The novel curcumin analog FLLL32 decreases STAT3 DNA binding activity and expression, and induces apoptosis in osteosarcoma cell lines
© Fossey et al; licensee BioMed Central Ltd. 2011
Received: 21 December 2010
Accepted: 28 March 2011
Published: 28 March 2011
Curcumin is a naturally occurring phenolic compound shown to have a wide variety of antitumor activities; however, it does not attain sufficient blood levels to do so when ingested. Using structure-based design, a novel compound, FLLL32, was generated from curcumin. FLLL32 possesses superior biochemical properties and more specifically targets STAT3, a transcription factor important in tumor cell survival, proliferation, metastasis, and chemotherapy resistance. In our previous work, we found that several canine and human osteosarcoma (OSA) cell lines, but not normal osteoblasts, exhibit constitutive phosphorylation of STAT3. Compared to curcumin, we hypothesized that FLLL32 would be more efficient at inhibiting STAT3 function in OSA cells and that this would result in enhanced downregulation of STAT3 transcriptional targets and subsequent death of OSA cells.
Human and canine OSA cells were treated with vehicle, curcumin, or FLLL32 and the effects on proliferation (CyQUANT®), apoptosis (SensoLyte® Homogeneous AMC Caspase- 3/7 Assay kit, western blotting), STAT3 DNA binding (EMSA), and vascular endothelial growth factor (VEGF), survivin, and matrix metalloproteinase-2 (MMP2) expression (RT-PCR, western blotting) were measured. STAT3 expression was measured by RT-PCR, qRT- PCR, and western blotting.
Our data showed that FLLL32 decreased STAT3 DNA binding by EMSA. FLLL32 promoted loss of cell proliferation at lower concentrations than curcumin leading to caspase-3- dependent apoptosis, as evidenced by PARP cleavage and increased caspase 3/7 activity; this could be inhibited by treatment with the pan-caspase inhibitor Z-VAD-FMK. Treatment of OSA cells with FLLL32 decreased expression of survivin, VEGF, and MMP2 at both mRNA and protein levels with concurrent decreases in phosphorylated and total STAT3; this loss of total STAT3 occurred, in part, via the ubiquitin-proteasome pathway.
These data demonstrate that the novel curcumin analog FLLL32 has biologic activity against OSA cell lines through inhibition of STAT3 function and expression. Future work with FLLL32 will define the therapeutic potential of this compound in vivo.
Osteosarcoma (OSA) is the most common form of malignant bone cancer in humans and dogs [1, 2]. Multidrug chemotherapy and aggressive surgical techniques have improved survival; however, the prognosis for human patients with metastatic disease remains extremely poor with survival rates of 10-20% . The disease in dogs occurs approximately 10 times more frequently than in people and treatment with surgery and adjuvant chemotherapy results in long-term survival rates of only 10-15% . Both clinical and molecular evidence suggest that human and canine OSA share several key features including early metastasis, chemotherapy resistance, altered expression of several proteins (e.g., ezrin, Met, PTEN), and p53 mutation, among others [4-10]. Given these similarities, canine OSA serves as a relevant model in which to evaluate the potential clinical utility of novel therapeutic targets for this disease.
