lines, abrams and osa  <dig> cells were treated with lll <dig> , doxorubicin  or both drugs in combination over a range of doses reflecting multiple concentrations of their respective ic <dig> concentrations ranging from  <dig> × to 4×.
dose–response curves and combination index  graphs were generated and analyzed using compusyn software .
the ci values were < <dig> in 12/ <dig> dose combinations in both os cell lines tested demonstrating that lll <dig> exhibits synergistic anti-proliferative effects with doxorubicin in these lines.
the dose reduction index , which determines the magnitude of dose reduction allowed for each drug when given in synergistic combination, as compared with the concentration of a single agent that is needed to achieve the same effect was  <dig> - <dig>  for lll <dig> and doxorubicin in the os lines.
these data further support the notion that lll <dig> and doxorubicin interact in a synergistic manner in os cell lines.
discussion
despite some advances in our understanding of the underlying molecular biology of os, treatment for this disease has not changed significantly over the last  <dig> years in dogs or people
<cit> .
surgical resection and aggressive chemotherapy protocols are effective, but have failed to improve the 5-year overall survival rate past 60-70% in humans
<cit> .
similarly, in dogs, limb amputation followed by adjuvant chemotherapy with doxorubicin or carboplatin results in a 1-year survival rate of less than 50% and a 2-year survival rate of approximately 10-20%
<cit> .
treatment with doxorubicin and platinum based compounds, the current standards of care in the field, also come with the potential for significant toxicities including myelosuppression, gastrointestinal toxicity, cardiotoxicity and in humans including ototoxicity and secondary malignancies
<cit> .
this is particularly relevant for pediatric patients in whom late toxicities can substantially affect quality of life.
clearly, new drugs and new therapeutic targets are needed to both improve the outcome of patients suffering from os and to reduce the long-term toxicities associated with the current standard of treatment.
our laboratory previously characterized constitutive stat <dig> activation in primary canine os tumor samples and canine os cell lines, and showed that direct downregulation of stat <dig> protein expression in os lines using sirna induced loss of cell viability and apoptosis
<cit> .
we similarly demonstrated that two earlier stat <dig> small molecule inhibitors  also impacted os cell viability and induced cell death in all cell lines evaluated
<cit> .
in concordance with our work, stat <dig> dysregulation has been demonstrated in os in humans, where high levels of stat <dig> correlated with metastasis and lower rates of overall survival
<cit> .
together, these data define stat <dig> as an important target for therapeutic intervention in os, particularly given the fact that stat <dig> function is dispensable in many normal cells.
the mechanism of persistent stat <dig> phosphorylation remain to be elucidated in os.
constitutive stat <dig> activation does not appear to occur through direct mutation in stat <dig> as it does with other known oncogenes
<cit> .
however, there are a multitude of ligands  and kinases  that initiate stat <dig> activation and thus there are many potential upstream drivers that could contribute to the observed dysregulation
<cit> .
in our prior studies we identified osm as a potential driver of stat <dig> phosphorylation in canine os tumor cells and found that inhibition of stat <dig> signaling disrupted osm induced biologic activities
<cit> .
it is also possible that a loss of stat <dig> regulatory mechanisms may play a role in sustained stat <dig> pathway signaling.
stat <dig> may also play a critical role in chemoresistance in a number of cancer types, including os
<cit> .
the mechanisms through which this may occur are not well understood, although available data suggests that upregulation of the drug-resistance and anti-apoptotic stat3-regulated genes survivin, mcl- <dig> and mdr <dig> may play a part.
indeed, research has shown that p-gp, the product of the mdr <dig> gene, can have its expression mediated by stat3
<cit> , providing a possible mechanism for stat3-mediated chemotherapy resistance.
experimental evidence generated by our laboratory and by others has clearly demonstrated that disruption of stat <dig> signaling inhibits the survival and proliferation of os cell lines and decreases the growth of os in mouse models of disease
<cit> .
