miR-125a-3p调控人结肠癌细胞浸润转移的作用及机制研究

目的:探讨miR-125a-3p在结肠癌细胞浸润与转移中的作用及其可能机制。方法:通过qRT-PCR方法检测miR-125a-3p在结肠癌细胞及组织样本中的表达;在结肠癌细胞过表达或沉默miR-125a-3p后,通过平板克隆实验、MTT实验、划痕实验、Transwell实验检测结肠癌细胞增殖、迁移及侵袭能力的变化;采用Western blot方法检测miR-125a-3p过表达后相关标志分子的表达水平变化情况。结果:miR-125a-3p在结肠癌细胞及组织呈现异常低表达;过表达miR-125a-3p抑制结肠癌细胞HCT116及SW480的增殖能力;过表达或沉默miR-125a-3p分别抑制或增强结肠癌细胞的迁移与侵袭能力;过表达miR-125a-3p在mRNA及蛋白水平均能够显著抑制Snail、N-cadherin及Vimentin的表达,而增加E-cadherin的表达。结论:miR-125a-3p参与调节结肠癌细胞浸润与转移,其机制可能是通过调控上皮间质转化途径介导的。     

miR-19a targets CLCA4 to regulate the proliferation,migration, and invasion of colorectal cancer cells

The role of miR-19a in colorectal cancer (CRC), a devastating disease with high mortality and morbidity, remains controversial. In the present study, we show that the level of miR-19a is significantly higher in clinical CRC tissue samples than in paracancerous tissue samples, and significantly higher in CRC cells lines HT29, SW480, and CaCO2 than in the normal human colon mucosal epithelial cell line NCM460. miR-19a mimics and inhibitors were synthesized and validated. Overexpression of miR-19a mimics significantly promoted, while miR-19a inhibitors inhibited, the proliferation, survival, migration, and invasion of SW480 and CaCO2 CRC cells. Furthermore, mRNA and protein levels of chloride channel accessory 4 (CLCA4) were lower in CRC cells and tissues. Bioinformatics and a luciferase reporter assay confirmed that CLCA4 was a miR-19a target. Further, miR-19a inhibition increased CLCA4 expression. The inhibitory effect of miR-19a on cell growth, survival, migration, and invasion was reversed by knockdown of CLCA4 expression. The data demonstrated that the miR-19a/CLCA4 axis modulates phospho-activation of the PI3K/AKT pathway in CRC cells. In conclusion, our results revealed that miR-19a overexpression decreases CLCA4 levels to promote CRC oncogenesis, suggesting that miR-19a inhibitors have potential applications for future therapeutic of CRC.Key words: Colorectal cancer, miR-19a, CLCA4, proliferation, migration, invasion     

pTyr421 Cortactin Is Overexpressed in Colon Cancer and Is Dephosphorylated by Curcumin: Involvement of Non-Receptor Type 1 Protein Tyrosine Phosphatase (PTPN1)

Cortactin (CTTN), first identified as a major substrate of the Src tyrosine kinase, actively participates in branching F-actin assembly and in cell motility and invasion. CTTN gene is amplified and its protein is overexpressed in several types of cancer. The phosphorylated form of cortactin (pTyr⁴²¹) is required for cancer cell motility and invasion. In this study, we demonstrate that a majority of the tested primary colorectal tumor specimens show greatly enhanced expression of pTyr⁴²¹-CTTN, but no change at the mRNA level as compared to healthy subjects, thus suggesting post-translational activation rather than gene amplification in these tumors. Curcumin (diferulolylmethane), a natural compound with promising chemopreventive and chemosensitizing effects, reduced the indirect association of cortactin with the plasma membrane protein fraction in colon adenocarcinoma cells as measured by surface biotinylation, mass spectrometry, and Western blotting. Curcumin significantly decreased the pTyr⁴²¹-CTTN in HCT116 cells and SW480 cells, but was ineffective in HT-29 cells. Curcumin physically interacted with PTPN1 tyrosine phosphatases to increase its activity and lead to dephosphorylation of pTyr⁴²¹-CTTN. PTPN1 inhibition eliminated the effects of curcumin on pTyr⁴²¹-CTTN. Transduction with adenovirally-encoded CTTN increased migration of HCT116, SW480, and HT-29. Curcumin decreased migration of HCT116 and SW480 cells which highly express PTPN1, but not of HT-29 cells with significantly reduced endogenous expression of PTPN1. Curcumin significantly reduced the physical interaction of CTTN and pTyr⁴²¹-CTTN with p120 catenin (CTNND1). Collectively, these data suggest that curcumin is an activator of PTPN1 and can reduce cell motility in colon cancer via dephosphorylation of pTyr⁴²¹-CTTN which could be exploited for novel therapeutic approaches in colon cancer therapy based on tumor pTyr⁴²¹-CTTN expression.     