The transcription factor STAT3 has been implicated as a key player in several features of malignant neoplasia including tumor cell survival, metastasis, and resistance to chemotherapy [11–13]. Our data and the work of others support the notion that STAT3 may be a relevant target for therapy in both human and canine OSA. In previous work, we demonstrated that human and canine OSA cell lines and tumors from canine patients exhibited constitutive activation of STAT3 . Loss of this expression after transfection with small interfering RNA targeting STAT3 or by reducing STAT3 DNA binding using LLL3 (a small molecule inhibitor) abrogated expression of STAT3 transcriptional targets and enhanced apoptosis . Increased levels of phosphorylated STAT3 have been identified in a subset of human OSA tissue samples and cell lines supportive of the role of this transcription factor in OSA . Suppression of this activated STAT3 with a dominant negative STAT3 led to decreased growth in these cell lines . Studies by Wang et al. showed that inhibition of STAT3 expression in OSA cells by siRNA decreased proliferation and enhanced apoptosis of these cells . Treatment of multidrug resistant OSA cell lines with a synthetic oleanane triterpenoid, C-28 methyl ester of 2-cyano-3,12-dioxoolen-1,9-dien-28-oic acid (CDDO-Me) downregulated STAT3 phosphorylation and nuclear translocation, subsequently inducing apoptosis . Indeed, overexpression of phosphorylated STAT3 was associated with a poor prognosis in patients with OSA  and high levels of STAT3 protein were associated with metastasis . Given the apparent role of STAT3 in the biology of OSA, clinically relevant therapies aimed at downregulating its activity would likely be therapeutically useful.
Curcumin (diferuloylmethane) is a naturally occurring compound found in the plant Curcuma longa that has numerous medicinal properties including anti-inflammatory and antitumor effects [18–20]. Curcumin has been investigated extensively as a potential therapeutic agent for the treatment of many different cancers, such as colorectal carcinoma [21, 22], head and neck squamous cell carcinoma , pancreatic cancer , and OSA [25, 26]. Curcumin is known to target multiple biochemical pathways, such as those mediated by Wnt/β-catenin , NF-κB , growth factor receptors like EGFR and HER2 , and JAK/STAT  enhancing its effect on cancer cells. Indeed, studies indicated that curcumin targets cellular transformation, invasion, angiogenesis, and metastasis [27, 29–32]. Recent work demonstrated that curcumin induced cell cycle arrest and apoptosis, and inhibited migration in human OSA cell lines [19, 33]. However, curcumin is not stable under physiologic conditions and is not readily absorbed after ingestion . Multiple modifications to the structure of curcumin have been investigated in an attempt to improve potency and biochemical properties [18, 35–37].
Recent work on improving both the target specificity and stability of curcumin by the College of Pharmacy at The Ohio State University produced the novel small molecule STAT3 inhibitor, FLLL32. As a diketone analog of curcumin, FLLL32 is more selective in its targeting than the parent compound due to the replacement of two hydrogen atoms on the central carbon of curcumin with a spiro-cyclohexyl ring . Improved interaction of FLLL32 with the Src homology-2 (SH2) domain of STAT3, a region instrumental in its dimerization and nuclear translocation, as well as greater stability, was predicted with these modifications as compared to curcumin [38, 39]. In subsequent work, FLLL32 was shown to promote apoptosis in multiple human cancer cell lines, inducing downregulation of STAT3 phosphorylation and DNA binding [38–40]. In human hepatocellular cancer cells, FLLL32 inhibited IL-6-induced STAT3 phosphorylation . FLLL32 was found to be more potent than some existing STAT3 inhibitors, including Stattic, S3I-201, and curcumin in colorectal, glioblastoma, multiple myeloma, rhabdomyosarcoma, and liver cancer cell lines [39, 42]. Together, these data demonstrate that FLLL32 exhibits improved efficacy at abrogating STAT3 functional activity and its effects in enhancing tumor cell survival in many cancer cell lines as compared to curcumin and other STAT3 inhibitors. Therefore, the purpose of this study was to explore the biologic activity of FLLL32 against canine and human OSA cell lines in vitro, delineate the mechanism of action of FLLL32, and compare the efficacy of FLLL32 to curcumin.