however, the challenge has been to develop a stat <dig> inhibitor that has good potential for future clinical application.
lll <dig> is an optimized analog of lll <dig>  a novel small molecule allosteric stat <dig> inhibitor that has been shown to inhibit proliferation and induce apoptosis in various cancer cell lines in vitro and in several mouse xenograft models, including os
<cit> .
lll <dig> works by binding to stat <dig> monomers at the phosphorylation site on y <dig> and thereby preventing stat <dig> dimerization and translocation into the nucleus.
similarly, anti-stat <dig> therapies, such as dominant negative stat <dig> molecules, rna interference and antisense oligonucleotides have been shown to be effective against a number of tumor types in vitro, but have yet to be tested in clinical trials, due in part to drug delivery issues including cell permeability, stability and solubility of the dna, rna and small molecules.
with respect to the small molecule inhibitors previously tested in canine os, flll <dig> suffered from lower activity than lll <dig> and solubility issues that precluded its further use in clinical trials and lll <dig>  while potent, did not bind directly to the py <dig> binding site of the stat <dig> monomer, unlike its optimized analog lll <dig>  which has a 10-fold increase in simulated binding energies to stat3
<cit> .
our collaborator  has demonstrated that lll <dig> has no off-target effects at concentrations used in this work , and does not inhibit any of the other stat family members
<cit> .
our current work shows that lll <dig> inhibits cell viability while inducing apoptosis in canine os cell lines expressing elevated levels of pstat <dig>  lll <dig> is quite potent, with ic50′s for the  <dig> canine os lines between  <dig>  μm and  <dig>  μm.
importantly, the ic <dig> generated for normal canine osteoblasts was  <dig>  μm, which demonstrates minimal toxicity in cells that lack constitutive activation of stat <dig>  with respect to the concentrations of drug used in these studies, preliminary pharmacokinetic data generated in mice indicate that exposures above  <dig> μm occur following intravenous and intraperitoenal administration of lll <dig> .
doxorubicin administration to dogs results in peak plasma levels of drug ranging from  <dig> - <dig>  μm, with drug concentrations above  <dig> - <dig>  μm lasting for 10– <dig> hours following a single iv bolus of drug given over  <dig> minutes
<cit> .
for the agents in combination, the ic <dig> of lll <dig> is reduced from  <dig> - <dig>  μm to  <dig> - <dig>  μm which are concentrations that are achievable in vivo; the ic <dig> of doxorubicin is reduced from  <dig> - <dig>  μm to  <dig> - <dig>  μm, which are also concentrations achievable in vivo.
therefore, we believe the drug concentrations used in this body of work are reflective of exposures obtainable in vivo.
stat <dig> transcriptional targets were all downregulated after only  <dig> hours of  <dig>  μm lll <dig> treatment, showing clear, rapid effects at biologically relevant concentrations.
protein expression of pstat <dig> and survivin were similarly downregulated under identical conditions.
while the timing of survivin downergulation lagged somewhat behind the loss of pstat <dig>  this was expected as stat <dig> is a transcriptional activator of survivin and existing transcript and protein would need to turn over first before a loss of survivin protein would be observed.
additionally, normal canine osteoblasts which have little to no pstat <dig> exhibit no loss of survivin protein at  <dig> hours of treatment, supporting the notion that stat <dig> and not other transcription factors are linked to the loss of survivin.
together, these results show that lll <dig> is more potent at inhibiting cell proliferation and decreasing pstat <dig> protein expression than both lll <dig> and flll <dig>
the synergy experiments in combination with doxorubicin show promise, with obvious clinical implications.
lll <dig> has strong activity against cells with constitutive pstat <dig> expression, but little effect on normal cells.
the significant dose reduction index seen when lll <dig> is used with doxorubicin could permit its use in the setting of lower doxorubicin doses, thereby potentially limiting some of the acute and long-term toxicities associated with dose intense doxorubicin.
this has particular relevance for dogs where cardiotoxicity limits the cumulative dose of doxorubicin to  <dig> mg/m <dig>  and the pediatric population where cognitive deficiencies, secondary neoplasia, and/or cardiac disease occur as long-term consequences following dose-intense treatment with doxorubicin.
