表没食子儿茶素没食子酸酯通过调控P53/miR-34a抑制鼻咽癌细胞的增殖

目的:观察表没食子儿茶素没食子酸酯(EGCG)对鼻咽癌细胞增殖的影响,并以微小RNA-34a(miR-34a)为靶点探讨其作用机制。方法:不同浓度的EGCG处理CNE-2Z细胞,CCK-8法、Ed U染色法和平板克隆形成实验检测细胞增殖能力的改变;流式细胞术检测细胞周期分布的改变;Western blot检测P53和Notch1蛋白水平的变化;real-time PCR检测miR-34a和Notch1 mRNA的表达。结果:EGCG处理后,CNE-2Z细胞的增殖受到明显抑制,克隆形成能力降低,细胞周期阻滞于G0/G1期,呈剂量依赖关系;随着EGCG浓度增高,P53和miR-34a的表达水平明显上调,而其下游Notch1 mRNA和蛋白的表达水平则明显下降。结论:EGCG可能通过调控P53/miR-34a/Notch1通路诱导细胞周期阻滞,抑制鼻咽癌细胞增殖。     

Antitumor activity of the pachymic acid in nasopharyngeal carcinoma cells

Objective: To investigate the antitumour efficacy of pachymic acid (PA), which is a fungal extract component, on nasopharyngeal carcinoma (NPC) cells CNE-1, CNE-2. Methods: We have chosen NPC cell line CNE-2 for the study, and the cells were treated with PA before the detection. CCK-8 assay was used to detect the proliferative ability, and Annexin V-PI double staining was used for the detection of apoptosis rate; and the nucleus damage was detected by transmission electron microscope, the protein expression of the DNA damage pathway were detected by Western blot. Results: PA can significantly inhibited proliferation of CNE-1, CNE-2 cells. The proportion of apoptotic cells of all cell lines gradually increased in a dose-dependent manner induced by PA, P < 0.05. Meanwhile, the nucleus could be caused morphological changes and the expression of DNA damage-related proteins was upregulated by PA in CNE-2. Conclusions: PA can significantly inhibit cell proliferation and increase the apoptosis rates and may induce the apoptosis of the human NPC cells.     

巨噬细胞移动抑制因子提高鼻咽癌细胞体外侵袭能力

目的　研究巨噬细胞移动抑制因子 (MIF)体外提高鼻咽癌细胞侵袭能力的原因 ,探讨鼻咽癌细胞早期发生侵袭和转移的机制。方法　采用微孔迁移分析法 (micron migrationassay)检测鼻咽癌细胞株CNE 1、CNE 2在细胞因子MIF诱导下通过 8μm直径微孔的细胞数量变化 ,采用Western蛋白印迹法、流式细胞术和酶联免疫吸附法检测侵袭转移相关因子基质金属蛋白酶 2、9(MMP2 ,MMP9)及白介素 8(IL 8)在此过程中的相应改变。结果　 (1)经MIF诱导后 ,CNE 1通过 8μm微孔的细胞数由原来的 2 3 7± 11 0 2增加至 113 7± 2 0 9,CNE 2则由原来的 3 4 7± 7 41增加至 3 11 3± 48 9,差异均呈显著性 (P =0 0 0 5,P =0 0 0 1)。 (2 )经MIF诱导后 ,癌细胞表达MMP9蛋白的细胞比例均明显增加 ,CNE 1细胞由 2 8 5%± 2 45%增加至 82 4%± 3 49% (P =0 0 0 1) ,而CNE 2细胞则由刺激前的 3 2 8%± 3 48%增加至 86 1%± 1 62 % (P =0 0 0 2 )。同时癌细胞MMP9蛋白表达强度也显著增强 ,CNE 1和CNE 2细胞MMP9蛋白的平均相对强度均比诱导前提高 3倍以上 (P <0 0 5)。但MIF刺激前后 ,MMP2蛋白的细胞表达数量和表达强度在两株细胞中却未见明显变化 (P值均大于 0 0 5)。 (3 )MIF诱导后 ,CNE 2细胞培养上清液中的IL 8浓度为 12     

