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These studies employed recently isolated OSCC cell lines and matched tumor-tissues to evaluate complementary chemopreventives with mechanisms that extend beyond pathway inhibition i.e.the synthetic vitamin A derivative fenretinide (4-HPR), the estrogen metabolite 2-methoxyestradiol (2-ME) and the humanized monoclonal IL6 receptor antibody tocilizumab (TOC). While previous data from our lab has confirmed the chemopreventive efficacy of 4-HPR [2], this agent combination was developed in response to experimental data derived from the OSCC tumor cells.
Our data show that 4-HPR+2-ME+TOC in combination suppress STAT3 activation and DNA binding of STAT3 and reduce release of sIL-6R. Although in vitro data are beneficial to identify molecular mechanisms, in vivo studies provide better assessment of therapeutic potential. Tumor xenograft studies which employ local delivery formulations are ongoing.
References:
1: Michael, A. Huber, Bundhit Tantiwongkosi. Oral and Oropharygneal Cancer. Med. Clin. N. Am. 98 (2014).
Figure 1: A. Tumor type characterization and immunohistochemistry for Pan Cytokeratin (epithelial origin) and Vimentin (epithelial-to-mesenchymal transition). B. Afatanib and Vargatef treatment after stimulation with VEGF, EGF, or combination. pSTAT3 levels persisted in treatment-refractory JSCC1/JSCC2 cell lines as evaluated by standard immunoblotting techiniques. C. Inter-cell line heterogeneity extends to OSCC-relevant autologous cytokine release, determined by ELISA analysis. Upon treatment and/or stimulation, JSCC1, JSCC2, & JSCC3 cell lines exhibited redundancies in STAT3 phosphorylation, providing mechanistic evidence of Afatanib and Vargatef treatment resistance.
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Figure 2: Cytosolic and nuclear proteins were harvested from sera deprived JSCC1 and JSCC2 cell lines following 24 h of treatment [(4-HPR, 5µM), (2-ME, 2.5 µM), LY5 (0.5 µM)] or vehicle (0.1% DMSO) control followed by standard immunoblotting techniques. A+B. 4-HPR treatment significantly reduced constitutive STAT3 phosphorylation and pSTAT3 nuclear translocation. C. JSCC3 cells (no constitutive STAT3) underwent 24h stimulation in base medium +10 ng/mL of IL-6 or 5 ng/mL of TGF-α, with or without 4-HPR, 2-ME or LY5 treatment. D. JSCC1 and JSCC2 cells were sera deprived for 24h, followed by additional 24 hour treatment in sera-free medium that contained: control (0.1% DMSO) or 5 µM 4-HPR. Medium was collected and analyzed by IL-6 ELISA (R&D Systems, MN) following manufacturer’s protocol. 4-HPR increased IL-6 release in both cell lines. As both NF-kB and STAT3 activate IL-6 expression, we introduced an FDA approved inhibitor of NF-kB: 2-ME. E. sIL-6R ELISAs of JSCC1, JSCC2, JSCC3, & 2095sc cells’ conditioned media showed significant inhibition of released sIL-6R by Tocilizumab (TOC) & 4-HPR+2-ME+Toc. Kruskal Wallis+Dunn’s Multiple Comparison test.
Figure 3: A+B. 2095sc, JSCC1, JSCC2 cells were sera deprived for 24h then treated as described above (control-0.1% DMSO). JSCC3 cells (24h base medium, 1h stimulation with10ng/mL of IL-6+5 ng/mL of TGF-α). Harvested proteins were evaluated by immunoblotting. (n=12 total with 3 repetitions for every individual cell line, Kruskal Wallis+Dunn’s Multiple Comparison test).