CONCLUSIONS
lll <dig>  a novel allosteric stat <dig> inhibitor, inhibited proliferation and promoted apoptosis in canine os cell lines.
lll <dig> decreased pstat <dig> and survivin expression and downregulated the stat3-mediated gene transcription of survivin, cyclin d <dig>  bcl- <dig> and mcl- <dig> within  <dig> hours of drug exposure in the nanomolar range.
these data support the clinical development of lll <dig> for the treatment of os and other cancers in which stat <dig> is known to be constitutively activated.
ethical support
all the studies we performed were in vitro with cell lines and as such, no iacuc or approval was necessary.
the cell lines have been available for several years and were previously published.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
jc carried out molecular experiments on os cell lines and drafted the manuscript.
mb participated in rt-pcr design and performance, as well as optimizing the experimental design for all the molecular experiments.
jl provided lll <dig>  wk assisted in experimental design.
sk performed the analysis of syngergy experiments.
mp designed and performed all statistical tests.
cl conceived the study, assisted in experimental design, and helped draft the manuscript.
all authors read and approved the final manuscript.

 the signal transducers and activators of transcription are a family of cell signaling proteins that play critical roles in inflammation, proliferation and differentiation. the stat family is comprised of isoforms with a variety of unique but also overlapping functions. stat proteins play critical roles in responding to extracellular signals from growth factors and cytokines, as well as regulating gene transcription in the nucleus. stat in particular has been shown to be dysregulated in many cancers including osteosarcoma and is frequently associated with malignant transformation and resistance to apoptosis in other tumor types. in the normal cell, activation of cell surface receptors induces phosphorylation of specific tyrosine residues on stat either through activation of receptor tyrosine kinase’s or janus kinases, depending on the nature of the signaling stimulus. the phosphorylated stat molecules then homodimerize via their sh- domains and subsequently translocate into the nucleus where binding to promoter elements of target genes acts to regulate their transcription. while stat activation is transient in normal cells due to a host of endogenous protein regulators, neoplastic cells often display constitutive stat activation, which contributes to increased angiogenesis, metastasis and chemotherapy resistance. although originally discovered as a protein involved in the pathway transducing a signal in response to interferon, stat was not linked to cancer until it was shown to be essential for v-src mediated cellular transformation. the importance of stat in tumor progression and survival is supported by the fact that overexpression of pstat has been linked to poor prognosis in several cancers and as such, has been proposed as a relevant target for therapeutic intervention. our work and that of others has demonstrated that both human and canine os cell lines and tumors constitutively express pstat and as such, stat represents a potential therapeutic target for this disease. the identification of novel therapeutic targets for os is critical given that approximately 40% of children and over 90% of dogs will die from os. to this end, several small molecule stat inhibitors have been developed and some have shown promising activity both in vitro and in mouse xenograft models. however, most of these inhibitors have suffered from issues such as poor solubility that preclude their clinical development. using structure based design, we have developed lll as a non-peptide small molecule inhibitor of stat that possesses good solubility and predictable oral bioavailability. lll binds to the phosphorylated tyrosine on stat monomers, blocking dimerization and subsequent translocation into the nucleus, abrogating its function as a transcription factor. the purpose of this study was to characterize the biologic activity of this new stat inhibitor, lll in canine os cells and evaluate its ability to inhibit stat and its downstream targets. methods cell lines and reagents canine os cell lines osa and osa were provided by jaime modiano, the canine d os cell line was purchased from american type cell culture collection, and the canine abrams os cell line was provided by doug thamm. osa osa and d were maintained in rpmi- supplemented with 10% fbs, non-essential amino acids, sodium pyruvate, penicillin, streptomycin, l-glutamine, and hepes -1-piperazineethanesulfonic acid) at 35°c, supplemented with 5% co the abrams cell line