We have previously reported inhibition of cell migration and proliferation induced by difluoromethylornithine (DMFO) and metformin (Met) combination in human melanoma and colon cancer cells. We further investigated the mechanism of cell death with the combination treatment in melanoma and colon cancer cells. The in vivo effect of the combination in melanoma was also determined.

Human colon cancer (HCT 116, HT 29) and melanoma (MEL1861, SK-23) were treated with Met, DFMO, or combination. Western blotting was performed to determine the expression of AMP kinase, mTOR, p70S6K and 4E-BP1. Apoptosis was measured by Annexin V assay. Autophagy was determined by fluorescence microscopy staining for LC3A/B and Western blot analysis of LC3A/B and Beclin-1 expression. For in vivo evaluation, SK-23 cells were injected SQ into BALB/c nu mice. After tumor nodule was established, 4 groups of 6 mice : group 1 - IP injections of vehicle and normal drinking water, group 2 - 2% (w/v) DFMO in drinking water; group 3 - IP Met (250 mg/kg/day), group 4 - DFMO plus Met. Mice body weight and tumor volume were measured every 3 days. Tumor weight was measured on Day 20 at necropsy. For comparison between groups, the student's t test was used and p< 0.05 was considered to be significant.

DFMO and Met induced apoptosis in colon cancer and melanoma cells in a dose- and time-dependent manner. Significant increase in apoptosis was noted in the combined treatment group compared with either one alone. Increased number of autolysosomes was observed in the combination group under fluorescence microscopy. Up-regulation of Beclin-1 and LC3A/B expression were also increased with combination treatment compared with either treatment alone. Increased expression of phosphor-AMPK, decreases expression of p70S6 and 4EBP1 were observed with the combination treatment. Both DFMO and Met alone have significant in vivo anti-proliferative effect on human melanoma cells compared with control. However, the anti-proliferative effect of the combination treatment was significantly better than either regimen alone (p< 0.001). Average SK-23 tumor weight was 100 mg for control group compared with 45 mg for group 2 (p<0.05), 35mg for group 3 (p< 0.05), and 25mg for the combination group (p< 0.001).

Our findings suggest that combination of DFMO and Met induced cancer cell death via apoptosis and autophagy though activation of AMPK pathway and suppression of the Akt/mTOR signaling pathway. DFMO and Met combination can have significant anti-tumor effect in vivo.

Citation Format: Yanping Zhang, Guangyong Peng, Eddy C. Hsueh. Induction of autophagy and apoptosis with polyamine synthesis inhibition and metformin in human melanoma and colon cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1418. doi:10.1158/1538-7445.AM2014-1418