Abstract 1977: Higher doses of BAMLET may be required to induce cell death in HPV16-infected cancer cells

Abstract

Abstract Introduction: Recent experiments showed that BAMLET (Bovine Alpha lactalbumin Made LEthal to Tumor cells), biochemically equivalent to HAMLET, administered at doses shown to induce volume reduction and clearance of cutaneous wart in humans was insufficient to clear DMBA-induced skin papillomas in C57/Bl6 mice expressing HPV16 E7 transgene on a K14 promoter. Gustufsson et al., reported >75% subjects and 96% of warts reduced lesion volume when 0.7 mM HAMLET solution was topically administered. Thus, BAMLET's limited effect on papilloma reduction led to an in vitro dose-response experiment evaluating exponentially increasing doses of BAMLET on HPV16-infected CaSki cells, a well described cancer cell line. Five doses were selected versus normal saline controls and the cell viability was evaluated at 4, 24 and 72 hours using trypan blue exclusion assay. Methods: Caski cells were cultured in 6-well plates, 2 x 105 cells/well were initialized and allowed to grow to confluence. The initiating dose was based on published data as the exposure dose for our initial mouse studies. Exponential extrapolations of the baseline dose were evaluated: BAMLET administered at 0, 17.5µM (1.0-fold), 31.1µM (1.8-fold), 55.3µM (3.2-fold), 98.4µM (5.6-fold) and 175.0µM (10-fold) at time 0 and allowed to grow. At 4, 24, and 72 hours, cells were typsinized, pelleted, and resuspended in 1 ml PBS. A 10µL aliquot of Trypan-blue (0.4% v/v) was mixed with 10 µL of resuspended cells and incubated for 3 minutes. Duplicate specimens were evaluated under microscopy (10X) using a hemocytometer. Viable (white) and non-viable (blue) cells were counted and the average prevalence of viable cells was estimated for each dose. Results: For 0, 17.5µM, 31.1µM and 55.3µM doses, viability at 4, 24, and 72 hours were closely approximated: 74-92%, 81-91% and 67-88%, respectively. However, at 98.4µM and 175.0µM doses, a significant reduction in viability was observed in a time-dependent manner. Specifically, 95% vs. 91%, 68% vs. 0% and 6% vs. 2% viability were each observed at 4, 24 and 72 hours for these doses, respectively. Conclusions: These data suggest that the sensitivity of HPV16 infected cells to BAMLET may differ significantly from low-risk viruses that often cause cutaneous warts. While these data sheds light on the ineffectiveness of BAMLET in our ongoing animal experiment, they should be similarly considered in trials of BAMLET in humans where more pathogenic HPVs are the causal agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1977. doi:1538-7445.AM2012-1977