Abstract 1330: A novel rosmarinic acid/blue light combination treatment inhibits cell proliferation and migration in head and neck squamous cell carcinoma
Authors: Waer Christi N. , Tumur Zohra , Hui Dee Dee , Le Bonnie , Guerra Carlos , Lewis Jill , Henson Bradley 
Published: July 2016

Abstract
Our labs have observed decreased proliferation of several cultured head and neck squamous cell carcinoma (HNSCC) cell lines when treated with rosmarinic acid or blue light. The purpose of this study was to test the hypothesis that the combination therapy would be more effective than either treatment alone, and to further characterize the cellular mechanisms responsible for these effects.
UM-SCC-6 oral squamous carcinoma cells were cultured in 24- or 96-well plates, and cells were exposed to blue light delivered from a Quartz-tungsten-halogen dental curing lamp (500 mW/cm2) for 90 or 120 sec, followed 1 h later by the addition of 80 μg/ml rosmarinic acid. Cell number was assessed at 24h using a WST-1 cell proliferation assay and cell migration was measured with an Oris Universal Migration Assay Kit. Reactive oxygen species (ROS) was quantified with each single treatment and combination using the CM-H2DCFH-DA assay. Western blot analysis was used to monitor changes in levels of cell signaling proteins known to be affected by each single treatment including phosphoAKT (pAKT) and AKT, NF-E2-related factor 2 (NRF2), heme oxygenase-1 (HO-1), and GAPDH (as a loading control).
As hypothesized, the combination of rosmarinic acid and blue light was more effective in reducing cell proliferation and migration than either single treatment alone. Both treatments also produced an early increase in ROS. Treatment with blue light significantly increased pAKT while rosmarinic acid decreased AKT activation. However, both treatments increased levels of Nrf2 and its downstream target, HO-1.
In order for the transcription factor NRF2 to translocate to the nucleus it needs to be stabilized and activated. AKT is an upstream kinase that is a known activator of NRF2 in response to an increase in ROS. Nuclear NRF2 binds to the antioxidant response element (ARE) within the promoters for phase II detoxifying/antioxidant genes, including that of HO-1. This signaling cascade has been shown to lead to anti-proliferative functions. Our results suggest that blue light induces this pathway by transiently increasing ROS that then significantly activates AKT. Subsequent NRF2 activation leads to increased levels of HO-1. Rosmarinic acid appears to use a different mechanism to upregulate NRF2 due to its downregulation of AKT. Importantly, both treatments activate NRF2, resulting in induction of antioxidant protein expression and ultimately decreasing ROS needed to promote tumor cell growth. This combination therapy may prove to be a useful, non-invasive treatment for HNSCC.
Citation Format: Christi N. Waer, Zohra Tumur, Dee Dee Hui, Bonnie Le, Carlos Guerra, Jill Lewis, Bradley Henson. A novel rosmarinic acid/blue light combination treatment inhibits cell proliferation and migration in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1330.