口虾蛄提取物对人鼻咽癌细胞端粒酶活性的影响

目的:研究口虾蛄提取物(EOS)对人低分化鼻咽癌细胞系(CNE-2Z)端粒酶活性的抑制作用及其可能机制。方法:MTT法检测不同浓度的EOS对CNE-2Z细胞增殖能力的影响;端粒酶重复序列扩增-酶联免疫吸附法(TRAP-ELISA)检测各组细胞端粒酶活性,RT-PCR法检测端粒酶逆转录酶(hTERT)mRNA表达,Western blotting检测c-Myc蛋白表达。结果:EOS对CNE-2Z细胞增殖具有抑制作用,且呈剂量依赖性(P0.01);EOS处理组CNE-2Z细胞端粒酶的活性、hTERT mRNA和c-Myc蛋白的表达水平均低于对照组(P0.01),也呈剂量依赖性,并且hTERT mRNA的下调与c-Myc的抑制存在相关性(P0.05)。结论:EOS可能通过下调细胞c-Myc表达而抑制了hTERT mRNA的转录,以致端粒酶的活性降低而抑制CNE-2Z细胞增殖。     

低氧-复氧对人鼻咽癌细胞增殖、细胞周期、黏附和侵袭能力的影响

目的: 研究细胞外微环境低氧-复氧对人鼻咽癌细胞(CNE-2Z)增殖、细胞周期、黏附和侵袭能力的影响。方法: 以混合气体(5% CO₂, 95% N₂, O₂<0.1%)置换法建立体外低氧培养模型;MTT法检测细胞增殖;流式细胞术检测细胞周期分布;同质黏附实验和细胞-基质黏附实验检测细胞黏附能力;Transwell实验检测细胞侵袭能力。结果: (1) 低氧24 h后抑制CNE-2Z细胞增殖(P<0.01),复氧后细胞快速增殖,在复氧第5 d A值与常氧对照组相比无显著差异(P>0.05);(2) 低氧24 h后细胞阻滞于G₁期(P<0.01),复氧后G₁期阻滞得到快速解除,在复氧24 h细胞周期分布与常氧对照组相比无显著差异(P>0.05);(3)低氧24 h后细胞的同质黏附能力下降(P<0.01)而细胞-基质黏附能力无明显改变(P>0.05),细胞侵袭能力降低(P<0.01);复氧24 h细胞同质黏附能力恢复至常氧对照组水平(P>0.05),而细胞-基质黏附能力(P<0.01)和细胞侵袭能力较常氧对照组显著增加(P<0.01)。结论: 鼻咽癌细胞能耐受微环境低氧的不利影响;低氧环境下存活的鼻咽癌细胞产生的适应性变化赋予肿瘤更强的增殖和转移潜能,从而促进肿瘤细胞的恶性演进。     

塞来昔布对鼻咽癌CNE-2Z细胞生物学行为的影响

目的 探讨塞来昔布对人鼻咽癌CNE-2Z细胞增殖、黏附、运动能力等生物学行为的影响.方法 不同浓度塞来昔布作用鼻咽癌细胞24 h后,运用平板克隆形成实验检测塞来昔布对鼻咽癌CNE-2Z细胞增殖的影响;应用细胞-细胞黏附实验、细胞-基质黏附实验和细胞运动实验检测塞来昔布对肿瘤细胞黏附和运动能力的影响;Western blot和免疫细胞化学检测鼻咽癌细胞COX-2蛋白表达变化.结果 塞来昔布可以明显抑制鼻咽癌CNE-2Z细胞的增殖,0、25、50、75、100 μmol/L组肿瘤细胞克隆形成数率分别为(85.68±1.83)%、(69.00±4.08)%、(59.03±2.43)%、(37.28±3.25)%和(12.58±3.69)%,各组间比较差异均有显著性(F=321.187,P＜0.01).细胞-细胞黏附实验结果显示,同一个时间点各组间细胞黏附率差异有显著性(F值分别为21.605、181.301,P均＜0.001),其中15 min时间点0、25、50 μmol/L组细胞间黏附率分别为(12.38±1.54)%、(19.30±1.49)%和(20.18±1.80)%,45 min时间点细胞间黏附率分别为(46.34±1.37)%、(58.72±1.68)%和(67.15±0.86)%;细胞-基质黏附实验结果表明,25、50 μmol/L组细胞异质黏附力明显低于无药组(t值分别为24.840和38.565,P均＜0.01);运动实验显示穿膜肿瘤细胞数分别为(124.3±8.02)个、(84.3±12.22)个和(55.3±9.29)个,组间差异有统计学意义,用药组穿膜细胞数明显减少(F＝36.010,P＜0.01);Western blot结果发现,25、50 μmol/L组COX-2蛋白的表达水平与对照组相比明显下降(t值分别为25.356和73.656,P均＜0.01);免疫细胞组织化学结果发现,0、25、50 μmol/L组COX-2阳性表达数分别为(380±11)个、(313±12)个、(213±8)个,各浓度组间差异有统计学意义(F＝298.578,P＜0.01).结论 塞来昔布可以抑制鼻咽癌细胞的增殖,增强鼻咽癌细胞间的黏附力,降低细胞与细胞外基质的黏附力及运动能力,机制可能与下调COX-2蛋白的表达水平有关.     