Cell Lines and Reagents
Canine OSA cell lines, OSA 8 and 16 were provided by Dr. Jaime Modiano (University of Minnesota, Minneapolis, MN) [43, 44]. The canine D17 OSA cell line and human OSA cell lines U2OS and SJSA were purchased from American Type Cell Culture Collection (ATCC, Manassas, VA). Cell line authentication of human OSA cell lines SJSA and U2OS was recently completed by The Ohio State University Comprehensive Cancer Center Molecular Cytogenetics Shared Resource by comparing the ATCC karyotype features with that of our cell lines. The canine lines and human line SJSA were maintained in RPMI-1640 supplemented with 10% fetal bovine serum, non-essential amino acids, sodium pyruvate, penicillin, streptomycin, L-glutamine, and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) at 35°C, supplemented with 5% CO2. The remaining human cell line U2OS was cultured in McCoy's medium with 10% FBS and the same supplements as listed for the canine lines. FLLL32 was synthesized and purified as described previously . Curcumin, the proteasome inhibitor MG132, and the pan-caspase inhibitor, Z-VAD-FMK, were purchased from EMD Chemicals (Gibbstown, NJ).
OSA cells (2 × 103) were seeded in 96-well plates overnight and incubated with DMSO, 10 μM curcumin, or increasing concentrations of FLLL32 (0.25, 0.75, 2.5, or 7.5 μM) for 72 hours. The volume of DMSO added to the vehicle treated wells was the same as that added to the drug treated wells. Each drug concentration was performed in four replicate wells. The media was removed, the wells were washed with PBS, and the plates were frozen at -80°C overnight before processing with the CyQUANT® Cell Proliferation Assay Kit (Molecular Probes, Eugene, OR) as described previously . Cell proliferation was calculated as a percentage of the DMSO- treated control wells with IC50 values derived after plotting proliferation values on a logarithmic curve.
Detection of Apoptosis/Caspase 3/7 Activity
OSA cells (1.1 × 104) were seeded in 96-well plates overnight and incubated with media, DMSO, 10 μM curcumin, or FLLL32 (2.5 or 7.5 μM) for 24 hours. Wells with media only were included as controls. Levels of caspase- 3/7 activity were determined using the SensoLyte® Homogeneous AMC Caspase- 3/7 Assay kit (Anaspec Inc, San Jose, CA) as described previously . To determine the effect of caspase activation on the loss of STAT3 protein, 1.1 × 104 OSA cells were pretreated for either 2 or 24 hours with 80 μM Z-VAD-FMK. Cells were then treated for 18 hours with media, DMSO, 80 μM Z-VAD-FMK, 10 μM FLLL32, or 10 μM FLLL32 and 80 μM Z-VAD-FMK. Caspase activation was measured as described previously .
To confirm that FLLL32 impaired STAT3 DNA binding, we used the Pierce LightShift Chemiluminescent EMSA kit (Thermo Fisher Scientific Inc, Rockford, IL) that employs a chemiluminescent detection system to detect protein:DNA interactions as described previously . Briefly, nuclear protein from human (0.8 μg/well) and canine (5 μg/well) OSA cell lines treated for four hours with media, DMSO, 10 μM curcumin, or 10 μM FLLL32 was collected using the NucBuster™ Protein Extraction kit (EMD Chemicals Inc, Gibbstown, NJ). Protein from cell lysates was collected from each group concurrently and processed for western blotting as described previously to confirm levels of STAT3 total protein and β-actin.
