K. E. Wong2,4, M. C. Mora2,4, S. S. Schneider4, K. P. Moriarty1, R. B. Arenas2,4, M. V. Tirabassi1 1Baystate Children’s Hospital,Department Of Pediatric Surgery,Springfield, MA, USA 2Baystate Medical Center,Department Of Surgery,Springfield, MA, USA 4Pioneer Valley Life Sciences Institute,Springfield, MA, USA
Introduction: Extracts derived from Rhodiola crenulata (RC), an adaptogenic plant from Tibet, is known to have anti-neoplastic properties in a variety of cancers. We have observed that RC exerts a striking cytotoxic and anti-proliferative effect on neuroblastoma cells in vitro and these effects are augmented in MYCN amplified neuroblastoma cells. The purpose of this study is to evaluate a possible mechanism by which RC causes cell death in neuroblastoma.
Methods: NB-1691 MYCN amplified neuroblastoma cells were transfected with a DNA vector containing a hypoxia inducible factor-1 (HIF-1) promoter on a firefly luciferase reporter gene. Following stable transfection, cells were treated with either 100ug/mL RC or ethanol vehicle control for 48 hours and a dual-luciferase reporting assay was performed to evaluate HIF-1 promoter activity. Quantitative RT-PCR was performed on RNA extracted from NB-1691 cells following 24 hour treatment with RC or ethanol to evaluate the expression of HIF-1α and its’ target genes including HK2, VEGF, MCT-1 and GLUT-1. Gene expression was normalized to GAPDH.
Results: HIF-1 transfected NB-1691 cells treated with RC exhibited an almost 50% increase in HIF-1 activity (p=0.009, figure 1A). HIF-1α mRNA level was observed to be significantly up-regulated upon treatment with RC treatment (p=0.019, figure 1B) in agreement with HIF-1 transfection results. While VEGF was also noted to be significantly up-regulated (p=0.023), VEGFa165, an inhibitory variant of VEGF, exhibited an upward trend as well. MCT-1, a cellular transporter exhibited a significant increase in expression (p<0.001, figure 1B), while other cellular transporters, including GLUT-1 exhibited no change and SLC7A-11 exhibited a downward trend. PKM2, involved in cellular glucose metabolism exhibited a signficant increase in expression (p=0.034, figure 1B) while HK2 exhibited a downward trend in activity. Of note, GAPDH amplification was noted to be slightly reduced upon treatment with RC.
Conclusion: We have observed an increase in both HIF-1 promoter activity and mRNA in response to treatment with RC suggestive of starvation of oxygen or an increase in glucose uptake. While several HIF1α targets were up-regulated with RC treatment, the notable exceptions were genes responsible for uptake of the nutrients needed to drive glycolysis. Taken together we hypothesize that the increased activity of HIF-1 reflects a starvation response which is unable to rescue the tumor cells due to lack of nutrients and leads to the rapid cell death we have observed previously. Future studies will evaluate the nutrient uptake, respiratory and glycolytic function in RC treated neuroblastoma cells.
