The MTT assay is one of the often used cell viability/cytotoxicity assays. However, when the methanol extracts of plants are used to test their cytotoxic potential, interference may occur, resulting in false-positive viability results. Therefore, in this study, the reliability of the MTT assay was investigated in the case of plant use. The methanol extracts of three different plants (Hypericum adenotrichum, Salvia kronenburgii, and Pelargonium quercetorum) were tested in breast cancer cell lines (MCF-7 and MDA-MB-231) using the MTT assay and the results were compared to the ATP assay, which is a much more sensitive and reliable assay due to its interference-free feature. Additionally, decreased cell density was confirmed with phase-contrast microscopy and fluorescence staining (Hoechst 33342 dye). Although both of the viability/cytotoxicity assays are considered as metabolic assays, viabilities (in %) in the MTT assay were found to be strikingly higher when compared to the results with the ATP assay. Even in the case of total death, the MTT assay still produced artificial/false increases in viability. The morphology-based evaluation of viability/cytotoxicity by phase-contrast microscopy and Hoechst 33342 staining were greatly compatible with the ATP assay results. Overestimated (false) viabilities in the MTT assay suggests a serious interference between the MTT assay itself and the extracts used. Some ingredients of plants may have reducing activity (like the dehydrogenase activity of the cells) that converts the MTT compound into the colored formazan that is the principle of the assay. Therefore, the MTT assay may not be a suitable assay for some plant extracts, urging great caution when plants are used.

Long non-coding RNAs (lncRNAs), a group of non-protein coding RNAs with lengths of more than 200 nucleotides, exert their effects by binding to DNA, mRNA, microRNA, and proteins and regulate gene expression at the transcriptional, post-transcriptional, translational, and post-translational levels. Depending on cellular location, lncRNAs are involved in a wide range of cellular functions, including chromatin modification, transcriptional activation, transcriptional interference, scaffolding and regulation of translational machinery. This review highlights recent studies on lncRNAs in the regulation of protein translation by modulating the translational factors (i.e, eIF4E, eIF4G, eIF4A, 4E-BP1, eEF5A) and signaling pathways involved in this process as wells as their potential roles as tumor suppressors or tumor promoters.

Purpose - Recent studies indicated that dysregulation of non-coding RNAs (ncRNAs) such as microRNAs (miRNAs) is involved in pathogenesis of various human cancers. However, the molecular mechanisms underlying miR-34a are not fully understood in triple-negative breast cancer (TNBC). Experimental Design- We performed in vitro functional assays on TNBC cell lines to investigate the role of miR-34a in FOXM1/eEF2K signaling axis. TNBC tumor xenograft models were used for in vivo therapeutic delivery of miR-34a. Results- In this study, we investigated the role of p53-driven ncRNA miR-34a and found that miR-34a is associated with significantly longer patient survival in TNBC and inversely correlated with levels of proto-oncogenic eEF2K, which was associated with significantly shorter overall patient survival. We showed that miR-34a directly binds to the 3`-untranslated region (3`-UTR) of eEF2K and FOXM1 mRNAs and suppresses its expression, leading to inhibition of TNBC cell proliferation, motility, and invasion. Notably, restoring miR-34a expression recapitulated the effects of inhibition of eEF2K and FOXM1, the transcription factor for eEF2K and the direct target of p53, in TNBC cell lines, whereas overexpression of eEF2K and FOXM1 rescued the effects and signaling pathways mediated by miR-34a. Moreover, in vivo therapeutic delivery of miR-34a nanoparticles by systemic intravenous administration delayed tumor growth of two different orthotopic TNBC tumor xenograft models by inhibiting eEF2K and FOXM1, intratumoral proliferation and angiogenesis and inducing apoptosis. Conclusions- Overall, our findings provide new insights into the tumor suppressor role of miR-34a by dual-targeting of FOXM1/eEF2K signaling axis and suggest that miR-34a-based gene therapy may be a potential therapeutic strategy in TNBC.