K. E. Wong1, M. C. Mora1, N. Sultana2, K. Moriarty3, R. Arenas1, N. Yadava2, S. Schneider2, M. Tirabassi3 1Baystate Medical Center,Department Of Surgery,Springfield, MA, USA 2Pioneer Valley Life Sciences Institute,Springfield, MA, USA 3Baystate Childrens Hospital,Springfield, MA, USA
Introduction: Rhodiola crenulata (RC) extracts are derived from the roots of adaptogenic Tibetan plants. The extracts are known to have anti-neoplastic properties in a variety of cancers. RC exerts a striking cytotoxic effect on MYCN amplified NB-1691 neuroblastoma cells. However, the mechanism of RC cytotoxicity is not yet known. Because rapidly proliferating cancer cells, such as neuroblastoma, are more dependent on glycolysis, we evaluated if RC cytotoxicity is mediated by alterations in cellular metabolism.
Methods:
NB-1691 cells were treated with 200ug/mL RC or ethanol vehicle control (VC) +/- 1mM pyruvate (PYR) in cell culture. Viability was evaluated using trypan blue exclusion with a cell viability counter following 24 hours of treatment. Pyruvate kinase (PK) activity, lactate dehydrogenase activity (LDH), NAD quantification and pyruvate dehydrogenase activity (PDH) assays were performed per protocol. NB-1691 cells were treated with either RC 200μg/mL or VC for two hours, metabolic assays were performed and differences in cells treated with VC and RC were evaluated.
Results:
Upon supplementation of media with PYR, the viability of NB-1691 was rescued from the cytotoxic effects of RC. RC treatment resulted in a 69% reduction in viability without PYR (p<0.001), while the addition of PYR with RC resulted in only 16.6% reduction in viability (p=0.0361) compared to VC (figure 1A). PK activity was significantly increased upon treatment with RC compared to VC (p=0.0081, figure 1B). LDH activity was significantly reduced upon treatment with RC compared to VC treated cells (p=0.0196, figure 1C). Total NAD (NAD++NADH) levels were reduced upon treatment with RC compared to VC (p<0.001). The total amount of NAD was observed to increase upon addition of PYR resulting in no significant difference between treatment groups in the presence of PYR (figure 1D). A significant increase in NADH generated by PDH was also observed upon treatment with RC compared to VC (p=0.03, figure 1E).
Conclusion:
RC extracts and the fractions contained within it hold promise as a potential chemotherapeutic option for neuroblastoma treatment given the cytotoxic effects it exerts upon NB-1691 cells in vitro. RC mediates neuroblastoma cytotoxicity through reprogramming optimal cellular metabolic functioning involving pyruvate metabolism. Reduced NAD levels observed upon RC treatment can halt glycolysis and limit intracellular pyruvate production by glycolysis made evident by the rescue of viability upon pyruvate supplementation. The changes in PK, PDH and LDH activities may be compensatory responses to pyruvate limitation following RC treatment. Together, these findings identify a potential mechanism of RC cytotoxicity upon treatment of NB-1691 cells in vitro.
