Rates of esophageal adenocarcinoma and the only known precursor lesion, Barrett's esophagus (BE), have markedly increased over the last three decades throughout the Westernized world. In the US 18,170 new incident cases and 15,450 deaths are estimated in 2014. These statistics reflect the poor 5-year survival rates (20%) and support the need for improved preventive efforts targeting this deadly malignancy. Our lab has been investigating the use of cranberry proanthocyanidins (C-PAC), as a natural product with cancer cell death inducing effects. Cranberry extracts have shown cancer inhibitory properties in a large number of in vitro studies utilizing diverse cancer cell lines; however, there is surprisingly little mechanistic information regarding the ability of cranberry constituents to inhibit cancers, especially in vivo.
In this study, we sought to characterize C-PACs effect on reactive oxygen species (ROS), autophagy and AKT/mTor/MAPK signaling utilizing one BE (CP-C) and three EAC (JHAD1, OE33, OE19) cell lines as well as OE19 tumor xenografts in athymic NU/NU mice. CellROX® Green reagent was used to monitor ROS levels following C-PAC treatment of cell lines. Lysates isolated from C-PAC treated cell lines and xenografts were analyzed for modulation of AKT/mTor/MAPK and autophagy signaling proteins, as well as various caspases.
We examined ROS levels of BE and EAC cell lines following exposure to C-PAC at 25 and 100 μg/ml alone and in combination with the pro-oxidant tert-Butyl hydroperoxide (TBHP) or the anti-oxidant N-acetyl cysteine (NAC). High dose C-PAC treatment significantly increased ROS levels at 3 hours (3.0 to 8.1-fold increase) and 6 hours (3.4 to 10.8-fold increase) in all EAC cells evaluated. In contrast, C-PAC significantly reduced ROS levels in CP-C BE cells (4-8-fold at 6 H); yet, C-PAC reduced viability of all cells 24-48 H post treatment. TBHP strongly induced ROS only in JHAD1 cells (21.2-fold) resulting in rapid death. NAC had the strongest mitigating effects in OE19 cells; the only cells which readily formed xenografts in vivo.
Orally delivered C-PAC [250 μg/day] significantly reduced OE19 tumor xenografts by 67%. Both in vitro and in vivo C-PAC inhibition was linked to inactivation AKT/mTor signaling networks (decreased AKT and p70S6K phosphorylation) and autophagy induction as evidenced by increased conversion of LC3B-I to LC3B-II. Furthermore, autophagy induction was Beclin-independent and cell death was largely caspase-independent. Together, these data suggest that C-PAC induces cancer cell death via ROS induction in EAC cells and ROS inhibition in premalignant cells, through autophagy induction and AKT/mTOR inactivation and modulation of MAPKs. Additional experiments using pathway-specific inhibitors and genetic approaches are ongoing to further assess C-PACs inhibitory effects against esophageal cancer.
Citation Format: Katherine M. Weh, Harini S. Aiyer, Amy B. Howell, Laura A. Kresty. Cranberry proanthocyanidins induce autophagic cell death via ROS generation and inhibition of AKT/mTOR/MAPK signaling pathways in esophageal adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1898. doi:10.1158/1538-7445.AM2015-1898