RT-PCR and qRT-PCR
Primers for human and canine reverse transcriptase polymerase chain reactions
5'- GGC CCA ATG GAA TCA GCT ACA G -3'
5'- GAA GGA ACT GCT TGA TTC TTC G -3'
5'- GGC CCA ATG GAA TCA GCT ACA G -3'
5'- GAA GAA ACT GCT TGA TTC TTC G -3'
5'- GAT GGC ACC CAT TTA CAC CTA C -3'
5'- GTC CTT GAA GAA GAA GAT CTC -3'
Primers for STAT3 quantitative reverse transcriptase polymerase chain reaction
5'-GGC CCA ATG GAA TCA GCT ACA G-3'
5'-GAA GGA ACT GCT TGA TTC TTC G-3'
5'-AAA TCC TTT AAC GAG GAT CCA TT-3'
5'-AAT ATA CGC TAT TGG AGC TGG A-3'
Protein lysates were prepared and quantified, separated by SDS-PAGE, and Western blotting was performed using previously described methods  on 2 × 106 OSA cells after treatment with either curcumin, FLLL32, or DMSO for 24 hours. The membranes were then incubated overnight with anti-p-STAT3 (Tyr705, Cell Signaling Technology, Danvers, MA), anti-p-ERK1/2 (Thr202/Tyr204, Cell Signaling Technology, Danvers, MA), anti-PARP (BD Biosciences, San Jose, CA), anti-VEGF (Calbiochem, Gibbstown, NJ), anti-ubiquitin (Cell Signaling Technology, Danvers, MA), or anti-survivin antibody (Novus Biologicals, Littleton, CO). The membranes were incubated with appropriate horseradish peroxidase linked secondary antibodies, washed, and exposed to substrate (SuperSignal West Dura Extended Duration Substrate, Pierce, Rockford, IL). Blots were stripped, washed, and reprobed for β-actin (Santa Cruz Biotechnology, Santa Cruz, CA), total STAT3 (Cell Signaling Technology, Danvers, MA) or total ERK1/2 (Cell Signaling Technology, Danvers, MA).
OSA cells (7 × 106) were serum starved for two hours then treated with DMSO, 10 μM curcumin, 10 μM FLLL32, or 10 μM MG132 for 4 hours. The volume of DMSO added to the vehicle treated wells was the same as that delivered to the drug treated wells. Cells were collected and lysate prepared as described previously . STAT3 antibody (Cell Signaling Technology, Danvers, MA) was added to lysates that had been precleared with Protein A- Agarose beads (Roche Diagnostics, Indianapolis, IN) and allowed to incubate overnight at 4°C. Protein A- Agarose beads were added to the protein lysate and antibody and incubated 1 hour at 4°C then washed three times in cold lysis buffer. This was spun down and supernatant separated by SDS-PAGE and transferred to a PVDF membrane (Thermo Scientific, Rockford, IL). Western blotting using an anti-ubiquitin antibody (Cell Signaling Technology, Danvers, MA) was performed after addition of the appropriate secondary antibody. The membrane was stripped and reprobed for total STAT3 (Cell Signaling Technology, Danvers, MA) or β-actin (Santa Cruz Biotechnology, Santa Cruz, CA). Images were scanned and analyzed using Image J (Rasband, W. S., Image J, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/, 1997-2009).
Proteasome Inhibition Assay
OSA cells (6 × 106) were serum starved for 2 hours then treated with DMSO, 10 μM curcumin, 10 μM FLLL32, or 10 μM MG132 for 4 hours. After treatment, cells were collected, washed with cold PBS, and lysed. Cell lysis buffer contained 50 mM HEPES (pH 7.5), 5 mM ethylenediaminetetraacetic acid (EDTA), 150 mM sodium chloride, and 1% Triton X-100. Proteasome enzyme was extracted and prepared for use in the 20S Proteasome Activity Assay Kit (Millipore, Billerica, MA). The 20S proteasome activity was measured in a 96-well plate. The assay is based on detection of the fluorophore 7-amino-4-methylcoumarin (AMC) after cleavage from labeled substrate LLVY-AMC. Samples were incubated for 1 hour at 37°C prior to detection of free AMC fluorescence using a 380/460 nm filter set in a SpectraMax microplate reader (Molecular Devices Corp., Sunnyvale, CA).
Statistical analysis of the CyQUANT® proliferation assays, caspase 3/7 activity, and real time PCR data was performed using the Student's t-test. P values of < 0.05 were considered statistically significant.
