ERK1／2抑制剂PD98059对大肠癌细胞侵袭和转移的影响

目的观察细胞外信号调节蛋白激酶（ERK1／2）抑制剂PD98059对大肠癌细胞侵袭和转移的影响,探讨其可能机制。方法PD98059不同浓度（0、10、20、40μmo1／L）干预大肠癌细胞24h。用Boyden小室检测其体外运动和侵袭能力,Western blot法检测ERK1／2磷酸化蛋白和ERK1／2蛋白表达情况。结果随着PD98059剂量增大,大肠癌细胞穿膜数逐渐减少（P〈0．05）,磷酸化ERK1／2蛋白水平逐渐降低（P〈0．05）,ERK1／2蛋白水平无明显变化（P〉0．05）。结论PD98059能抑制大肠癌细胞的运动和侵袭力;其机制与PD98059抑制磷酸化ERK1／2有关。     

Sphingosine kinase 1 enhances colon cancer cell proliferation and invasion by upregulating the production of MMP-2/9 and uPA via MAPK pathways

Purpose

Sphingosine kinase (SphK) 1 is an oncogenic enzyme promoting transformation, proliferation, and survival of a number of human tumor cells. However, its effect on colon cancer cell behavior has not been fully clarified.

Methods

SphK1 plasmid or SphK1 shRNA transfection and N,N-dimethylsphingosine (DMS) was used to regulate the expression and activity of SphK1 in colon cancer line LOVO. Cell proliferation, apoptosis, invasion, and protein expression were detected by MTT, flow cytometry, transwell chambers model, and western blot. The levels of metalloproteinases-2/9 (MMP-2/9) and urokinase plasminogen activator (uPA) were detected by ELISA.

Results

Overexpression of SphK1 after plasmid transfection markedly enhanced LOVO cell viability and invasiveness and reduced cell apoptosis. In contrast, inhibition of SphK1 by DMS and shRNA significantly suppressed cell viability and invasiveness but promoted cell apoptosis. SphK1 increased the constitutive expression of extracellular signal-regulated kinase1/2 (ERK1/2) but reduced the constitutive expression of p38 mitogen-activated protein kinase (MAPK). Blocking ERK1/2 pathway inhibited the biological effects induced by overexpression of SphK1. Blocking p38 MAPK pathway reversed the effects of DMS and SphK1 shRNA. Moreover, SphK1 was required for the production of MMP-2/9 and uPA in tumor cells, which was suppressed by ERK1/2 inhibitor U0126, but enhanced by the p38 MAPK inhibitor SB203580.

Conclusions

SphK1 enhances colon cancer cell proliferation and invasiveness, meanwhile suppressing cell apoptosis. SphK1 promoting the secretion of MMP-2/9 and uPA via activation of ERK1/2 and suppression of p38 MAPK pathways maybe the molecular mechanisms for its regulation of the malignant behavior of colon cancer cell.     

Function of chloride intracellular channel 1 in gastric cancer cells

AIM: To investigate the effect of chloride intracellular channel 1 (CLIC1) on the cell proliferation, apoptosis, migration and invasion of gastric cancer cells.METHODS: CLIC1 expression was evaluated in human gastric cancer cell lines SGC-7901 and MGC-803 by real time polymerase chain reaction (RT-PCR). Four segments of small interference RNA (siRNA) targeting CLIC1 mRNA and a no-sense control segment were designed by bioinformatics technology. CLIC1 siRNA was selected using Lipofectamine 2000 and transfected transiently into human gastric cancer SGC-7901 and MGC-803 cells. The transfected efficiency was observed under fluorescence microscope. After transfection, mRNA expression of CLIC1 was detected with RT-PCR and Western blotting was used to detect the protein expression. Proliferation was examined by methyl thiazolyl tetrazolium and apoptosis was detected with flow cytometry. Polycarbonate membrane transwell chamber and Matrigel were used for the detection of the changes of invasion and migration of the two cell lines.RESULTS: In gastric cancer cell lines SGC-7901 and MGC-803, CLIC1 was obviously expressed and CLIC1 siRNA could effectively suppress the expression of CLIC1 protein and mRNA. Proliferation of cells transfected with CLIC1 siRNA3 was enhanced notably, and the highest proliferation rate was 23.3% (P = 0.002) in SGC-7901 and 35.55% (P = 0.001) in MGC-803 cells at 48 h. The G2/M phase proportion increased, while G0/G1 and S phase proportions decreased. The apoptotic rate of the CLIC1 siRNA3 group obviously decreased in both SGC-7901 cells (62.24%, P = 0.000) and MGC-803 cells (52.67%, P = 0.004). Down-regulation of CLIC1 led to the inhibition of invasion and migration by 54.31% (P = 0.000) and 33.62% (P = 0.001) in SGC-7901 and 40.74% (P = 0.000) and 29.26% (P = 0.002) in MGC-803. However, there was no significant difference between the mock group cells and the negative control group cells.CONCLUSION: High CLIC1 expression can efficiently inhibit proliferation and enhance apoptosis, migration and invasion of gastric cancer cells in vitro. CLIC1 might be a promising target for the treatment of gastric cancer.     