was cultured in dmem medium with 10% fbs and l-glutamine. normal canine osteoblasts were cultured in canine osteoblast medium. lll was synthesized and purified as described previously. the following antibodies were used for western blotting experiments: pstat, total stat, survivin and β-actin. cell proliferation os cells were seeded in triplicate in 96-well plates overnight in 10% fbs supplemented medium and incubated with dmso or increasing concentrations of lll doxorubicin, or both for hours. the medium was removed and the plates were frozen at −80°c overnight before processing with the cyquant® cell proliferation assay kit according to the manufacturer’s instructions. cell proliferation was calculated as a percentage of the dmso-treated control wells and ic values derived after plotting proliferation values on a logarithmic curve. each experiment was repeated times. detection of apoptosis os cells were seeded in triplicate in 96-well plates overnight in 10% fbs supplemented medium and incubated with medium only, dmso or lll at increasing concentrations for hours. caspase 3/ activity was determined using the sensolyte® homogeneous amc caspase 3/ assay kit according to manufacturer’s instructions. to further assess apoptosis, 2× cells were plated in a t plate and allowed to grow overnight before being treated with dmso or lll for hours. the cells were then harvested and incubated with fitc conjugated annexin v and propidium iodide dye following the manufacturer’s protocol before evaluation by flow cytometry. cellquest software was used to analyze the samples for early and late apoptosis. western blotting os cells or canine osteoblasts in 1% fbs medium were treated with dsmo or μm lll for hours. normal canine osteoblasts were serum starved for hours prior to identical treatment. protein lysates were prepared and quantified, separated by sds-page, and western blotting was performed using previously described methods. the membranes were incubated overnight with anti-pstat or anti-survivin antibodies, then incubated with appropriate horseradish peroxidase linked secondary antibodies, washed, and exposed to substrate. blots were stripped, washed, and reprobed for total stat or β-actin, respectively. rt-pcr and qrt-pcr total rna was extracted from canine os cells in 10% fbs supplemented medium following hours of treatment with dmso or μm lll using rneasy mini kits according to the manufacturer’s instructions. after rna extraction, samples were treated with dnase i using rq rnase-free dnase. cdna was generated from μg of total rna using superscript iii reverse transcriptase kit according to the manufacturer’s instructions. for each pcr reaction, 1/ of the resultant cdna was used in a total volume of μl. primers designed and utilized for canine survivin, cyclin d bcl- vegfa, mcl- and s are listed in table as are the annealing temperatures for each reaction. standard pcr was performed with all primer sets and amplicon length verified through agarose gel electrophoresis and visualization of products using the alpha imager system. to quantitatively measure the effect of lll treatment on stat downstream targets, total rna was collected as described above. real-time quantitative pcr was performed using applied biosystem’s stepone plus real-time pcr system. canine survivin, cyclin d bcl- vegfa, mcl- and s mrna were detected using fast sybr green pcr master mix according to the manufacturer’s protocol. all reactions were performed in triplicate and included non-template controls for each gene. relative expression was calculated using the comparative threshold cycle method. experiments were repeated times using samples in triplicate. drug combination analysis experiments were performed in 96-well plates. os cells were seeded at a density of × cells per well in rpmi medium containing 10% fcs. stock solutions of lll and doxorubicin were generated and serial dilutions for each compound were prepared, with the concentration range from.0625x to 4x the ic value of each drug. to assess potential synergistic interactions, the treatment regimen involved simultaneous treatment of cells with lll and doxorubicin for hours, in addition to controls consisting of cells treated with the individual compounds alone for hours. all treatments were performed in triplicate wells. following drug treatment, the number of viable cells in each well was determined using cyquant® as described previously. drug interactions were analyzed using compusyn., which is based on the median effect model of chou and talalay. statistical analysis all the values reported are mean ± sd. delta cts from qrt-pcr were compared using two sample t-tests and holm’s method was used to control type-i error across tests of multiple genes. the jonckhere-terpstra test was used to test for a monotone trend in cell proliferation and caspase activity with dose of drug. if the jt test was insignificant, we performed the mack-wolfe test for a non-monotone, or umbrella, dose–response. all analyses were performed using sas version. the mack-wolfe test was performed using the mwuspu and mwuspk sas macros developed by juneau.  