Effect of RSK4 on biological characteristics of colorectal cancer

Background

This study aimed to investigate the expression of P90 Ribosomal Protein S6 kinase 4 (RSK4) in colorectal cancer cells and its biological function.

Methods

We selected early SW480 and HCT116 colorectal cancer cell lines, using Lipofectamine? 2000 transfection reagent carrying RSK4 gene transfected into cells to establish the colorectal cancer cell lines with high expression of RSK4. RT-PCR and western blot (WB) analysis confirmed RSK4 expression in SW480 and HCT116 cancer cell lines. We used methylthiazoltetrazolium (MTT) assay and flow cytometry to detect the proliferation of colorectal cancer cells. After transfection of RSK4, the effect of RSK4 on the RNA levels associated with epithelial–mesenchymal transition (EMT) of colorectal cancer cells was analyzed by real-time fluorescence quantitative PCR and the expression of EMT-related protein was detected by WB analysis.

Results

After transfection of RSK4 overexpression, the MTT assay detected that RSK4 could significantly inhibit the growth of colorectal cancer cells in vitro; flow cytometry detected that S-phase cells decreased significantly, and G0/1 cells increased significantly (P?<?0.05). The invasion ability of SW480 and HCT116 cells transfected with RSK4 was markedly lower than that in the control group, and the difference was statistically significant (P?<?0.05). Fluorescent quantitative PCR and WB analysis showed that the expression of EMT-associated molecular E-cadherin was remarkably increased and the expression of Snail was significantly decreased (P?<?0.01).

Conclusion

RSK4 gene in colorectal cancer cell lines with low expression of RSK4 after transfection can inhibit the growth and invasion of tumor cells. RSK4 gene may inhibit EMT and inhibit metastasis of colorectal cancer cells, may be a potential tumor suppressor gene and inhibit tumor distant metastasis, and may provide the biological basis for new therapeutic targets.

     

Liriodenine induces G1/S cell cycle arrest in human colon cancer cells via nitric oxide- and p53-mediated pathway

Liriodenine is an aporphine alkaloid compound extracted from the leaves of Michelia compressa var. lanyuensis. It had been reported to have an anti-colon cancer effect, but the mechanism remains unclear. In the present study, the antiproliferative mechanisms of liriodenine were investigated in the human colon cancer SW480 cells. Flow cytometry analysis indicated that liriodenine notably induced the G1/S phase arrest. The G1/S phase cycle-related proteins analysis illustrated that the expressions of cyclin-dependent kinase (CDK) 2, CDK4 and CDK6, and of cyclin D1 and A, as well as the phosphorylation of retinoblastoma tumor suppressor protein (ppRB) were found to be markedly reduced by liriodenine, whereas the protein levels of the CDK inhibitors (CKIs), p21 and p27 were increased. Moreover, the intracellular nitric oxide (NO) production, protein levels of inducible NO synthase (iNOS) and, p53 were increased. The p53 overexpression was a downstream event of NO production in liriodenine-induced G1/S-arrested SW480 cells, and the up-regulation of p21 and p27 was found to be mediated by a p53-dependent pathway. The inhibition of p53 by pifithrin-α (PFT-α), down-regulation of p21 and p27 by siRNA, or NO reduction by S-ethylisothiourea (ETU) entirely abolished the liriodenine-induced G1/S phase arrest. We concluded that liriodenine potently inhibited the cell cycle of SW480 cancer cells via NO- and p53-dependent G1/S phase arrest pathway. These results suggest that liriodenine might be a powerful agent against colon cancer.     

