The Effects of Rottlerin and Genistein on Cell Proliferation, Invasion and Cell Death/Cycle in Neuroblastoma Cancer Cell Lines
Project no. 24 · Status: Kapanmış · 2016
Summary
Objectives and Aim: Neuroblastoma (NB) is the most common extracranial solid cancer in childhood and the most common cancer in infancy in the world. Tyrosine kinase signaling networks play a major role in governing cell differentiation, including in neuroblastoma. Rottlerin, a naturally occurring polyphenolic compound derived from Mallotus philipinensis (Euphorbiaceae), appears to have great potential in cancer therapy because of its effects on several cellular processes such as proliferation and apoptosis. Genistein is a phytoestrogen and acts as tyrosine kinase inhibitor. Genistein have been found to inhibit the uncontrolled cell growth of cancer, most likely by inhibiting cell division and cell survival in several cancers such as prostate, cervix, brain and breast. Recently, we learned that Eukaryotic elongation factor-2 kinase (EF2K), an atypical kinase, is dramatically up-regulated in many cancer cells and promotes cell survival and proliferation, its inhibition significantly reduces cell growth and colony formation. Rottlerin and Genistein also showed inhibitory effects on this kinase in another solid tumours like Panc Ca. However, the role that Rottlerin and Genistein in this cancer and effects of these drugs on pathways regulating cell proliferation, colony formation, survival, invasion/metastasis and resistance and relationship between EF2K and these drugs remains are largely unknown. After all of these knowledge, we investigated the effects of Rottlerin and Genistein separately and in combination on these cell behaviours in Neuroblastoma cells. Methods: In this study, the human neuroblastoma cancer cell lines (SH-SY5Y, Kelly) were used. Rottlerin and Genistein were also employed for therapy. As in vitro experiments, cell proliferation, colony formation, invasion, wound-healing tests, western blot, cell cycle and apoptosis analysis by flow cytometry were performed. Results: Our results showed that Rottlerin and Genistein treatments caused a significant reduction in cell proliferation, colony formation, invasion/wound-healing capacity in Neuroblastoma cells at concentrations of 5 µM and 30 µM, respectively (p<0,0001). The combination of these doses also empowered the level of inhibition in these analysis (p<0,0001). Additionally, these drugs also increased the level of apoptosis in Neuroblastoma cell lines (p<0,0001). Additionally, our agents caused G1 cell cycle arrest in these cells (p<0,0001). We showed that Rottlerin and Genistein treatments markedly inhibit EF2K overexpression and due to this downregulation inhibit Neuroblastoma cell proliferation, colony formation and invasion/migration in vitro. Our western blot data suggested that EF2K may enhance tumorigenesis and metastasis through the upregulation of pro-tumorigenic, metastatic proteins and pathways in Neuroblastoma cells and our agents probably showed their anti-proliferative, anti-metastatic and apoptotic effects through EF2K downregulation. Conclusion: In conclusion, it was revealed with all these results that Rottlerin and Genistein have important effects on cell proliferation, colony formation, invasion, and cell cycle/apoptosis in Neuroblastoma cells. Moreover, it has been shown that Rottlerin and Genistein may show these effects through downregulation of EF2K as a novel potential therapeutic target in this cancer. Overall, treatment with Rottlerin and Genistein in combination may be a viable approach and beneficial to neuroblastoma patients with use in new nano-drug delivery systems.