Bufalin Inhibits the Differentiation and Proliferation of Cancer Stem Cells Derived from Primary Osteosarcoma Cells through Mir-148a

Background/Aims: Prior studies have shown that bufalin inhibits cellular proliferation and induces apoptosis in various human cancers. MicroRNA-203 (miR-203) has been shown to function as an important regulator of tumor progression at various stages. In this study, we investigated the effect of miR-203 expression and bufalin treatment on glioma cell proliferation and stem cell-like phenotypes. Methods: We used cell viability assay, colony formation assay, cell apoptosis assay and neurosphere formation assay to dectect the treatment effect of bufalin on U251 and U87 cells. Cells were transfected with the miR-203 mimic without bufalin treatment or cells were transfected with anti-miR-203 under bufalin treatment, the above expreiments were repeated. RT-PCR was employed to quantify miR-203 expression. Western blot was performed to detect the stem cell-like (CSC) markers, OCT4 and SOX2. Luciferase activity assay was used to determine whether the SPARC is the target of miR-203. Results: Bufalin treatment inhibited cell proliferation, colony formation, and CSC phenotypes and increased cell apoptosis and expression of miR-203. Furthermore, overexpression of miR-203 led to similar outcomes as bufalin treatment with respect to the cell viability, colony formation, cell apoptosis and the phenotypes of glioma cells. While anti-miR-203 attenuated the inhibitory effects of bufalin as promoting cell proliferation, colony formation and CSC phenotyes and inhibiting cell apoptosis. In addition, we identified SPARC as a novel target gene of miR-203. Conclusions: These findings suggest that miR-203 plays an important role in bufalin’s ability to inhibit the growth of glioma cells and the development of stem cell-like phenotypes.

Section: Discussionmentioning

confidence: 99%

Bufalin Inhibits Cellular Proliferation and Cancer Stem Cell-Like Phenotypes via Upregulation of MiR-203 in Glioma

Liu

Weng

et al. 2017

“…In recent years, increasing studies have demonstrated that bufalin could inhibit cell proliferation and induce apoptosis in a variety of tumor cells [15-22]. However, the detailed mechanisms of bufalin-induced apoptosis in NSCLC cells have not been fully elucidated.…”

Section: Introductionmentioning

confidence: 99%

Degradation of Mcl-1 through GSK-3β Activation Regulates Apoptosis Induced by Bufalin in Non-Small Cell Lung Cancer H1975 Cells

Kang

Zhang

Qing-qin

et al. 2017

Background/Aims: Mcl-1, an anti-apoptotic Bcl-2 family member, is often overexpressed in non-small cell lung cancer (NSCLC). Bufalin has been reported to induce apoptosis in various tumor cells. However, there is no report showing that bufalin could downregulate Mcl-1 expression in NSCLC. Methods: Cell proliferation was analyzed by cell counting kit-8 (CCK-8) assay in H1975 cells. Cell apoptosis was detected by flow cytometry. Mcl-1 mRNA was detected by RT-PCR. The expression of apoptosis-associated proteins in H1975 cells was detected by western blotting. The levels of Mcl-1 ubiquitination and NOXA were analyzed by Immunoprecipitation assay. Results: Cell growth was inhibited by bufalin in a time and dose-dependent manner. Bufalin induced apoptosis in NSCLC cells by activating caspase cascades and downregulating Mcl-1 expression. However, overexpression of Mcl-1 diminished bufalin-induced apoptosis. Furthermore, bufalin did not reduce Mcl-1 mRNA expression in H1975 cells, but strongly promoted Mcl-1 protein degradation. Proteasome inhibitor MG132 markedly prevented the degradation of Mcl-1 and blocked bufalin-induced Mcl-1 reduction. Bufalin did not significantly affect NOXA protein levels, but downregulated the expression of p-GSK-3β. GSK-3 inhibitor and GSK-3β siRNA resulted in increased levels of Mcl-1 and reversed the bufalin-induced Mcl-1 degradation. Conclusion: Bufalin induced cell apoptosis in H1975 cells may be through downregulation of Mcl-1. Proteasomal degradation of Mcl-1 via GSK-3β activation was involved in bufalin-induced apoptosis.

“…MiRNAs regulate the protein translation via its base-pairing with the 3′-untranslated region (3′-UTR) of the mRNA of the target genes to affect cell proliferation, apoptosis and differentiation [26,27,28]. MiRNAs have been well defined as a regular for tumorigenesis [29,30,31,32,33,34,35]. Among all miRNAs, miR-21 was recently reported as an aberrantly expressed miRNA in NSCLC [36,37,38,39,40], but the alteration of miR-21 during the Carboplatin treatment is not well defined.…”

Section: Introductionmentioning

confidence: 99%

MicroRNA-21 Regulates Non-Small Cell Lung Cancer Cell Invasion and Chemo-Sensitivity through SMAD7

Lin

et al. 2016

Background/Aims: SMAD7 is a key inhibitor of transforming growth factor β (TGFβ) receptor signaling, which regulates the alteration of cancer cell invasiveness through epithelial-mesenchymal cell conversion. Carboplatin is a commonly used drug in the chemotherapy for non-small cell lung cancer (NSCLC). Nevertheless, the molecular mechanisms underlying its suppressive effects on the NSCLC cell invasion are not completely understood. In the current study, we addressed this question by analyzing the effects of Carboplatin on microRNA-regulated SMAD7. Methods: We used Carboplatin to treat NSCLC cell lines. We performed bioinformatics analyses on the binding of microRNA-21 (miR-21) to the 3'-UTR of SMAD7 mRNA, and verified the biological effects of this binding using promoter luciferase reporter assay. The effects of Carboplatin or miR-21-modification on NSCLC cell invasion were evaluated in either a transwell cell invasion assay, or a scratch wound healing assay. Results: We found that Carboplatin inhibited the NSCLC cell invasion, in either a transwell cell invasion assay, or a scratch wound healing assay. Moreover, Carboplatin increased the levels of SMAD7 protein, but not mRNA, in NSCLC cells, suggesting presence of post-transcriptional control of SMAD7 by Carboplatin. Furthermore, expression of miR-21 was found to be inhibited by Carboplatin, and bioinformatics analyses showed that miR-21 targeted the 3'-UTR of SMAD7 mRNA to inhibit its translation, which was confirmed by luciferase reporter assay. Conclusion: Carboplatin may upregulate SMAD7 through suppression of miR-21 to inhibit TGFβ receptor signaling mediated NSCLC cell invasion.