MMP2 role in breast cancer brain metastasis development and its regulation by TIMP2 and ERK1/2

Matrix metalloproteinase 2 (MMP2) is important in breast cancer (BC) invasion and metastasis. We previously reported that BC brain metastases, in a rat syngeneic model developed in our laboratory, have high expression and activity of MMP2. The MMP2 mechanism of action in the brain is still under intense scrutiny. To study the role of MMP2 in the development of BC brain metastasis we transfected ENU1564 rat mammary adenocarcinoma cells with tissue inhibitor of MMP2 (TIMP2). Animals inoculated with ENU1564-TIMP2 cells had decreased orthotopic tumor growth, decreased orthotopic metastastic behavior and did not develop brain metastases. These results were associated with decreased MMP2 activity, demonstrated by gel zymography. Mitogen activated protein kinase (MAPK) pathway components, such as ERK1/2, have been correlated to MMP expression and/or astrocyte activity. We found that BC brain metastases have peripheral astrocyte reactivity and higher expression of glial fibrillary acidic protein (GFAP) and phosphorylated-ERK1/2 (p-ERK1/2). Additionally, rat astrocyte-conditioned media increased in vitro invasion of ENU1564 cancer cells and increased expression of MMP2 and p-ERK1/2. Blockage of ERK1/2 phosphorylation by treatment with MEK inhibitor (PD98059) decreased the expression of MMP2 in cancer cells grown in rat astrocyte-conditioned media. Our results are highly suggestive that MMP2 plays a role in the development of BC metastases, in particular to the brain. Furthermore, our results suggest that astrocyte factors and the ERK1/2 signaling pathway may be associated with BC brain metastasis development; and that ERK1/2 may regulate MMP2 in a way that is modifiable by astrocyte factors.     

Functional polymorphisms and haplotypes in the promoter of the MMP2 gene are associated with risk of nasopharyngeal carcinoma

Matrix metalloproteinases (MMPs) play important roles in cancer initiation and development. Several polymorphisms in the promoters of a number of MMP genes, which can affect the respective MMP production in an allele-specific manner, have been well characterized. We examined whether these functional polymorphisms were related to the risk of nasopharyngeal carcinoma (NPC) in Chinese populations. Eight polymorphisms in the promoter of MMP1, MMP2, MMP3, MMP7, MMP9, MMP12, and MMP13 were genotyped in two independent case-control populations; one is from Guangxi province (593 patients with NPC and 480 controls), and the other is from Guangdong province (239 patients and 286 controls). We observed significantly increased susceptibility to NPC for the MMP2 -1306CC (rs243865:C>T) (odds ratio [OR] = 2.01, 95% confidence interval [CI] = 1.30-3.10) and -735CC (rs2285053:C>T) (OR = 1.56, 95% CI = 1.17-2.09) genotype carriers compared with noncarriers in the Guangxi population. This association was confirmed in the Guangdong population (for -1306CC: OR = 2.19, 95% CI = 1.21-3.96; for -735CC: OR = 1.60, 95% CI = 1.13-2.28). The C(-1306)-C(-735) haplotype was also significantly associated with increased susceptibility to NPC in both the Guangxi (OR = 1.64, 95% CI = 1.35-1.99) and Guangdong population (OR = 1.68, 95% CI = 1.29-2.19). Furthermore, stratified analysis indicated that the increased susceptibility to NPC related to the -1306CC and -735CC genotype and the C(-1306)-C(-735) haplotype was more pronounced in heavier smokers. Our findings suggest that the genetic polymorphisms or haplotype in the MMP2 promoter may play a role in mediating the susceptibility to NPC in Chinese populations.     

