Abstract
Chemotherapeutic regimens containing camptothecin (CPT), 5-fluorouracil, and oxaliplatin are used to treat advanced colorectal cancer. We previously reported that an indole derivative, 3-(2-bromoethyl)indole (BEI-9), inhibited the proliferation of colon cancer cells and suppressed NF-κB activation. Here, we show that a combination of BEI-9 with either CPT or tumor necrosis factor alpha (TNFα) enhances cell death. Using colorectal cancer cells, we examined the activation of NF-κB by drugs, the potential of BEI-9 for inhibiting drug-induced NF-κB activation, and the enhancement of cell death by combination treatments. Cells were treated with the chemotherapeutic drugs alone or in combination with BEI-9. NF-κB activation, cell cycle profiles, DNA-damage response, markers of cell death signaling and targets of NF-κB were evaluated to determine the effects of single and co-treatments. The combination of BEI-9 with CPT or TNFα inhibited NF-κB activation and reduced the expression of NF-κB-responsive genes, Bcl-xL and COX2. Compared to CPT or BEI-9 alone, sequential treatment of the cells with CPT and BEI-9 significantly enhanced caspase activation and cell death. Co-treatment with TNFα and BEI-9 also caused more cytotoxicity than TNFα or BEI-9 alone. Combined BEI-9 and TNFα enhanced cell death through caspase activation and cleavage of the switch-protein, RIP1 kinase. BEI-9 reduced the expression of COX2 both alone and in combination with CPT or TNF. We postulate that BEI-9 enhances the effects of these drugs on cancer cells by turning off or redirecting NF-κB signaling. Therefore, the combination of BEI-9 with drugs that activate NF-κB needs to be evaluated for clinical applications.
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Acknowledgements
This study was supported by grants from the National Institutes of Health through National Institute of General Medical Sciences NIGMS grant #SC3109314, National Institute of Minority Health and Health Disparities NIMHD grant #U54MD008149, and RCMI Core Facility Grant #G12MD007585 to Tuskegee University. The reported content is solely the responsibility of the authors and does not necessarily represent the official view of the NIH.
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10495_2017_1427_MOESM1_ESM.pptx
Supplementary Fig. 1: A. Photomicrographs of SW620 cells treated with BEI (1, 2, or 5 µM), TNFα, or with BEI plus TNF combinations as shown. Arrows point to cells showing membrane blebbing phenotypes suggestive of apoptosis. B. Phase contrast (upper row) and fluorescent images (lower row) of SW620 cells treated as shown. The spots in the lower row show pseudo-colored images of caspase-positive (FITC-VAD-FMK reactive) cells, which fluoresce green. In this experiment, CPT+BEI treatment was simultaneous, and did not result in enhanced cell death. Supplementary Fig. 2: A. Morphology of LoVo cells treated for 24 hr with vehicle, BEI, TNF, or a combination of the two drugs. B. A caspase 3/7 activation fluorescent assay that shows the caspase-activating effect of the TNFα – BEI combination. C. Morphology of Colo205 cells treated as shown. D. Cell cycle analysis of Colo205 cells treated as in panel C. The bars indicate the percentages of cells at each phase of the cell cycle shown on the X-axis. The percentage of sub-G1 cells increases with BEI and with the combination BEI and TNFα. Supplemental Fig. 3: Annexin -V/PI staining profiles of HCT116 cells treated as shown for 24 hr. The percentages of Annexin V-positive or Annexin -V/PI double-positive cells are increased by combination treatment compared to single agent treatments. (PPTX 18005 KB)
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Chowdhury, R., Gales, D., Valenzuela, P. et al. Bromoethylindole (BEI-9) redirects NF-κB signaling induced by camptothecin and TNFα to promote cell death in colon cancer cells. Apoptosis 22, 1553–1563 (2017). https://doi.org/10.1007/s10495-017-1427-6
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DOI: https://doi.org/10.1007/s10495-017-1427-6