lll inhibits the proliferation of canine os cell lines canine os cell lines were treated with increasing concentrations of lll for hours and effects on cell proliferation were assessed. lll significantly reduced cell proliferation at concentrations as low as μm with the calculated ic concentrations in the nanomolar range for all cell lines. normal canine osteoblasts were comparatively resistant to the anti-proliferative effects of lll with an approximately 7-fold higher calculated ic of μm. lll promotes apoptosis of canine os lines to determine if lll growth inhibition was mediated via apoptosis, canine os cell lines were treated with dmso or lll for hours, and caspase 3/ activity was measured. in all cell lines, caspase 3/ activity was increased at hours post treatment with lll at concentrations of - μm. 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 hour exposure to μm lll there was an increase in the proportion of early apoptotic and late apoptotic cells. this correlated with data generated from the caspase assay. normal canine osteoblasts were treated and analyzed by flow cytometry as described above, and were far less sensitive to the apoptosis inducing effects of lll. lll treatment decreases pstat and survivin expression in canine os lines canine os cells and normal canine osteoblasts were treated with dmso, μm lll or μm lll for or hours to determine the time and dose dependence of its effect on stat phosphorylation and survivin expression. western blot analysis revealed pstat was completely downregulated following treatment with μm lll for only hours, with a concomitant downregulation of survivin expression. as expected, these results were time- and dose-dependent. importantly, normal canine osteoblasts treated identically to their os counterparts had significantly lower pstat expression and demonstrated no change in survivin expression following μm lll treatment at hours. lll treatment decreases stat3-mediated gene transcription to assess the effects of lll on transcriptional targets of stat the expression of cyclin d bcl- mcl- and survivin was assessed using quantitative rt-pcr. standard pcr was run with all primer sets and amplicon length verified prior to quantitative analysis. expression of the stat regulated genes evaluated was significantly downregulated in all os cell lines after hours of treatment with μm lll when compared to dmso treated cells supporting the notion that inhibition of pstat by lll affects its transcriptional activity. lll enhances the antiproliferative effects of doxorubicin in canine os cells to assess whether inhibition of pstat would enhance the biologic activity of chemotherapy in os cell lines, abrams and osa cells were treated with lll, doxorubicin or both drugs in combination over a range of doses reflecting multiple concentrations of their respective ic concentrations ranging from × to 4×. dose–response curves and combination index graphs were generated and analyzed using compusyn software. the ci values were < in 12/ dose combinations in both os cell lines tested demonstrating that lll exhibits synergistic anti-proliferative effects with doxorubicin in these lines. the dose reduction index, which determines the magnitude of dose reduction allowed for each drug when given in synergistic combination, as compared with the concentration of a single agent that is needed to achieve the same effect was - for lll and doxorubicin in the os lines. these data further support the notion that lll and doxorubicin interact in a synergistic manner in os cell lines. discussion despite some advances in our understanding of the underlying molecular biology of os, treatment for this disease has not changed significantly over the last years in dogs or people. surgical resection and aggressive chemotherapy protocols are effective, but have failed to improve the 5-year overall survival rate past 60-70% in humans. similarly, in dogs, limb amputation followed by adjuvant chemotherapy with doxorubicin or carboplatin results in a 1-year survival rate of less than 50% and a 2-year survival rate of approximately 10-20%. treatment with doxorubicin and platinum based compounds, the current standards of care in the field, also come with the potential for significant toxicities including myelosuppression, gastrointestinal toxicity, cardiotoxicity and in humans including ototoxicity and secondary