Treatment with curcumin or FLLL32 decreased proliferation of OSA cell lines
FLLL32 induced activation of caspase 3/7, PARP cleavage, and apoptosis of OSA cell lines
FLLL32 decreased STAT3 DNA binding in OSA cell lines
Downregulation of STAT3 via FLLL32 treatment decreased expression of VEGF, MMP2, and survivin
Treatment with FLLL32 decreased pSTAT3 and total STAT3 expression in canine and human OSA
STAT3 downregulation after FLLL32 treatment occurred through the ubiquitin/proteasome pathway
Inhibition of caspase activation did not affect loss of STAT3 following FLLL32 treatment
Curcumin has a long history of use as a medicinal compound and is known to have multiple anti-inflammatory and anti-cancer properties; however, blood levels that can be achieved after oral administration are low, which limits its potential clinical value [20, 27, 34]. Curcumin also affects a broad range of cellular targets including STAT3 [53, 54] in addition to a host of other signaling molecules such as Wnt/β-catenin , NF-κB , and HER2 , and the proteasome . Given the number of targets affected by curcumin and its poor bioavailability, efforts have been directed at improving its chemical properties by complexing it with lipids/phospholipids [55, 56] and developing more specific derivatives [35, 57–59]. Interestingly, many of these analogues have demonstrated greater stability and more potent activity against several tumor cell lines, including those derived from breast, prostate, pancreas, and colon cancers when compared to curcumin [35, 57–59]. Curcumin has been found to be well-tolerated in healthy individuals and OSA patients , most recently when given as a solid lipid particle formulation. However, peak plasma levels reached only 22.43 ng/mL (approximately 60 nM), well below concentrations known to have biologic effects against OSA cells in vitro.
During the development of novel curcumin analogs, our collaborators determined that one of these compounds, FLLL32, was particularly effective at suppressing the growth of pancreatic and breast cancer cells . To produce FLLL32, the two hydrogen atoms on the central carbon of curcumin were replaced with a spiro-cyclohexyl ring. It was proposed that this alteration would confer greater stability and specificity for STAT3 than curcumin . Recent work with FLLL32 showed that it induced apoptosis in human melanoma, multiple myeloma, glioblastoma, pancreatic, breast, and colorectal cancer cell lines and inhibited STAT3 phosphorylation and DNA binding [38–40]. The compound also exhibited higher potency at inhibiting proliferation and STAT3 DNA binding activity than curcumin and other JAK/STAT3 inhibitors in human rhabdomyosarcoma cells . Indeed, FLLL32 has been shown to be more potent than other STAT3 inhibitors in promoting growth inhibition of multiple cancer cell lines, and the drug is improved in its specificity as demonstrated by kinase profile assays that revealed almost no activity against tyrosine kinases such as Lck, Syk, Lyn, Yes, and Abl-1 . Given the superior specificity and efficacy of FLLL32 as compared to curcumin in a variety of cancer cell lines, the purpose of this study was to evaluate the biologic activity of this compound against OSA cell lines.
Previous studies have explored the activity of curcumin against OSA both in vitro and in human clinical trials [18, 20, 27]. OSA cell lines experienced cell cycle arrest, reduced proliferation, and underwent apoptosis following treatment with curcumin [19, 33, 60]. Prior work in our laboratory demonstrated that STAT3 is constitutively activated in OSA cell lines and that inhibition of STAT3 through STAT3 siRNAs or the small molecule STAT3 inhibitor LLL3 resulted in loss of proliferation and apoptosis . Data presented in this study showed that FLLL32 inhibited proliferation of OSA cell lines and promoted apoptosis via caspase 3/7 activation at lower concentrations than curcumin. This is consistent with recent work demonstrating apoptosis via caspase activation in human multiple myeloma, glioblastoma, liver cancer, colorectal, and melanoma cell lines after FLLL32 exposure [39, 40]. Cleavage of PARP, an indicator of caspase-3-mediated apoptosis, was also seen in many of these human cancer cell lines upon treatment with FLLL32 . Interestingly, loss of messenger RNA and protein expression of survivin, an inhibitor of apoptosis, as well as decreased STAT3 DNA binding activity was observed in human rhabdomyosarcoma cells treated with FLLL32 . The concurrent reduction in STAT3 transcriptional activity of targets such as survivin through decreased DNA binding and loss of STAT3 phosphorylation likely both played a role in the reduced survival of OSA tumor cells observed following exposure to FLLL32.
