C. Padmanabhan1, C. W. Lindsley5,6, A. G. Waterson5,6, R. D. Beauchamp1,2,3,4 1Vanderbilt University Medical Center,Surgery,Nashville, TN, USA 2Vanderbilt University Medical Center,Cell And Developmental Bioogy,Nashville, TN, USA 3Vanderbilt University Medical Center,Cancer Biology,Nashville, TN, USA 4Vanderbilt University Medical Center,Ingram Cancer Center,Nashville, TN, USA 5Vanderbilt University Medical Center,Chemistry,Nashville, TN, USA 6Vanderbilt University Medical Center,Pharmacology,Nashville, TN, USA
Introduction:
Colorectal cancer (CRC) is the third most common cancer diagnosis and the third most common cause of cancer related death in the United States. The inability of chemotherapy to eradicate quiescent cancer stem cells is a leading hypothesis for cancer recurrence. New therapies that do not rely on cellular proliferation for cytotoxic effect must be developed. The tumor necrosis factor related apoptosis inducing ligand (TRAIL), which selectively induces apoptosis in cancer cells irrespective of cell proliferation, was thought to be such a therapy but the anti-cancer effect identified in preclinical models was not realized in clinical trials due to resistance. Our lab has identified a novel small molecule (ML327) that sensitizes colorectal cancer cell lines to TRAIL. We hypothesize this effect is due to loss of the anti-apoptotic protein c-FLIP, a potent inhibitor of the extrinsic apoptosis pathway initiated by TRAIL.
Methods:
CRC cell lines were pre-treated with ML327 or vehicle control at a 10-μM concentration for 24 hours. TRAIL was then added at 100 ng/mL for 4 hours. Cells were lysed and Western blot was performed with antibodies against cFLIP and cleaved caspase 3. Cells were also fixed with 70% ethanol and stained with propidum iodide for FACS analysis. Cells were then treated with ML327 at a 10-μM concentration for up to 4 hours and lysed at hourly increments. Western blot was performed with antibodies against cFLIP.
Results:
24 hours of ML327, but not vehicle, pre-treatment increased TRAIL-induced cleaved caspase 3 protein levels (Fig. A). cFLIP protein level was abundant in vehicle treated cells but was nearly undetectable after 24 hour ML327 treatment (Fig. B). These findings were associated with increased cell death after TRAIL exposure as confirmed by a statistically significant increase (p = 0.002) in the Sub G0 population on FACS analysis (Fig. C). ML327-induced reductions in cFLIP protein occurred quickly, with reduced levels evident by 1 hour and complete loss by 4 hours (Fig. D).
Conclusion:
ML327 is a novel small molecule that sensitizes CRC cells to the TRAIL ligand as evident by increased apoptosis and subsequent cell death. This sensitization is associated with loss of cFLIP. Ongoing analysis will determine whether this is a cause and effect association and further elucidate the exact mechanism of cFLIP loss. It will be of interest to determine the in vivo efficacy of ML327 induced TRAIL sensitization. With these experiments, we hope to develop a novel therapeutic that will induce cell death in cancer cells irrespective of cell proliferation.