Expression of MMP2, MMP9 and MMP3 in Breast Cancer Brain Metastasis in a Rat Model

In order to study the expression of MMP2, MMP3 and MMP9 in breast cancer brain metastasis, we used a syngeneic rat model of distant metastasis of ENU1564, a carcinogen-induced mammary adenocarcinoma cell line. At six weeks post inoculation we observed development of micro-metastasis in the brain. Immunohistochemistry and Western Blotting analyses showed that MMP-2, -3 and -9 proteins expressions are consistently significantly higher in neoplastic brain tissue compared to normal brain tissue. These results were confirmed by RT-PCR. In situ zymography revealed gelatinase activity within the brain metastasis. Gel zymography showed increase in MMP2 and MMP3 activity in brain metastasis. Furthermore, we were able to significantly decrease the development of breast cancer brain metastasis in animals by treatment with PD 166793, a selective synthetic MMP inhibitor. In addition, PD 166793 decreased the in vitro invasive cell behavior of ENU1546. Together our results suggest that MMP-2, -3 and -9 may be involved in the process of metastasis of breast cancer to the brain.     

Effect of DAPK1 gene on proliferation,migration, and invasion of carcinoma of pancreas BxPC-3 cell line

DAPK1 can induce apoptosis in several cells; to determine the effect of DAPK1 would provide a new potential therapeutic strategy for treating pancreatic cancer. The aim of the present study was to investigate the effect of DAPK1 gene on proliferation, migration, and invasion of carcinoma of pancreas BxPC-3 cell line and explore the possible mechanisms. In our study, DAPK1 over-expressed cells were established by using the lentiviral transfection method, and DAPK1 obviously increased in BxPC-3 cells after transient transfection. Cell Counting Kit-8 (CCK-8) assay was used to determine the BxPC-3 cells proliferation after transfection. Apoptosis of the BxPC-3 cells was determined by using flow cytometry analysis. In addition, cell adhesion assay and in vitro invasion assay were performed. Western blotting was used to determine the protein expressions of caspase-3, DAPK1, VEGF, PEDF, MMP2, AKT, P-AKT, P-ERK, Bcl2, and Bax. Our results demonstrated that DAPK1 gene over-expression can suppress the proliferation, migration, and invasion of carcinoma of pancreas BxPC-3 cell line, and the possible mechanisms may be correlated to induction of mitochondria-mediated apoptosis, down-regulations of MMP-2 and VEGF, up-regulations of PEDF, through the PI3K/Akt and ERK pathways.     

miR-21 inhibitor suppresses proliferation and migration of nasopharyngeal carcinoma cells through down-regulation of BCL2 expression

This study is to investigate the expression of miR-21 in nasopharyngeal carcinoma (NPC) cells, and the effect of miR-21 in the biological behavior and expression of B-cell lymphoma 2 (BCL2) in NPC cells. Paired NPC and adjacent non-tumor tissues were obtained from 53 patients who underwent primary surgical resection of NPC tissues. Luciferase reporter assay was performed to test whether BCL2 is a direct target of miR-21. Methylthiazolyl blue tetrazolium assay and colony assay were used to evaluate the effect of miR-21 on NPC cell proliferation. Transwell and wound-healing assays were carried out to test the effect of low expression of miR-21 on cancer cell migration and invasion. QRT-PCR and Western blotting were used to measure the levels of mRNA and protein expression, respectively. Tumor tissues showed a positive correlation between the levels of miR-21 and BCL2 protein expression. Cells transfected with miR-21 inhibitor healed slower compared the control (P < 0.05). In addition, cell migration was notably inhibited by the down-regulation of miR-21 in vitro (P < 0.05). The reduction in miR-21 expression showed a remarkable effect on the biological behavior of NPC cell clone formation (P < 0.05). Low expression of miR-21 by transfection with miRNA expression plasmid led to a decrease in BCL2 expression, which was accompanied by reduced migration and proliferation of the cancer cells. Our results demonstrated that miR-21 inhibitor down-regulated BCL2 expression level, suggesting that BCL2 might be a target gene for the initiation and development of NPC cells.     