RNF2 promotes the progression of colon cancer by regulating ubiquitination and degradation of IRF4

Ring finger protein 2 (RNF2), as a well-known E3 ligase, has an oncogenic role in various cancers. The role of RNF2 in colon cancer is still unknown. The aim of this work is to determine the biological role of RNF2 in colon cancer. We first examined the expression of RNF2 and interferon regulatory factor 4 (IRF4) in colon cancer patients and colon cancer cell lines (SW480 and HCT116). Compared with normal tumor-adjacent tissues, RNF2 was up-regulated whereas IRF4 was down-regulated in the colon cancer tissues. RNF2 was also up-regulated in colon cancer cells with respect to human fetal colon epithelial cells. RNF2 overexpression enhanced the ability of proliferation, migration and invasion of SW480 cells, whereas RNF2 knockdown caused an opposite result in HCT116 cells. Furthermore, a tumor xenograft model was constructed to verify the impact of RNF2 overexpressed-SW480 cells on tumor growth. RNF2 up-regulation elevated Ki-67 proliferation index, accelerated the growth of tumor tissues, and led to severe colon tissue damage in the tumor xenograft mice. In addition, RNF2 interacted with IRF4, and repressed IRF4 protein expression. IRF4 was a substrate of RNF2, and RNF2 promoted the ubiquitination and degradation of IRF4. RNF2 overexpression increased the ability of proliferation, migration and invasion in SW480 cells by promoting the ubiquitination and degradation of IRF4. In conclusion, this work demonstrated that RNF2 promoted tumor growth in colon cancer by regulating ubiquitination and degradation of IRF4. Thus, RNF2 may be served as a potential therapeutic target for colon cancer.     

Concentration- and stage-specific effects of nitrite on colon cancer cell lines

Colorectal cancer (CRC) is the second leading cause of cancer-related death in the United States. Nitrite in cured meats is thought to contribute to increased incidence of colon cancer. We sought to determine the effect of nitrite on human colon cancer cell lines at different stages. Our results indicate nitrite has no effect on proliferation of stage 1 SW116 colon cancer cells, while nitrite inhibits proliferation of stage 2 SW480 at 10 nM–100 μM and inhibits stage 3 HCT15 proliferation at 100 nM–1 μM, but promotes a significant increase in proliferation on stage 4 COLO205 cells at 100 μM. Furthermore, nitrite inhibited invasion into Matrigel® of stage 3 SW480 colon cancer cells in a concentration-dependent manner. However, it significantly promotes the invasion of stage 4 cells at 100 μM. Our FACS data demonstrated that nitrite decreased cell cycle progression in SW480 and HCT15 with arrested G2/M transition and delayed G1 phase entry in a concentration-dependent manner. However, 100 μM nitrite promoted cell cycle progression in COLO205 cells with increased S-phase entry. Taken together, our data indicate nitrite inhibits cancer cell progression at low concentrations and early stage but promotes cancer cell progression at higher concentrations in cells representing stage 4 colon carcinomas.     

Erlotinib inhibits colon cancer metastasis through inactivation of TrkB-dependent ERK signaling pathway