ERK1/2信号通路阻断剂PD98059对胃癌细胞MGC-803中RECK、MMP-9基因表达的影响

目的:探讨ERK1/2信号通路在RECK基因抑制侵袭转移中的作用.方法:体外培养人胃癌MGC-803细胞,采用MTT法观察胃癌细胞MGC-803增殖情况,Western blot检测P-ERK的蛋白表达,RT-PCR法和Western blot法检测RECK和基质金属蛋白酶9(MMP-9) mRNA和蛋白表达.结果:M...     

Modulation of colon cancer cell invasiveness induced by deoxycholic acid]

BACKGROUND/AIMS: Deoxycholic acid (DCA), a secondary bile acid, has been implicated to promote colon cancer growth and progression. However, its molecular mechanisms are largely unknown. In this study, we investigated the effects of DCA on proliferation, migration, and invasiveness of colon cancer cells (HT-29). METHODS: HT-29 cells were incubated with either medium (control) only or DCA for 24-48 hours. Time courses of RT-PCR for vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF)-1alpha mRNA expression, Western blotting for VEGF and matrix metalloproteinase (MMP)-9, zymography for MMP-9 activation, and wound-migration assay were determined after various concentrations of DCA (0-80 microM) treatment. Moreover, these experiments were reassessed after pretreatments (2-6 hours) with specific inhibitors of various signal pathways. RESULTS: DCA enhanced HIF-1alpha mRNA expression, VEGF mRNA and VEGF protein expression, MMP-9 protein expression/activation, and cell migration ability in a dose-related manner. DCA-induced VEGF protein expression was inhibited by pretreatment with NS-398 (COX-2 inhibitor), PDTC (NF-kappaB inhibitor), or tauroursodeoxycholic acid (TUDC). DCA-induced cell migration ability was inhibited by pretreatment of GF109203X, a protein kinase C inhibitor. DCA-induced MMP-9 protein expression/activation was inhibited by pretreatment with SB203580, U0126, or PDTC. CONCLUSIONS: DCA significantly upregulates invasive and angiogenic potentials of human colon cancer cells through multiple signal transduction pathways.     

丹参酚酸B靶向SOX2OT/miRNA-34a/SOX2轴抑制大肠癌细胞侵袭转移

目的:验证SOX2OT/miRNA(miR)-34a/SOX2信号轴对人大肠癌LOVO细胞侵袭转移的影响以及丹参酚酸B(SalB)通过该信号轴浓度依赖性抑制LOVO细胞侵袭转移.方法:双荧光素酶报告基因检测SOX2OT与miR-34a、miR-34a与SOX2的靶向结合;瞬时转染SOX2OT质粒和miR-34a mim...     

