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.