malignancies. this is particularly relevant for pediatric patients in whom late toxicities can substantially affect quality of life. clearly, new drugs and new therapeutic targets are needed to both improve the outcome of patients suffering from os and to reduce the long-term toxicities associated with the current standard of treatment. our laboratory previously characterized constitutive stat activation in primary canine os tumor samples and canine os cell lines, and showed that direct downregulation of stat protein expression in os lines using sirna induced loss of cell viability and apoptosis. we similarly demonstrated that two earlier stat small molecule inhibitors also impacted os cell viability and induced cell death in all cell lines evaluated. in concordance with our work, stat dysregulation has been demonstrated in os in humans, where high levels of stat correlated with metastasis and lower rates of overall survival. together, these data define stat as an important target for therapeutic intervention in os, particularly given the fact that stat function is dispensable in many normal cells. the mechanism of persistent stat phosphorylation remain to be elucidated in os. constitutive stat activation does not appear to occur through direct mutation in stat as it does with other known oncogenes. however, there are a multitude of ligands and kinases that initiate stat activation and thus there are many potential upstream drivers that could contribute to the observed dysregulation. in our prior studies we identified osm as a potential driver of stat phosphorylation in canine os tumor cells and found that inhibition of stat signaling disrupted osm induced biologic activities. it is also possible that a loss of stat regulatory mechanisms may play a role in sustained stat pathway signaling. stat may also play a critical role in chemoresistance in a number of cancer types, including os. the mechanisms through which this may occur are not well understood, although available data suggests that upregulation of the drug-resistance and anti-apoptotic stat3-regulated genes survivin, mcl- and mdr may play a part. indeed, research has shown that p-gp, the product of the mdr gene, can have its expression mediated by stat3, providing a possible mechanism for stat3-mediated chemotherapy resistance. experimental evidence generated by our laboratory and by others has clearly demonstrated that disruption of stat signaling inhibits the survival and proliferation of os cell lines and decreases the growth of os in mouse models of disease. however, the challenge has been to develop a stat inhibitor that has good potential for future clinical application. lll is an optimized analog of lll a novel small molecule allosteric stat inhibitor that has been shown to inhibit proliferation and induce apoptosis in various cancer cell lines in vitro and in several mouse xenograft models, including os. lll works by binding to stat monomers at the phosphorylation site on y and thereby preventing stat dimerization and translocation into the nucleus. similarly, anti-stat therapies, such as dominant negative stat molecules, rna interference and antisense oligonucleotides have been shown to be effective against a number of tumor types in vitro, but have yet to be tested in clinical trials, due in part to drug delivery issues including cell permeability, stability and solubility of the dna, rna and small molecules. with respect to the small molecule inhibitors previously tested in canine os, flll suffered from lower activity than lll and solubility issues that precluded its further use in clinical trials and lll while potent, did not bind directly to the py binding site of the stat monomer, unlike its optimized analog lll which has a 10-fold increase in simulated binding energies to stat3. our collaborator has demonstrated that lll has no off-target effects at concentrations used in this work, and does not inhibit any of the other stat family members. our current work shows that lll inhibits cell viability while inducing apoptosis in canine os cell lines expressing elevated levels of pstat lll is quite potent, with ic50′s for the canine os lines between μm and μm. importantly, the ic generated for normal canine osteoblasts was μm, which demonstrates minimal toxicity in cells that lack constitutive activation of stat with respect to the concentrations of drug used in these studies, preliminary pharmacokinetic data generated in mice indicate that exposures above μm occur following intravenous and intraperitoenal administration of lll. doxorubicin administration to dogs results in peak plasma levels of drug ranging from - μm, with drug concentrations above - μm lasting for 10– hours following a single iv bolus of