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CUGBP2, a translation inhibitor, induces colon cancer cells to undergo apoptosis. Mcl-1, an antiapoptotic Bcl-2 family protein, interferes with mitochondrial activation to inhibit apoptosis. Here, we have determined the effect of CUGBP2 on Mcl-1 expression. We developed a HCUG2 cell line by stably expressing CUGBP2 in the HCT-116 colon cancer cells. HCUG2 cells demonstrate decreased levels of proliferation and increased apoptosis, compared with HCT-116 cells. Flow cytometry analysis demonstrated higher levels of cells in the G2-M phase. Western blot analyses demonstrated that there was decreased Bcl-2 and Mcl-1 protein but increased expression of Bax, cyclin B1, and Cdc2. Immunocytochemistry also demonstrated increased levels of cyclin B1 and Cdc2 in the nucleus of HCUG2 cells. However, there was colocalization of phosphorylated histone H3 with transferase- mediated dUTP nick-end labeling (TUNEL). Furthermore, immunostaining for α-tubulin demonstrated that there was disorganization of microtubules. These data suggest that CUGBP2 expression in HCUG2 cells induces the cells to undergo apoptosis during the G2-M phase of the cell cycle. We next determined the mechanism of CUGBP2- mediated reduction in Mcl-1 expression. Mcl-1 protein, but not Mcl-1 mRNA, was lower in HCUG2 cells, suggesting translation inhibition. CUGBP2 binds to Mcl-1 3′-untranslated region (3′-UTR) both in vitro and in HCUG2 cells. Furthermore, CUGBP2 increased the stability of both endogenous Mcl-1 and luciferase mRNA containing the Mcl-1 3′-UTR. However, luciferase protein expression from the luciferase-Mcl-1 3′-UTR mRNA was suppressed. Taken together, these data demonstrate that CUGBP2 inhibits Mcl-1 expression by inhibiting Mcl-1 mRNA translation, resulting in driving the cells to apoptosis during the G2 phase of the cell cycle. [ABSTRACT FROM AUTHOR]

The cancer stem cell (CSC) hypothesis is an evolving oncogenesis concept. CSCs have a distinct ability to self-renew themselves and also give rise to a phenotypically diverse population of cells. Targeting CSCs represents a promising strategy for cancer treatment. Plant-derived compounds are potent in restricting the expansion of CSCs. DCLK1 has been already reported as a colon CSC specific marker. Nanoparticles can effectively inhibit multiple types of CSCs by targeting specific markers. We have synthesized DCLK1 functionalized folic acid conjugated hesperetin encapsulated chitosan nanoparticles (CFH-DCLK1), specifically to target CSCs. In this regard, we have performed proliferation assay, colony formation assay, cell migration assay, apoptosis assay, flow cytometry analysis, real-time RT- PCR and western blot analyses to determine the effect of CFH-DCLK1 and CFH nanoparticles in HCT116-colon cancer cells. In our study, we have determined the median inhibitory concentration (IC50) of CFH (47.8 µM) and CFH-DCLK1 (4.8 µM) nanoparticles in colon cancer cells. CFH-DCLK1 nanoparticles induced apoptosis and inhibited the migration and invasion of colon cancer cells. Real time PCR and western blot results have demonstrated that the treatment with CFH-DCLK1 nanoparticles significantly reduced the expression of CSC markers such as DCLK1, STAT1 and NOTCH1 compared to the CFH alone in HCT116 colon cancer cells. Finally, in the 3D spheroid model, CFH-DCLK1 nanoparticles significantly inhibited the colonosphere growth. Overall, our results highlight the effectiveness of CFH-DCLK1 nanoparticles in targeting the colon cancer cells and CSCs. This study would lead to the development of therapies targeting both cancer cells and CSCs simultaneously using nanoformulated drugs, which could bring changes in the current cancer treatment strategies. [Display omitted] • Cancer stem cells drive tumor initiation, metastasis, drug resistance and recurrence. • Nanoparticles can target cancer cells and cancer stem cells specifically. • Targeting doublecortin like kinase 1 can inhibit cancer stem cells self-renewal. • Folate receptor overexpression in cancer cells can be used for targeting therapy. [ABSTRACT FROM AUTHOR]