Evaluation of MMP2 and Caspase-3 expression in 107 cases of papillary thyroid carcinoma and its association with prognostic factors

Papillary thyroid carcinoma (PTC), including its variants and widely varying behavior, constitutes about 80% of all thyroid malignancies. Increased knowledge regarding molecular alterations has led to attempts to identify diagnostic or prognostic factors for a reliable preoperative approach to the classification of patients according to risk of recurrence. In this study, 107 cases of PTC with known histological properties, including vascular or capsular invasion, were assessed for expression of MMP2 and Caspase-3 using immunohistochemistry. Considering 10% as a cutoff to discriminate cases with invasive behavior from the non-invasive group, there was no relationship between expression of MMP2 or Caspase-3 in tumor cells and the presence of capsular invasion (p = 0.45 and 0.64, respectively), as well as for the expression of Caspase-3 and vascular invasion (p = 0.43). In case of MMP2, a borderline correlation was found between the positive reaction of tumor cells with the presence of vascular invasion (p = 0.05). So the evaluation of MMP2 in thyroid PTC appears to be of some benefit to the prediction of tumor behavior while Caspase-3 as a marker of prediction seems to be of no use.     

CircRNA has_circ_0001806 promotes hepatocellular carcinoma progression via the miR-193a-5p/MMP16 pathway

Reportedly, circular RNAs (circRNAs) are crucial regulators in cancer progression. Nonetheless, the molecular mechanism of circRNAs in hepatocellular carcinoma (HCC) has not been fully clarified. Gene expression omnibus (GEO) database was employed to screen out the differentially expressed circRNAs in HCC. qRT-PCR and western blot were executed to detect circ_0001806 expression, miR-193a-5p expression, and MMP16 mRNA and protein expressions in HCC. The effect of circ_0001806 on HCC was analyzed by the CCK-8 method and Transwell experiment. RIP assay, pull-down experiment, and dual-luciferase reporter gene experiment were applied to validate the targeting relationships among circ_0001806, miR-193a-5p, and MMP16. Circ_0001806 was up-modulated in HCC tissues and cell lines. Knockdown of circ_0001806 impeded the multiplication, migration, and invasion of HCC cells. Circ_0001806 could up-regulate MMP16 expression through repressing miR-193a-5p, thereby facilitating the malignant biological behaviors of HCC. Circ_0001806 promoted HCC progression by regulating miR-193a-5p/MMP16 axis.     

RhoB enhances migration and MMP1 expression of prostate cancer DU145

Rho family protein regulates variety of cellular functions as cytoskeletal organization, cell proliferation and apoptosis. In the present study, we demonstrate that RhoB-overexpressed prostate cancer cells showed an enhanced cell motility and the administration of the GSK-3 inhibitors inhibited this increase in migration. Among the extracellular matrix and adhesion-related molecules, MMP1 RNA expression was increased in RhoB-overexpressed cells, administration of MMP inhibitor suppressed the collagen gel invasion in these cells. This is the first report evaluating RhoB function and the downstream signaling events in prostate cancer cell. Our results indicate that RhoB promotes cell motility and invasion in a metastatic prostate cancer cell.     

The invasive behavior of two human nasopharyngeal carcinoma (NPC) cell lines CNE-1, CNE-2Z in vitro]

Method of co-culture of tumor cells with precultured heart fragments (PHF) in vitro was used in studying the invasive behavior of human nasopharyngeal carcinoma (NPC) cell lines CNE-land CNE-2Z respectively. This improved method was based on the gyrotory shaker organ culture technique originally established by Mareel in order to show NPC cell invasiveness in a rather identical image. Histopathological sections revealed that the tumor cells might invade into the cell layer of fibroblasts surrounding the heart tissue. This would make the heart muscle cells degenerated, atrophied and necrotic and finally displaced by the fibrotic tissue. Data also pointed out that CNE-2Z cells showed more active invasive ability than that of the CNE-1 cells in the organ culture.     

