We herein report that sulforaphane (SFN), a potent anti-cancer and well-tolerated dietary compound, inhibits cancer stem-like cell (CSC) properties and enhances therapeutic efficacy of cisplatin in human non-small cell lung cancer (NSCLC). SFN exerted these functions through upregulation of miR-214, which in turn targets the coding region of c-MYC. This finding was further corroborated by our observations that plasmid or lentiviral vector-mediated expression of 3'UTR-less c-MYC cDNA and cisplatin- or doxorubicin-induced endogenous c-MYC accumulation was similarly suppressed by either SFN or miR-214. Further, we showed that the reported inhibitory effects of SFN on β-catenin are also mediated by miR-214. SFN/miR-214 signaling inhibited CSC properties and enhanced the cytotoxicity of chemotherapeutic drugs in vitro. Experiments with nude mice carrying xenograft tumors showed that SFN sensitized NSCLC cells to cisplatin's efficacy, which is accompanied by inhibition of cisplatin-induced c-MYC accumulation in tumor tissues. Our results provided strong evidence and mechanisms to support consideration of SFN or synthetic derivatives as a therapeutic agent in combination with cisplatin for the treatment of patients with NSCLC and, potentially, other types of c-MYC-addicted tumors.
Cancer stem cells (CSCs) form spheres in vitro in serum-free suspension culture. Sphere formation is particularly useful to enrich the potential CSC subpopulations as a functional approach. Few reports are currently available on tumorspheres in esophageal cancer (EC). The present study focused on evaluating the cancer stem-like properties and analyzing the difference between spheroid and adherent cells of the Eca109 human EC cell line. Immunofluorescence and immunoblotting analysis revealed that EC tumorspheres expressed the stem cell markers Nanog and Oct4 more highly, but showed a decreased expression of the differentiation marker CK5/6. The spheroids were chemoresistant to cisplatin compared to the adherent cells (32.5 vs. 135.8 µM in IC50). Side population cells increased in tumorspheres compared to adherent cells (0.7 vs. 5.6%). A marked upregulation of drug-resistant genes (ABCG2 and MDR1) was observed in sphere-forming cells. We compared the profiles of adherent and spheroid cells by microarrays and obtained one representative differentially expressed gene, aldehyde dehydrogenase (ALDH). We also verified that the cancer stem-like cells of EC contained a high ALDH enzymatic activity. ALDH-positive cells were enriched by 11- to 12-fold in spheroids, compared to adherent cells (2.5 vs. 28.6%). Immunofluorescence and immunoblotting analysis also revealed a higher expression of ALDH in EC tumorspheres. In conclusion, our study verified that sphere-forming culturing can be utilized to demonstrate the putative esophageal CSCs, and identified a potential esophageal CSC surface marker, ALDH.
Non–small cell lung cancer (NSCLC) is the main type of lung malignancy. Early diagnosis and treatments for NSCLC are far from satisfactory due to the limited knowledge of the molecular mechanisms regarding NSCLC progression. Long noncoding RNA (lncRNA) ZNFX1 antisense RNA1 (ZFAS1) has been implicated for its functional role in the progression of malignant tumors. This study aimed to determine the ZFAS1 expression from lung cancer clinical samples and to explore the molecular mechanisms underlying ZFAS1‐modulated NSCLC progression. Experimental assays revealed that clinical samples and cell lines of lung malignant tumors showed an upregulation of ZFSA1. ZFAS1 expression was markedly upregulated in the lung tissues from patients with advanced stage of this malignancy. The loss‐of‐function assays showed that knockdown of ZFAS1‐suppressed NSCLC cell proliferative, as well as invasive potentials, increased NSCLC cell apoptotic rates in vitro and also attenuated tumor growth of NSCLC cells in the nude mice. Further experimental evidence showed that ZFAS1 inversely affected miR‐150‐5p expression and positively affected high‐mobility group AT‐hook 2 (HMGA2) expression in NSCLC cell lines. MiR‐150‐5p inhibition or HMGA2 overexpression counteracted the effects of ZFAS1 knockdown on NSCLC cell proliferative, invasive potentials and apoptotic rates. In light of examining the clinical lung cancer samples, miR‐150‐5p expression was downregulated and the HMGA2 expression was highly expressed in the lung cancer tissues compared with normal ones; the ZFAS1 expression showed a negative correlation with miR‐150‐5p expression but a positive correlation with HMGA2 expression in lung cancer tissues. To summarize, we, for the first time, demonstrated the inhibitory effects of ZFAS1 knockdown on NSCLC cell progression, and the results from mechanistic studies indicated that ZFAS1‐mediated NSCLC progression cells via targeting miR‐150‐5p/HMGA2 signaling.
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