Enhanced migration of tissue inhibitor of metalloproteinase overexpressing hepatoma cells is attributed to gelatinases： Relevance to intracellular signaling pathways

AIM: To study the effect of gelatinases (especially MMP-9) on migration of tissue inhibitor of metalloproteinase (TIMP-1) overexpressing hepatoma cells. METHODS: Wild type HepG2 cells, cells stably transfected with TIMP-1 and TIMP-1 antagonist (MMP-9-H401A, a catalytically inactive matrix metalloproteinase (MMP) which still binds and neutralizes TIMP-1) were incubated in Boyden chambers either with or without Galardin (a synthetic inhibitor of MMP-1, -2, -3, -8, -9) or a specific inhibitor of gelatinases. RESULTS: Compared to wild type HepG2 cells, the cells overexpressing TIMP-1 showed 115% migration (P<0.05) and the cells overexpressing MMP-9-H401A showed 62% migration (P<0.01). Galardin reduced cell migration dose dependently in all cases. The gelatinase inhibitor reduced migration in TIMP-1 overexpressing cells predominantly. Furthermore, we examined intracellular signal transduction pathways of TIMP-1-dependent HepG2 cells. TIMP-1 deactivates cell signaling pathways of MMP-2 and MMP-9 involving p38 mitogen-activated protein kinase. Specific blockade of the ERK pathway suppresses gelatinase expression either in the presence or absence of TIMP-1. CONCLUSION: Overexpressing functional TIMP-1-enhanced migration of HepG2-TIMP-l cells depends on enhanced MMP-activity, especially MMP-9.     

氯喹增强LOVO细胞对5-氟尿嘧啶化疗敏感性的作用

目的研究结肠癌细胞LOVO在氯喹(CQ)作用下对化疗药物5-氟尿嘧啶(5-FU)的敏感性变化及机制。方法用MTT法分别检测CQ和5-FU作用24 h和48 h对LOVO细胞的增殖抑制作用。实验分为4组:空白对照组、CQ组、5-FU组和联合作用组。CQ、5-FU及两者联合作用于LOVO细胞后,通过细胞划痕实验和Transwell分别检测细胞迁移和侵袭的变化,采用流式细胞术和TUNEL方法检测细胞凋亡,采用Western blot检测细胞自噬及凋亡相关蛋白的表达情况。结果在不同浓度CQ、5-FU作用下LOVO细胞增殖受到抑制,且呈现剂量依赖性,48 h时CQ和5-FU的IC50分别为11.8μg/ml和2.5μmol/L。与空白对照组相比,CQ、5-FU作用后细胞迁移率和细胞侵袭能力显著降低,且两药联用组的抑制效应更显著。流式细胞术检测显示,CQ组、5-FU组细胞凋亡率分别为(17.85±1.04)%、(17.36±0.96)%,显著高于空白对照组(4.11±0.23)%,两药联用组凋亡率为(25.03±2.27)%,两药联用组与CQ、5-FU单用组相比,细胞凋亡率更高,差异均有统计学意义(P<0.01)。TUNEL检测显示,CQ组、5-FU组细胞凋亡指数显著高于空白对照组,两药联用组与CQ、5-FU单用组相比细胞核凋亡指数更高。Western blot检测显示CQ和5-FU处理后导致P53、Bax、caspase3、caspase9和P62蛋白表达水平显著升高,而Bcl-2、LC3和Beclin-1蛋白表达水平则显著降低,差异均有统计学意义(P<0.01)。结论 CQ能抑制结直肠癌细胞LOVO的增殖、迁移、侵袭和自噬,促进其凋亡,并能增强细胞对化疗药物5-FU的敏感性。     

槲皮素对人胃癌细胞侵袭和MMP-2表达的影响

目的:观察槲皮素(Quercetin,Que)对人胃癌细胞侵袭的影响,并探讨其可能机制.方法:采用不同浓度的槲皮素处理胃癌BGC-823细胞后,以软琼脂集落培养试验检测癌细胞锚着不依赖性增殖,以Boyden小室模型方法检测癌细胞侵袭能力,采用荧光实时定量PCR检测癌细胞基质金属蛋白酶-2(matrix metallopeptidase-2,MMP-2)基因mRNA水平,以Western blot方法检测癌细胞MMP-2基因蛋白水平变化.结果:不同浓度的胃癌BGC-823细胞经槲皮素处理后,恶性增殖和侵袭能力均明显下降,且呈剂量依赖性(P<0.005,P<0.005).槲皮素处理组MMP-2基因mRNA和蛋白水平均明显下调,且呈时间和浓度依赖性,即随着作用时间的延长和槲皮素作用浓度的增加,MMP-2的mRNA和蛋白水平逐渐下降,差异均有统计学意义(P<0.001,P<0.001).结论:槲皮素可明显抑制胃癌BGC-823细胞侵袭能力,其机制可能与下调MMP-2基因表达有关.     

