Suppression of human lung cancer cell growth and migration by berbamine

The purpose of this study is to investigate the effects of berbamine (BER), a naturally occurring small-molecule compound from Traditional Chinese Medicine (TCM) Berberis amurensis, on the growth and migration of human lung cancer A549 cell line. This cell line is the non–small cell lung cancer (NSCLC) which constitutes 80% of lung cancer cases and remains an aggressive lung cancer associated with a poor patient survival. Our present results have shown that BER significantly suppressed the in vitro and ex vivo growth of A549 cells in dose- and time-dependent manners. Furthermore, Western blot analysis confirmed that BER dose-dependently down-regulated the expression of anti-apoptotic protein Bcl-2 and up-regulated the level of pro-apoptotic protein Bax, eventually leading the reduction of Bcl-2/Bax protein ratio in A549 cells. In addition, BER significantly inhibited the A549 cell migration at the low concentrations without restraining the cell growth. More importantly, BER significantly enhanced the anticancer activity of anticancer agents such as trichostatin A (the histone deacetylase inhibitor) and celecoxib (the inhibitor of cyclooxygenase-2) by strongly reducing the viability and/or the Bcl-2/Bax protein ratio in A549 cells. Our findings suggest that BER may have the wide therapeutic and/or adjuvant therapeutic application in the treatment of human NSCLC.     

Effects of matrine against the growth of human lung cancer and hepatoma cells as well as lung cancer cell migration

The purpose of this study is to investigate in vitro and ex vivo effects of matrine on the growth of human lung cancer and hepatoma cells and the cancer cell migration as well as the expressions of related proteins in the cancer cells. Matrine significantly inhibited the in vitro and ex vivo growth of human non-small cell lung cancer A549 and hepatoma SMMC-7721 cells. Matrine induced the apoptosis in A549 and SMMC-7721 cells. Western blot analysis indicated that matrine dose-dependently down-regulated the expression of anti-apoptotic protein Bcl-2 and up-regulated the level of pro-apoptotic protein bax, eventually leading the reduction of ratios of Bcl-2/Bax proteins in A549 and SMMC-7721 cells. Furthermore, matrine significantly suppressed the A549 cell migration without reducing the cell viability. In addition, matrine dramatically reduced the secretion of vascular endothelial growth factor A in A549 cells. More importantly, matrine markedly enhanced the anticancer activity of anticancer agent trichostatin A (the histone deacetylase inhibitor) by strongly reducing the viability and/or the ratio of Bcl-2/Bax protein in A549 cells. Our findings suggest that matrine may have the broad therapeutic and/or adjuvant therapeutic application in the treatment of human non-small cell lung cancer and hepatoma.     

Inhibitory effects of norcantharidin against human lung cancer cell growth and migration

The effects of norcantharidin (NCTD), an anticancer drug in China, on the growth and migration in human lung cancer cells were investigated by in vitro and ex vivo assays. NCTD significantly inhibited the in vitro and ex vivo growth of human non-small cell lung cancer A549 cells in dose- and time-dependent manners. Western blot analysis indicated that NCTD dose-dependently down-regulated the expression of anti-apoptotic protein Bcl-2 and up-regulated the level of pro-apoptotic protein Bax, eventually leading the reduction of ratio of Bcl-2/Bax proteins in A549 cells. Moreover, NCTD significantly suppressed the A549 cell migration in the case of without reducing the cell viability. More importantly, NCTD significantly enhanced the anticancer activity of anticancer agents such as trichostatin A (the histone deacetylase inhibitor), celecoxib (the inhibitor of cyclooxygenase-2) and lovastatin (the inhibitor of HMG-CoA reductase) by strongly reducing the viability and/or the ratio of Bcl-2/Bax protein in A549 cells. Our findings suggest that NCTD may have the wide therapeutic and/or adjuvant therapeutic application in the treatment of human lung cancer.     

Downregulation of miR-16 promotes growth and motility by targeting HDGF in non-small cell lung cancer cells

MicroRNAs play important roles in the development and progression of non-small cell lung cancer (NSCLC). miR-16 functions as a tumor-suppressor and is inhibited in several malignancies. Herein, we validated that miR-16 is downregulated in NSCLC tissue samples and cell lines. Ectopic expression of miR-16 significantly inhibited cell proliferation and colony formation. Moreover, miR-16 suppressed cell migration and invasion in NSCLC cells. Hepatoma-derived growth factor (HDGF) was found to be a direct target of miR-16 in NSCLC cell lines. Rescue experiments showed that the suppressive effect of miR-16 on cell proliferation, colony formation, migration, and invasion is partially mediated by inhibiting HDGF expression. This study indicates that miR-16 might be associated with NSCLC progression, and suggests an essential role for miR-16 in NSCLC.     