白藜芦醇诱导鼻咽癌细胞CNE-2Z凋亡过程中caspase-6的活化

目的:探讨白藜芦醇(Res)诱导鼻咽癌细胞CNE-2Z凋亡过程中是否有caspase-6的活化。方法:用Western-blot分析caspase-6酶原的裂解,半定量RT-PCR检测caspase-6mRNA表达的改变,比色法测定caspase-6活性的变化。结果:用Res 0(对照)、25、50、100、200μmol/L处理细胞24小时,caspase-6酶原的表达随药物浓度的增加而减少,100 μmol/L时开始出现活性裂解片段P20(20 kD),200μmol/L时P20增加;caspase-6 mRNA的表达呈浓度依赖性的增加(P<0.01);caspase-6活性呈浓度和时间依赖性的升高(P<0.01)。结论:Res诱导CNE-2Z细胞凋亡过程中有caspase-6的活化。     

Silencing SATB1 influences cell invasion,migration, proliferation,and drug resistance in nasopharyngeal carcinoma

Special AT rich sequence binding protein 1 (SATB1) play an important role in many cancers, but the role of SATB1 in nasopharyngeal carcinoma (NPC) is still not full understand. Immunofluorescence staining showed that SATB1 was mainly localized in the nuclei in CNE-2 cell. After successful down-regulation of SABT1 in NPC cell line CNE-2 by shRNA, compared to parental CNE-2 and control shRNA group, the capacity of the proliferation, migration, invasion and drug resistance of CNE-2 cell was reduced, which indicated that SATB1 may be involved in NPC development and progression. SATB1 may be a promising therapeutic target for nasopharyngeal carcinoma.     

反义PKCα对CNE-2Z细胞周期的影响

目的:观察蛋白激酶Cα亚型(PKCα)的反义寡核苷酸对鼻咽癌CNE-2Z细胞生长、细胞周期和cyclinE表达的影响。方法:脂质体法转染反义PKCα,MTT法测细胞生长,流式细胞仪检测细胞周期,免疫细胞化学及点印迹法检测cyclinE表达。结果:①随着反义PKCα的浓度增加,细胞生长指数逐渐降低(P＜0.01)。②反义PKCα使细胞G₁期百分比增高(P＜0.05)。③反义PKCα使细胞cyclinE的表达减弱,扫描定量反义PKCα组为对照组的66.5％±18.4％(P＜0.05)。结论:反义PKCα可通过抑制cyclinE表达、阻滞细胞于G₁期从而抑制CNE-2Z的生长。     

G6PD upregulates Cyclin E1 and MMP9 to promote clear cell renal cell carcinoma progression

Background: Clear cell renal cell carcinoma (ccRCC) is a cell metabolic disease with high metastasis rate and poor prognosis. Our previous studies demonstrate that glucose-6-phosphate dehydrogenase (G6PD), the first and rate-limiting enzyme of the pentose phosphate pathway, is highly expressed in ccRCC and predicts poor outcomes of ccRCC patients. The aims of this study were to confirm the oncogenic role of G6PD in ccRCC and unravels novel mechanisms involving Cyclin E1 and MMP9 in G6PD-mediated ccRCC progression.Methods: Real-time RT-PCR, Western blot and immunohistochemistry were used to determine the expression patterns of G6PD, Cyclin E1 and MMP9 in ccRCC. TCGA dataset mining was used to identify Cyclin E1 and MMP9 correlations with G6PD expression, relationships between clinicopathological characteristics of ccRCC and the genes of interest, as well as the prognosis of ccRCC patients. The role of G6PD in ccRCC progression and the regulatory effect of G6PD on Cyclin E1 and MMP9 expression were investigated by using a series of cytological function assays in vitro. To verify this mechanism in vivo, xenografted mice models were established.Results: G6PD, Cyclin E1 and MMP9 were overexpressed and positively correlated in ccRCC, and they were associated with poor prognosis of ccRCC patients. Moreover, G6PD changed cell cycle dynamics, facilitated cells proliferation, promoted migration in vitro, and enhanced ccRCC development in vivo, more likely through enhancing Cyclin E1 and MMP9 expression.Conclusion: These findings present G6PD, Cyclin E1 and MMP9, which contribute to ccRCC progression, as novel biomarkers and potential therapeutic targets for ccRCC treatment.