Fibroblast-derived CXCL12/SDF-1α promotes CXCL6 secretion and co-operatively enhances metastatic potential through the PI3K/Akt/m TOR pathway in colon cancer

AIM To investigate the underlying mechanism by which CXCL12 and CXCL6 influences the metastatic potential of colon cancer and internal relation of colon cancer and stromal cells. METHODS Western blotting was used to detect the expression of CXCL12 and CXCL6 in colon cancer cells and stromal cells. The co-operative effects of CXCL12 and CXCL6 on proliferation and invasion of colon cancer cells and human umbilical vein endothelial cells(HUVECs) were determined by enzyme-linked immunosorbent assay,and proliferation and invasion assays. The angiogenesis of HUVECs through interaction with cancer cells and stromal cells was examined by angiogenesis assay. We eventually investigated activation of PI3K/Akt/m TOR signaling by CXCL12 involved in the metastatic process of colon cancer.RESULTS CXCL12 was expressed in DLD-1 cancer cells and fibroblasts. The secretion level of CXCL6 by colon cancer cells and HUVECs were significantly promoted by fibroblasts derived from CXCL12. CXCL6 and CXCL2 could significantly enhance HUVEC proliferation and migration(P 0.01). CXCL6 and CXCL2 enhanced angiogenesis by HUVECs when cultured with fibroblast cells and colon cancer cells(P 0.01). CXCL12 also enhanced the invasion of colon cancer cells. Stromal cell-derived CXCL12 promoted the secretion level of CXCL6 and co-operatively promoted metastasis of colon carcinoma through activation of the PI3K/Akt/m TOR pathway.CONCLUSION Fibroblast-derived CXCL12 enhanced the CXCL6 secretion of colon cancer cells,and both CXCL12 and CXCL6 co-operatively regulated the metastasis via the PI3K/Akt/m TOR signaling pathway. Blocking this pathway may be a potential anti-metastatic therapeutic target for patients with colon cancer.     

circPUM1通过调控miR-524-5p表达对结肠癌细胞恶性生物学行为的影响

目的探讨环状RNA PUM1(circPUM1)对结肠癌细胞增殖、凋亡、迁移、侵袭的影响及其分子机制。方法实时荧光定量聚合酶链反应(qRT-PCR)检测结肠癌组织、癌旁组织中circPUM1、微小RNA-524-5p(miR-524-5p)的表达量。体外培养人结肠癌细胞株SW620,分别将si-NC、si-circPUM1、miR-NC、miR-524-5p mimics、si-circPUM1与anti-miR-NC、si-circPUM1与anti-miR-524-5p转染至SW620细胞。甲基噻唑基四唑(MTT)检测细胞增殖;Transwell小室实验检测细胞迁移、侵袭能力;流式细胞术检测细胞凋亡率;双荧光素酶报告实验验证circPUM1是否能够结合miR-524-5p;蛋白免疫印迹法(Western blotting)检测细胞周期蛋白1(Cyclin D1)、p21、基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)、B淋巴细胞瘤-2(Bcl-2)、B淋巴细胞瘤-2相关蛋白(Bax)表达量。结果结肠癌组织circPUM1的表达水平显著高于癌旁组织(P<0.05),miR-524-5p的表达水平显著降低(P<0.05);干扰circPUM1表达或miR-524-5p过表达后,细胞活力显著降低(P<0.05),迁移细胞数与侵袭细胞数显著减少(P<0.05),细胞凋亡率显著升高(P<0.05),Cyclin D1、MMP-2、MMP-9、Bcl-2蛋白表达显著降低(P<0.05),p21、Bax蛋白表达显著升高(P<0.05);双荧光素酶报告实验证实circPUM1可靶向结合miR-524-5p的作用位点;抑制miR-524-5p表达可减弱干扰circPUM1表达对SW620细胞增殖、凋亡、迁移、侵袭的作用。结论circPUM1可通过海绵吸附miR-524-5p促进结肠癌细胞增殖、迁移、侵袭,抑制细胞凋亡。