CDA-2 induces cell differentiation through suppressing Twist/SLUG signaling via miR-124 in glioma.

Cell differentiation agent-2 (CDA-2) is an extraction from healthy human urine consisting of primary organic acids and peptides, and it has been demonstrated to inhibit growth and induce differentiation in glioma and other cell lines. But the mechanism of CDA-2 remains unclear. In this study, we demonstrated that CDA-2 inhibited cell growth and induced differentiation of glioma cells, accompanied with decreased expression of SLUG, Twist and Vimentin in both SWO-38 and U251 cell lines. Overexpression of SLUG or Twist greatly eliminated the efficiency of CDA-2 in inducing differentiation. Further study showed that CDA-2 treatment resulted in great changed microRNAs (miRNAs) detected by quantitative PCR, in which miR-124 was one of the most changed miRNAs and its level was increased by fourfold. The result of miRNA target prediction showed that miR-124 could regulate hundreds of genes which were relative to cell differentiation, such as SLUG, Vimentin, actin cytoskeleton, focal adhesion, tight junction. Inhibition of miR-124 up-regulated SLUG, Twist and Vimentin proteins, and partly eliminated the function of CDA-2 on these mesenchymal markers. Our findings demonstrated for the first time that CDA-2 induced cell differentiation through suppressing Twist and SLUG via miR-124 in glioma cells.

Cell Differentiation Agent-2 (CDA-2) Inhibits the Growth and Migration of Saos-2 Cells via miR-124/MAPK1.

BACKGROUND: CDA-2 (cell differentiation agent 2), isolated from healthy human urine, exerts antitumor effects in multiple types of cancer cells. However, its role in osteosarcoma has not been studied. METHODS: The MTT assay was used to examine the cell proliferation rate. A colony formation assay was used to examine cell growth. The Transwell assay was used to examine cell migration ability. A real-time PCR assay was used to examine the expression levels of miR-124 and MAPK1. A Western blot assay was used to examine protein expression levels. MAPK1 was selected as a possible target of miR-124, and the targeting relationship was examined by a luciferase reporter assay. RESULTS: We revealed that CDA-2 decreased the growth, migration and invasion ability of the osteosarcoma cell line Saos-2. Further study revealed that CDA-2 elevated the expression level of miR-124. MAPK1 was identified as a downstream target of miR-124. Knockdown of miR-124 or overexpression of MAPK1 counteracted CDA-2's effects on cell growth and invasion. CONCLUSION: Our data revealed that the miR-124/MAPK1 axis mediated CDA-2's function in Saos-2 cells. CDA-2 can be used as a new treatment strategy for osteosarcoma.

Sulforaphane inhibits cancer stem-like cell properties and cisplatin resistance through miR-214-mediated downregulation of c-MYC in non-small cell lung cancer.

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 ß-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.

Therapeutic effect and apoptosis mechanism of lung-tonifying and expectorant decoction on lung cancer rats with Qi deficiency and blood stasis.

OBJECTIVE: To explore the effect and specific mechanism of lung-tonifying and expectorant decoction on lung cancer rats with Qi deficiency and blood stasis, and aim to provide a new idea on treating the disease with traditional Chinese medicine based on syndrome differentiation. METHODS: A total of 60 C57BL/6J male rats were included in the study. The model of Qi deficiency and blood stasis was established in 60 rats by using multiple-factor stimulation. About 10 rats were randomly taken to verify whether the model establishment was successful and the rest of 50 rats were divided into 5 groups with 10 rats each: blank control group, cisplatin group, low dose group, medium dose group and high dose group. The blank control group was treated with normal saline, and cisplatin group was treated with cisplatin while the other three groups were treated with lung-tonifying and expectorant decoction at different doses. The volume change in transplanted tumor, tumor inhibition rate, apoptosis rate, and expression of Bcl-2, Bax, cleaved caspase-3 and cleaved caspase-9 in 5 groups were compared. RESULTS: The rapidest growth rate of transplanted tumor volume was observed in blank control group and the slowest in cisplatin group. The growth rate was gradually decreased with the increasing dose of lung-tonifying and expectorant decoction, and the difference in growth of tumor volume among groups was statistically significant (P < 0.05). The cisplatin group showed the highest tumor inhibition rate, with dose-dependent increase (P < 0.05). The apoptosis rate in low dose group was higher than blank control group but lower than high dose group (P < 0.05). The apoptosis rate in medium dose group was significantly higher than blank control group (P < 0.05). The apoptosis rate in high dose group was significantly higher than control group (P < 0.05). The positive expression rates of Bcl-2 and Bax in all groups showed statistically significant difference (P < 0.05), while expression of cleaved caspase-3 and cleaved caspase-9 in 5 groups was significantly different, with dose-dependent increase (P < 0.05). CONCLUSIONS: The lung-tonifying and expectorant decoction inhibits the proliferation of tumor cells by inducing and activating the cell apoptosis in treatment of lung cancer with Qi deficiency and blood stasis, probably with good clinical therapeutic effect.

Esophageal cancer tumorspheres involve cancer stem-like populations with elevated aldehyde dehydrogenase enzymatic activity.

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.

[Expression of peroxisome proliferator-activated receptor gamma in glioma].

OBJECTIVE: To investigate the expression and significance of peroxisome proliferators-activated receptor gamma (PPAR gamma) in human glioma. METHODS: Immunohistochemical staining for PPAR gamma was performed using biopsy specimens of human glioma of various histological types. Expression of PPAR gamma and GFAP in glioma cell lines SWO-38, U251 and SHG-44 were analyzed using Western blotting and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Immunohistochemical study showed that PPAR gamma was expressed in glioma tissues with positive rate of 37.5%. Western blotting and RT-PCR showed that PPAR gamma was expressed in both glioma cell lines SWO-38 and U251, but not in SHG-44 cells. However, high expression of GFAP was detected in SHG-44 cells. CONCLUSION: PPAR gamma is associated with carcinogens of glioma. Actived PPAR gamma by agonist may be a novel approach to the treatment of glioma.

[Effects of exogenous human chorionic gonadotropin on nude mice bearing endometrial carcinoma].

OBJECTIVE: To observe the effects of exogenous human chorionic gonadotropin (hCG) on nude mice bearing transplanted endometrial carcinoma. METHODS: Human endometrial carcinoma xenograft was transplanted in nude mice, and the effects of hCG injection on the tumor growth was evaluated according to tumorigenesis and xenograft weights. The expression of Ki-67 in the tumor was determined by immunohistochemistry, and HE staining was performed for morphological observation and measurement of the necrosis area in the tumor. The effect of hCG on fibrosis in the tumor was evaluated with Masson staining. RESULTS: Compared to normal saline-treated tumor-bearing mice, the mice with hCG treatment showed increased tumor weight. HE staining for tumors in HCG-treatment group visualized tumor cell arrangement in glandular structure with smaller necrosis area, and Masson staining identified thick and compact collagen fibers as compared with the thin and loosely arranged fibers in saline-treated group. No significant difference was found in the Ki-67 expression in the tumors between the two groups. CONCLUSION: Exogenous hCG can promote the differentiation of the endometrial carcinoma cells in vivo.