drug given over minutes. for the agents in combination, the ic of lll is reduced from - μm to - μm which are concentrations that are achievable in vivo; the ic of doxorubicin is reduced from - μm to - μm, which are also concentrations achievable in vivo. therefore, we believe the drug concentrations used in this body of work are reflective of exposures obtainable in vivo. stat transcriptional targets were all downregulated after only hours of μm lll treatment, showing clear, rapid effects at biologically relevant concentrations. protein expression of pstat and survivin were similarly downregulated under identical conditions. while the timing of survivin downergulation lagged somewhat behind the loss of pstat this was expected as stat is a transcriptional activator of survivin and existing transcript and protein would need to turn over first before a loss of survivin protein would be observed. additionally, normal canine osteoblasts which have little to no pstat exhibit no loss of survivin protein at hours of treatment, supporting the notion that stat and not other transcription factors are linked to the loss of survivin. together, these results show that lll is more potent at inhibiting cell proliferation and decreasing pstat protein expression than both lll and flll the synergy experiments in combination with doxorubicin show promise, with obvious clinical implications. lll has strong activity against cells with constitutive pstat expression, but little effect on normal cells. the significant dose reduction index seen when lll is used with doxorubicin could permit its use in the setting of lower doxorubicin doses, thereby potentially limiting some of the acute and long-term toxicities associated with dose intense doxorubicin. this has particular relevance for dogs where cardiotoxicity limits the cumulative dose of doxorubicin to mg/m and the pediatric population where cognitive deficiencies, secondary neoplasia, and/or cardiac disease occur as long-term consequences following dose-intense treatment with doxorubicin.  lll a novel allosteric stat inhibitor, inhibited proliferation and promoted apoptosis in canine os cell lines. lll decreased pstat and survivin expression and downregulated the stat3-mediated gene transcription of survivin, cyclin d bcl- and mcl- within hours of drug exposure in the nanomolar range. these data support the clinical development of lll for the treatment of os and other cancers in which stat is known to be constitutively activated. ethical support all the studies we performed were in vitro with cell lines and as such, no iacuc or approval was necessary. the cell lines have been available for several years and were previously published. competing interests the authors declare that they have no competing interests. authors’ contributions jc carried out molecular experiments on os cell lines and drafted the manuscript. mb participated in rt-pcr design and performance, as well as optimizing the experimental design for all the molecular experiments. jl provided lll wk assisted in experimental design. sk performed the analysis of syngergy experiments. mp designed and performed all statistical tests. cl conceived the study, assisted in experimental design, and helped draft the manuscript. all authors read and approved the final manuscript. 
we evaluated the effects of lll treatment on canine os cell lines and found that lll inhibited proliferation, induced apoptosis, reduced stat phosphorylation, and decreased the expression of several transcriptional targets of stat in these cells. the present study sought to characterize the biologic activity of a novel allosteric stat inhibitor, lll in canine os cell lines. lll exhibits biologic activity against canine os cell lines through inhibition of stat related cellular functions supporting its potential use as a novel therapy for os. lastly, lll exhibited synergistic anti-proliferative activity with the chemotherapeutic doxorubicin in the os lines.
 stat has been shown to be dysregulated in nearly every major cancer, including osteosarcoma. constitutive activation of stat 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 stat inhibitor, lll in canine os cell lines.  we evaluated the effects of lll treatment on canine os cell lines and found that lll inhibited proliferation, induced apoptosis, reduced stat phosphorylation, and decreased the expression of several transcriptional targets of stat in these cells. lastly, lll exhibited synergistic anti-proliferative activity with the chemotherapeutic doxorubicin in the os lines.  lll exhibits biologic activity against canine os cell lines through inhibition of stat related cellular functions supporting its potential use as a novel therapy for os. stat3osteosarcomacanine 