IMCIDU 2019 FULLTEXT & ABSTRACT BOOK, vol.1, no.1, pp.561-566, 2019 (National Conference Book)
INTRODUCTION: Neuroblastoma is one of the most common solid tumours in children less than 1 year of age. Intensive multimodal treatments provide a small improvement in survival rates, but patients with refractory and relapsed disease have a significant percentage. Therefore, novel molecular targets and therapeutic strategies are needed to prolong patient survival. Rottlerin, a naturally occurring polyphenolic compound derived from Mallotus philipinensis, 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 cell division and survival in several cancers. Recently, we learned that Eukaryotic elongation factor-2 kinase(EF2K) 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 effects of these drugs on in vitro cell behaviours, regulator pathways and relationship between EF2K and these drugs remains are largely unknown. Thus, we investigated the effects of Rottlerin and Genistein separately and in combination on neuroblastoma cells. MATERIAL-METHODS: In this study, the human neuroblastoma cancer cell lines (SH-SY5Y,Kelly) were used. Rottlerin and Genistein were also employed for therapy. The viability and/or proliferation of cells were detected by MTS and Colony formation assays after drug treatments. In-vitro invasion and wound-healing assays were used to assess cell motility and the ability of migration/invasion. Apoptosis and cell cycle were assessed by Annexin-V and propidium iodide assays in flow cytometry, respectively. Western blot analysis was performed to evaluate protein changes. One-way ANOVA with post-hoc Tukey test was performed for statistical analysis. Significance level was accepted as p<0.05. This study was supported by EgeUniversity-BAP(16-TIP-006).
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 5μM and 30μM, 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). Moreover, 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 this downregulation may be responsible for in vitro effects. 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 neuroblastoma. 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.