Recent work has shown that expression of high levels of STAT3 in human OSA tumor samples correlated to poor differentiation, metastasis, and lower rates of overall and relapse-free survival . Overexpression of phosphorylated STAT3 in OSA has also been linked to poor prognosis . STAT3 is known to enhance tumor cell invasion, metastasis, and angiogenesis through enhanced expression of VEGF and MMP2 . Human patients with OSA whose tumors had higher VEGF expression as shown by immunohistochemistry had a significantly worse prognosis and had lung metastasis [62, 63]. Previous work revealed that treatment of OSA cell lines with curcumin inhibited their migration . Mouse xenograft models of pancreatic and colorectal cancer treated with curcumin exhibited suppression of tumor angiogenesis and tumor growth inhibition . In more recent studies, FLLL32 inhibited vascularity and tumor growth in chicken embryo xenografts and reduced tumor volume in mouse xenografts of breast cancer . Our data demonstrate that in the OSA cell lines we tested, VEGF mRNA and protein and MMP2 mRNA were expressed and treatment with 10 μM FLLL32 downregulated the expression of these STAT3 transcriptional targets following 24 hours of drug exposure. Interestingly, VEGF mRNA expression appeared to increase over baseline in both the OSA8 and SJSA lines after curcumin exposure, although this did not correlate with the findings obtained by Western blotting in which VEGF protein was absent in OSA8 cells and unchanged in SJSA cells. The mechanism for this observed discrepancy is not clear, although there are several possible explanations. Curcumin may somehow interfere with translation of VEGF mRNA, directly enhance degradation of VEGF protein, or alternatively, given its diversity of cellular targets, affect proteins other than STAT3 that in turn alters VEGF expression. Further investigation of these potential mechanisms is needed. Given the putative role of both VEGF and MMP2 in the process of tumor growth and metastasis and recent data demonstrating the ability of FLLL32 to abrogate breast cancer xenograft growth in mice, future work assessing the effects of FLLL32 in mouse models of OSA is warranted.
Treatment of OSA cell lines with FLLL32 promoted loss of both pSTAT3 and total STAT3. This loss of STAT3 correlated with the presence of mono- and polyubiquitinylated STAT3, indicating that proteasome-mediated degradation was likely responsible for the observed decrease in protein. Interestingly, curcumin has been shown to inhibit activities of the proteasome in certain cancer cells ; however we detected no evidence for this activity after treating the OSA cell lines with curcumin or FLLL32 at the doses and time points examined. Although modulation of STAT3 protein levels is known to occur in part through caspase cleavage  a pan-caspase inhibitor did not affect the observed loss of STAT3 after FLLL32 treatment. Additionally, we did not see a significant decrease in STAT3 mRNA 24 hours after FLLL32 treatment, indicating that loss of STAT3 mRNA could not be primarily responsible for the protein downregulation that occurs after FLLL32 exposure. These data support the assertion that in addition to blocking STAT3 function, FLLL32 acts to promote downregulation of STAT3 protein, thereby enhancing the functional consequences of this small molecule inhibitor.
The novel small molecule STAT3 inhibitor FLLL32 downregulated proliferation and promoted apoptosis of OSA cells. FLLL32 inhibited STAT3 DNA binding and induced proteasome mediated degradation of STAT3 resulting in a subsequent loss of VEGF, MMP2, and survivin expression. These data support the notion that STAT3 is a relevant target for therapeutic intervention in OSA and that FLLL32 and similar analogs may have clinical utility for the treatment of OSA.
Acknowledgements and Funding
This work was supported by grants from the Morris Animal Foundation, the American Kennel Club, and NIH K01 (1K01RR025393-01). The authors would like to thank Tim Vojt of The Ohio State University College of Veterinary Medicine Biomedical Media Services for his assistance in figure preparation.
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