The distal metastasis is the main cause of death in patients with colon cancer. Tyrosine receptor kinase B (TrkB) and ERK signals may be the potential targets for the treatment of colon cancer metastasis. This study aims to investigate whether erlotinib inhibits distant metastasis of colon cancer by regulating TrkB and ERK signaling pathway. Human colon adenocarcinoma cell lines (SW480 and Caco-2) pretreated with exogenous C-X-C motif chemokine ligand 8 (CXCL8) were used to assess the suppressive effect of erlotinib on tumor metastasis, including anoikis, epithelial-mesenchymal transformation (EMT), migration, and invasion. Through TrkB overexpression, Akt suppression, and ERK suppression, the roles of TrkB, Akt, and ERK in erlotinib-induced metastasis inhibition of colon cancer cells were explored. The results showed that erlotinib alleviated CXCL8-induced metastasis of the colon cancer cells. Overexpression of TrkB in colon cancer cells eliminated the effect of erlotinib on anoikis, inhibition of EMT, migration, and invasion, and downregulation of p-ERK and p-Akt. Furthermore, the inhibition of ERK activation instead of Akt activation was found to participate in erlotinib-mediated metastasis resistance, including anoikis, inhibition of EMT, migration, and invasion. In conclusion, erlotinib inhibits colon cancer cell anoikis resistance, EMT, migration, and invasion by inactivating TrkB-dependent ERK signaling pathway.     

TMEM180 contributes to SW480 human colorectal cancer cell proliferation through intra-cellular metabolic pathways

TMEM180, a novel colon cancer–specific protein with a 12-transmembrane topology, is upregulated at low oxygen. Previously, we established a humanized monoclonal antibody against TMEM180 aimed at clinical trials. Prior to such trials, it is necessary to clarify the function of TMEM180 in cancer. To compare SW480 human colon cancer cells and their TMEM180-knockdown derivatives, we analyzed proliferation and oxygen consumption, and also performed phosphorylation proteomics, metabolomics, and next-generation sequencing (NGS). The preliminary results revealed that TMEM180 appeared to promote the growth of colon cancer but had almost no effect on oxygen consumption or expression of phosphorylated proteins. By contrast, glycolysis differed dramatically between SW480 and TMEM180-knockdown cells. The NGS analysis revealed that TMEM180 promotes enzyme expression in nitric oxide (NO) synthesis system, suggesting that it promotes glucose and glutamine metabolism, thereby contributing to cancer growth. Overall, the results of this study warrant further basic studies of TMEM180 molecule.     

miR‐4709 overexpression facilitates cancer proliferation and invasion via downregulating NR3C2 and is an unfavorable prognosis factor in colon adenocarcinoma

To date, microRNA‐4709 (miR‐4709) has not been studied in colon adenocarcinoma (COAD) on the basis of experiments. In our study, we aimed to investigate the biological roles and clinical significance of miR‐4709 in COAD. The expression difference between miR‐4709 and NR3C2 was measured based on The Cancer Genome Atlas database and cells. Kaplan‐Meier and logrank tests were applied to determine the overall survival (OS) differences according to the miR‐4709 and NR3C2 expression levels. To measure whether the miR‐4709 level was associated with COAD cell growth, migration, and invasion, we respectively conducted 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide, wound healing, and transwell assays. A luciferase reporter assay was applied to confirm the relationship between miR‐4709 and its predicted target. High expression of miR‐4709 was found in COAD tissues and cells. The OS rate in the miR‐4709 low expression group was significantly higher than that in the miR‐4709 high expression group. Univariate and multivariate analyses exhibited that miR‐4709 expression was an independent adverse prognostic factor. Exogenous miR‐4709 overexpression promoted proliferation, migration, and invasion of LOVO and SW480 cells. Bioinformatics analysis and luciferase assay demonstrated that miR‐4709 directly binds to the 3′‐untranslated region of NR3C2. NR3C2 was lowly expressed in COAD and high expression of NR3C2 had a better prognosis compared with that in the low expression of NR3C2. Correlation analysis showed that there is a close association between the level of expression of NR3C2 and miR‐4709. Accordingly, miR‐4709 may function as an oncogene in COAD and provide a preclinical proof for candidate management to target new miR‐4709‐NR3C2 signaling for COAD therapy.     

