The novel curcumin analog FLLL32 decreases STAT3 DNA binding activity and expression, and induces apoptosis in osteosarcoma cell lines

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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 <it>in vivo</it>.</p

Gene expression analysis of cell death induction by Taurolidine in different malignant cell lines

<p>Abstract</p> <p>Background</p> <p>The anti-infective agent Taurolidine (TRD) has been shown to have cell death inducing properties, but the mechanism of its action is largely unknown. The aim of this study was to identify potential common target genes modulated at the transcriptional level following TRD treatment in tumour cell lines originating from different cancer types.</p> <p>Methods</p> <p>Five different malignant cell lines (HT29, Chang Liver, HT1080, AsPC-1 and BxPC-3) were incubated with TRD (100 μM, 250 μM and 1000 μM). Proliferation after 8 h and cell viability after 24 h were analyzed by BrdU assay and FACS analysis, respectively. Gene expression analyses were carried out using the <it>Agilent </it>-microarray platform to indentify genes which displayed conjoint regulation following the addition of TRD in all cell lines. Candidate genes were subjected to <it>Ingenuity Pathways Analysis </it>and selected genes were validated by qRT-PCR and Western Blot.</p> <p>Results</p> <p>TRD 250 μM caused a significant inhibition of proliferation as well as apoptotic cell death in all cell lines. Among cell death associated genes with the strongest regulation in gene expression, we identified pro-apoptotic transcription factors (EGR1, ATF3) as well as genes involved in the ER stress response (PPP1R15A), in ubiquitination (TRAF6) and mitochondrial apoptotic pathways (PMAIP1).</p> <p>Conclusions</p> <p>This is the first conjoint analysis of potential target genes of TRD which was performed simultaneously in different malignant cell lines. The results indicate that TRD might be involved in different signal transduction pathways leading to apoptosis.</p