Evodiamine inactivates NF-κB and potentiates the antitumor effects of gemcitabine on tongue cancer both in vitro and in vivo Qi Guo,1 Yanmei Liu,1 Jiayuan Zhao,1 Jing Wang,1 Yue Li,1 Yunqing Pang,1 Jian Chen,2 Jing Wang1 1Department of Periodontology, School of Stomatology, Lanzhou University, Lanzhou, Gansu, China; 2Department of Pediatric Surgery, The First Hospital of Lanzhou University, Lanzhou, Gansu, China Objective: The aim of this study was to investigate whether evodiamine (EVO) could potentiate the antitumor activity of gemcitabine (GEM) in tongue cancer cells and determine its potential underlying mechanisms. Materials and methods: Human Tca8113 and CAL-27 tongue squamous carcinoma cell lines were treated with EVO and GEM in different sequences and doses, after which cell proliferation was measured. Drug interactions were analyzed using the Chou–Talalay method with CompuSyn software. Clonality, apoptosis, and migration were measured using the plate clone formation assay, annexin V/propidium iodide (PI) staining, Hoechst 33342 staining, and the wound-healing test. The activity of the nuclear factor kappa light-chain enhancer of activated B cell (NF-κB) p65 subunit and its downstream proteins was quantified by Western blotting. The effects of the drug combination in vivo were assessed using a CAL-27 heterotopic xenograft model. Results: EVO and GEM had synergistic effects on CAL-27 and Tca8113 cell lines in time- and concentration-dependent manners. Combination of drugs inhibited cell proliferation and migration and reduced the expression of NF-κB p65, B cell lymphoma 2 (Bcl-2), and B cell lymphoma extra large (Bcl-xl) compared with the control and either drug alone. In vivo, combination treatment of the xenograft model with EVO and GEM led to a significant reduction in tumor volume growth and inhibited the activation of NF-κB p65 with no obvious adverse reactions. Conclusion: The results of this study showed that EVO may inhibit cancer cells by suppressing NF-κB activity, and in combination with GEM, it may increase the chemosensitivity of tongue squamous cancer cells, thereby improving the treatment response. Keywords: tongue cancer, evodiamine, gemcitabine, combination index, apoptosis, NF-κB

Objective: To use gene chip technology to study the effects of evodiamine (EVO) on the gene expression profile of tongue squamous cell carcinoma (TSCC) CAL‐27 cell line, for the purpose of analyzing the mechanisms underlying the effects of EVO on gene expression and functional regulation of TSCC cells at the gene level.Method: Differentially expressed genes in CAL‐27 cells treated with EVO were detected using gene chip technology and analyzed using ingenuity pathway analysis.Results: Microarray results showed that there were 1243 differentially expressed genes following treatment with CAL‐27 cells; 684 genes were upregulated and 559 were downregulated. Classical pathway analysis revealed a total of 89 signal transduction pathways with upregulated gene set enrichment, including lipopolysaccharide/interleukin (IL)‐1‐mediated inhibition of retinoid X receptor (RXR) function, agrin interactions at neuromuscular junctions, cholecystokinin/gastrin‐mediated signaling, toll‐like receptor signaling, and IL‐6 signaling. A total of 39 signal transduction pathways were enriched for the downregulated genes, including interferon signals, liver X receptor/RXR activation signals, and RhoGDI signals. In the disease and function analysis, the upregulated genes were enriched in viral infection, RNA virus replication, viral replication, cancer cell invasion, cell invasion, and other related functions, while downregulated genes were enriched in neuromuscular diseases, and leukocyte differentiation, antiviral response, connective tissue cell death and other functions.Conclusions: Gene chip analysis offers an effective means of screening differential gene expression between EVO‐treated TSCCs and controls, thus providing a sound basis for further research.

Zinc finger MIZ-type containing 1 (ZMIZ1) is a transcriptional coactivator related to the protein inhibitors of activated STATs (PIAS) family. Mounting evidence suggests that ZMIZ1 plays a crucial role in the occurrence and development of cancers. The function of ZMIZ1 in tongue squamous cell carcinoma (TSCC) and the mechanisms underpinning its role in this disease have not been fully clarified. We performed qualitative ZMIZ1 protein expression analyses using immunohistochemistry in 20 patient-derived, paraffin-embedded TSCC tissue sections. We used RNAi to knock down ZMIZ1 expression in the CAL-27 TSCC cell line and quantified the impact of ZMIZ1 knock down on proliferation, migration and apoptosis via CCK-8, scratch assay and flow cytometry, respectively. We used qRT-PCR and western blotting to investigate the role of ZMIZ1 in this cell line. Finally, we established a model of lung metastasis in nude mice to replicate the in vitro results. ZMIZ1 protein was significantly more abundant in TSCC case tissue samples. ZMIZ1 knockdown reduced the invasion and metastases of TSCC tumor cells and promoted apoptosis. ZMIZ1 knockdown was associated with the down-regulation of Notch signaling pathway related factors Jagged1 and Notch1, and invasion and metastasis related factors MKP-1, SSBP2 and MMP7 in vitro and in vivo, at the mRNA level. In vitro and in vivo data suggest that knock down of ZMIZ1 may inhibit TSCC invasion and metastasis by modulating Notch signaling. ZMIZ1 inhibition may therefore represent a new therapeutic target for TSCC.

BackgroundAs kidney cancer affects children primarily, Wilms tumor (WT) is known as pediatric embryonic cancer stemmed from pluripotent embryonic renal precursor cells. Among many studied genes, ZMIZ1 has been implicated in cancer etiology, but its specific role in tumor development and progression remains questionable. MethodsThe expression of ZMIZ1 in WT was evaluated by searching the bioinformatics database. Clinical samples of WT tissue were collected, and the expression of ZMIZ1 in tumor and adjacent normal tissues was detected by immunohistochemistry. Lentivirus was applied to downregulate ZMIZ1 in human WT cells. The effect of ZMIZ1 gene on invasion and metastasis of tumor cells was analyzed by wound healing assays and Transwell assays. QRT-PCR and Western blot were used to detect the expression of key genes in the Notch signaling pathway. Finally, to investigate the function of ZMIZ1 gene in vivo, a nude mouse lung metastasis model was constructed, and immunohistochemistry was performed.ResultsImmunohistochemistry revealed a significant up-regulation of ZMIZ1 in WT tissues compared to adjacent normal tissues. A thorough analysis of the Oncomine database yield similar results, supporting the role of ZMIZ1 in WT. Both in vitro and in vivo experiments showed that the knockdown of ZMIZ1 inhibited the invasion and metastasis of WT. Moreover, further studies demonstrated that ZMIZ1-mediated invasion and metastasis of WT depended on the Notch signaling pathway. ConclusionIn a word, ZMIZ1 may be a potential target to affect the invasion and metastasis, which might provide a new promising therapeutic strategy for WT.