Dictyopteris undulata extract induces apoptosis in human colon cancer cells

The present study investigated the cytotoxic and apoptotic effects of an ethanol extract derived from the marine brown alga Dictyopteris undulata against human colon adenocarcinoma cells. The Dictyopteris undulata extract (DUE) showed cytotoxic activity against SW480 cells in a dose-dependent manner, with 50% inhibition of cell viability at a concentration of 40 μg/mL. DUE also induced programmed cell death in SW480 cells, as evidenced by apoptotic body formation, DNA fragmentation, an increase in the population of apoptotic sub-G1 phase cells, and mitochondrial membrane depolarization. Moreover, DUE significantly modulated the expression of apoptosisassociated proteins, resulting in a decrease in B cell lymphoma-2 expression and an increase in Bcl-2-associated X protein expression, as well as the activation of caspase-9 and caspase-3. Furthermore, DUE showed apoptotic cell death in two other colon cancer cell lines, SNU407 and HT29. These observations suggest that DUE may prove useful as a therapeutic agent for the attenuation of colon cancer.     

胰岛素样生长因子1（IGF-I）通过PI-3K/Akt途径对结肠癌细胞凋亡的影响

目的：探讨胰岛素样生长因子-1（IGF-I）通过磷酯酰肌醇3-激酶/蛋白激酶B（PI-3K/Akt）信号通路对结肠癌细胞株SW480凋亡率的影响及其凋亡抑制蛋白survivin表达水平的变化。方法：培养结肠癌SW480细胞株,实验分成三组：未加IGF-I空白组、IGF-I刺激组、IGF-I＋LY294002阻断组,检测阻断剂LY294002是否阻断PI-3K/Akt通路（Western Blot检测三组P-Akt表达情况）;Western Blot及免疫荧光观察三组survivin蛋白表达变化;MTT法检测细胞增殖活性,流式细胞术检测细胞凋亡情况。结果：Western blot结果显示LY294002可抑制IGF-I诱导的p-Akt的表达（P〈0.05）;阻断IGF-I诱导的PI-3K/Akt通路后MTT显示结肠癌细胞SW480增殖抑制率升高（P〈0.05）,流式细胞术分析显示凋亡率明显上升（P〈0.05）;Western blot及免疫荧光结果显示LY294002可抑制IGF-I诱导的survivin的表达（P〈0.05）。结论：IGF-I可通过PI-3K/Akt通路诱导survivin表达,从而抑制结肠癌细胞SW480的凋亡。     

Silibinin triggers apoptotic signaling pathways and autophagic survival response in human colon adenocarcinoma cells and their derived metastatic cells

Silibinin, a flavonolignan isolated from the milk thistle plant (Silybum marianum), possesses anti-neoplastic properties. In vitro and in vivo studies have recently shown that silibinin inhibits the growth of colorectal cancer (CRC). The present study investigates the mechanisms of silibinin-induced cell death using an in vitro model of human colon cancer progression, consisting of primary tumor cells (SW480) and their derived metastatic cells (SW620) isolated from a metastasis of the same patient. Silibinin induced apoptotic cell death evidenced by DNA fragmentation and activation of caspase-3 in both cell lines. Silibinin enhanced the expression (protein and mRNA) of TNF-related apoptosis-inducing ligand (TRAIL) death receptors (DR4/DR5) at the cell surface in SW480 cells, and induced their expression in TRAIL-resistant SW620 cells normally not expressing DR4/DR5. Caspase-8 and -10 were activated demonstrating the involvement of the extrinsic apoptotic pathway in silibinin-treated SW480 and SW620 cells. The protein Bid was cleaved in SW480 cells indicating a cross-talk between extrinsic and intrinsic apoptotic pathway. We demonstrated that silibinin activated also the intrinsic apoptotic pathway in both cell lines, including the perturbation of the mitochondrial membrane potential, the release of cytochrome c into the cytosol and the activation of caspase-9. Simultaneously to apoptosis, silibinin triggered an autophagic response. The inhibition of autophagy with a specific inhibitor enhanced cell death, suggesting a cytoprotective function for autophagy in silibinin-treated cells. Taken together, our data show that silibinin initiated in SW480 and SW620 cells an autophagic-mediated survival response overwhelmed by the activation of both the extrinsic and intrinsic apoptotic pathways.