Niclosamide exerts anti-tumor activity through generation of reactive oxygen species and by suppression of Wnt/ β-catenin signaling axis in HGC-27, MKN-74 human gastric cancer cells

Introduction Gastric carcinoma (GC) remains a therapeutic challenge despite having many potent drugs to treat. Various studies emphasized the role of dysregulated Wnt/β-catenin pathway in cancer. In the present study, we examined the anti-cancer effect of Niclosamide and its effect on the dysregulated β-catenin pathway in human gastric carcinoma cell lines. Methods Cytotoxicity of compound to gastric cancer cell line was assessed by MTT cell viability assay, cell cycle analysis, and apoptosis assay was done using standard kits of Muse™ Cell Analyser. Reactive oxygen species (ROS) generation and mitochondrial membrane potential were analyzed by 2′,7′-Dichlorodihydrofluorescein diacetate (DCFDA) and tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining respectively. Protein expression studies were carried out by standard western blotting protocols. Results Niclosamide treatment resulted in a dose-dependent inhibition of viability of the gastric carcinoma cell-lines induced cell cycle arrest in the G0/G1 phase and strongly induced apoptosis in a concentration-dependent manner by downregulating Cyclin-D1 and CDK4 levels, critical proteins required for G1-S phase progression. DCFDA and JC-1 staining results indicated that Niclosamide enhanced intracellular ROS generation and disrupted mitochondrial membrane potential. Furthermore, niclosamide treatment decreased the expression of NF-KB, Bcl-2 and increased the expression of Bax protein. Niclosamide treatment significantly decreased the β-catenin mediated transcriptional activity and down-regulated β-catenin levels and its downstream proteins cyclinD1, CDK-4, and c-myc expression and also impeded Akt phosphorylation, a common internode in the Wnt and Akt/mTOR signaling in HGC-27 cells. Conclusion This study demonstrated that Niclosamide might become a promising therapeutic agent for the management of gastric cancer and further warrants its clinical trials in gastric cancer patients.

MicroRNA-221-3p Plays an Oncogenic Role in Gastric Carcinoma by Inhibiting PTEN Expression

Gastric carcinoma is one of the most common malignancies in men, and microRNA plays a critical role in regulating the signaling networks of gastric carcinoma tumorigenesis and metastasis. We first report the functional characteristics of miR-221-3p in gastric carcinoma. Quantification in gastric carcinoma cell lines and tumor samples reveals significantly increasing miR-221-3p expression. Moreover, a high level of miR-221-3p is correlated with a poor prognosis for gastric carcinoma patients. Ectopic miR-221-3p expression significantly promotes gastric carcinoma cell proliferation, invasion, and sphere formation, while silencing miR-221-3p significantly inhibits these abilities in gastric carcinoma cells. Tests in vivo showed that miR-221-3p significantly promotes tumor growth in xenograft mouse models. In this study, we reveal that miR-221-3p targets PTEN mRNA and downregulates PTEN, which is the possible mechanism of miR-221-3p-induced oncogenic properties. Collectively, we reveal a critical role for miR-221-3p in gastric carcinoma development and progression.

Epigenetic regulation and functional roles of SHP1 in gastric carcinoma cell lines.

61 Background: The SH2-containing protein tyrosine phosphatase 1 (SHP1) is an important negative regulator in cytokine-mediated signal transduction and cell cycling. Recent studies demonstrated that promoter methylation of SHP1 is frequently observed in gastric adenocarcinoma tissues. We tried in this in vitrostudy to reveal promoter hypermethylation and to investigate the effects of SHP1 in gastric carcinoma cell lines. Methods: We performed RT-PCR, Western blot, methylation specific PCR (MSP) and bisulfite pyrosequencing to demonstrate promoter hypermethylation of SHP1. To evaluate functional roles of SHP1, we transfected SHP1 plasmid and analyzed WST-1 assay, wound closure assay and Matrigel invasion assay. Results: We observed that both gene and protein expression of SHP1 were negative in 8 of 10 gastric cancer cell lines (SNU-1, SNU-5, SNU-16, SNU-638, SNU-719, MKN-28, MKN-45, AGS). MSP showed methylation-specific band only in all 10 gastric cancer lines. Bisulfite pyrosequencing revealed 96.5% and 97.3% of methylation frequency in AGS and SNU-719 cells. When treating SNU-719, MKN-28 and AGS cells with 5-Aza-2’-deoxycytidine (5-Aza-dc), SHP1 was re-expressed in all three cells. SHP1 expression is known to be correlated with Janus kinase (JAK) and signal transducers and activators of transcription (STAT) signaling pathway in epithelial cells. When introducing exogenous SHP1 in SNU-719 and MKN-28 cells by transient transfection, protein expression of constitutive phospho-JAK2 (Tyr 1007/1008) and phospho-STAT3 (Tyr 705) were substantially down-regulated both, which in turn decreased target gene expression of STAT3, including cyclin D1, MMP-9, VEGF and survivin. Induction of SHP1 significantly inhibited cell proliferation, migration and invasion in SNU-719 and MKN-28 cells. Conclusions: Epigenetic silencing of SHP1 is frequently caused by promoter hypermethylation in gastric carcinoma cell lines. Overexpression of SHP1 down-regulates JAK2/STAT3 pathway to modulate various target genes and inhibit cell proliferation, migration and invasion in gastric cancer cells.

RNF43 Inhibits Cancer Cell Proliferation and Could be a Potential Prognostic Factor for Human Gastric Carcinoma

Background/Aims: RNF43 is a member of transmembrane E3 ubiquitin ligases and plays important roles in tumor formation progression. In current study, we aimed to explore RNF43 expression and analyze its role in gastric carcinoma. Methods and Results: The level of RNF43 was detected in 77 cases of gastric carcinoma and matched normal tissues by real-time PCR, western blotting and immunohistochemistry. We found that the expression of RNF43 was significantly down-regulated in the gastric carcinoma tissues compared to the normal mucosae (all P<0.001). In addition, RNF43 was significantly correlated with histological differentiation (P = 0.001), T-stage cancer (P<0.001), depth of invasion (P<0.001), metastasis of regional lymph nodes (P<0.001), pTNM stage (P<0.001) and survival (P = 0.021). We further explored the biological functions of RNF43 in gastric carcinoma cell lines. Both gain- and loss-function assays show that RNF43 could suppress cell proliferation while promotes cell apoptosis. Further, we found that RNF43 was positively correlated with p53 and cleaved-caspase3 and negatively correlated with Ki67 and Lgr5. Concolusion: In conclusion, RNF43 might act as a tumor suppressor in gastric carcinoma and might be a potential indicator for the clinical assessment of gastric cancer prognosis