Novel hybrids of podophyllotoxin and formononetin inhibit the growth,migration and invasion of lung cancer cells

In this study, three hybrids of podophyllotoxin and formononetin were synthesized and evaluated for anticancer efficacy. Some of the derivatives exhibited potent cytotoxicity against a panel of human and mouse cancer cell lines, with IC₅₀ values in the low micromolar to submicromolar range. Evaluation against A549 lung tumor cell line identified that the IC₅₀ value of compound 10a was 0.753 μM, indicating that 10a was 2.568-fold more efficacious than parent podophyllotoxin. Mechanistic studies revealed that 10a induced A549 cell apoptosis mainly via caspase pathway, as well as disrupted the microtubule organization by occupying the colchicine binding site of the tubulin. Moreover, wound healing assay and transwell invasion assay indicated that 10a displayed potent inhibitory effects on invasion and migration in A549 cancer cells. In additiona, a decrease in vimentin immunostaining was also observed in A549 cells after treatment with 10a. Overall, hybrid 10a might be a promising candidate for the potential treatment of human lung carcinoma.     

Andrographolide could inhibit human colorectal carcinoma Lovo cells migration and invasion via down-regulation of MMP-7 expression

Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess multiple pharmacological activities. In our previous study, Andro had been shown to have potent anti-cancer activity against human colorectal carcinoma Lovo cells by inhibiting cell-cycle progression. To further investigate the mechanism for the anti-cancer properties of Andro, it was used to examine the effect on migration and invasion of Lovo cells. The results of wound-healing assay and in vitro transwell assay revealed that Andro inhibited dose-dependently the migration and invasion of Lovo cells under non-cytotoxic concentrations. Using zymographic assay and RT-PCR, the results revealed that Andro diminished the activity and the mRNA and protein levels of MMP-7, but not MMP-2 or MMP-9. The down-regulation of MMP-7 appeared to be via the inactivation of activator protein-1 (AP-1) since the treatment with Andro suppressed the nuclear protein level of AP-1, which was accompanied by a decrease in DNA-binding level of the factor. Taken together, these results indicated that Andro reduces the MMP-7-mediated cellular events in Lovo cells, and provided a new mechanism for its anti-cancer activity.     

Different effects of estradiol and various antiestrogens on TNF-alpha-induced changes of biochemical markers for growth and invasion of human breast cancer cells

In the field of estrogen therapy breast cancer risk is one of the most controversially discussed topic. Actually, the as yet largest placebo-controlled study, the Women's Health Initiative, rather showed a risk reduction, in contrast to observational studies. In the present study we have investigated the effect of estradiol on TNF-alpha-induced changes of various markers in human breast cancer cells and compare it with the effect of the antiestrogens tamoxifen and 2-methoxyestradiol. MCF-7 cells were used for the experiments, the incubation time was 96 h. TNF-alpha elicited a 3-4-fold increase of monocyte-attracting protein-1 (MCP-1) and interleukin-8 (IL-8) as compared to the control value, matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) were enhanced by 30 to 40%. E2 alone had no effect on MCP-1, slightly reduced the synthesis of MMP-9 and increased VEGF concentrations by about 20%. In combination with TNF, E2 induced a further stimulation of MCP-1, IL-8 and VEGF, whereby the MMP-9 synthesis was not changed. Tamoxifen and the endogenous estradiol metabolite 2-methoxyestradiol seem to be able to partly inhibit the action of TNF-alpha and estradiol. Our results suggest that estrogens may slightly increase tumor growth and spreading beyond the effect of chemokines such as TNF-alpha. However, the magnitude of this E2 effect seems to be marginal as compared to the effect of TNF-alpha alone. The risk of recurrence of breast cancer in patients taking hormone therapy after breast cancer may be slightly enhanced by estrogens, but seems mainly to be driven by the potency of still existing tumor cells to secrete chemokines which can stimulate tumor growth and spreading.     

circ_0000218通过靶向吸附miR-1182对宫颈癌HeLa细胞增殖、迁移和侵袭的影响

目的探讨环状RNA 0000218(circ_0000218)是否通过靶向吸附miR-1182从而影响宫颈癌HeLa细胞增殖、迁移和侵袭。方法采用实时荧光定量PCR(RT-qPCR)技术分析43例宫颈癌患者癌组织、癌旁组织中circ_0000218和miR-1182的表达水平。根据转染序列不同分为si-NC组、si-circ_0000218组、miR-NC组、miR-1182组、pcDNA组、pcDNAcirc_0000218组、si-circ_0000218+anti-miR-NC组、si-circ_0000218+anti-miR-1182组。运用细胞计数试剂盒(CCK-8)法、Transwell实验分析circ_0000218和miR-1182表达对HeLa细胞增殖、迁移和侵袭的影响。蛋白质印迹法检测Ki-67、基质金属蛋白酶2(MMP-2)和MMP9蛋白表达。双荧光素酶报告实验和RT-qPCR分析circ_0000218和miR-1182的靶向关系。癌旁组织与宫颈癌组织比较采用配对t检验,两组间比较采用独立样本t检验进行统计学分析。结果宫颈癌组织中circ_0000218表达量高于癌旁组织(4.17±0.32比1.00±0.05),而miR-1182表达量低于癌旁组织(0.33±0.03比1.00±0.05),差异具有统计学意义(P均<0.001)。与si-NC组比较,si-circ_0000218组HeLa细胞增殖活力(0.86±0.04比0.37±0.03)、迁移数量[(86.73±7.13)个比(38.52±3.19)个]和侵袭数量[(66.80±4.95)个比(26.58±2.55)个]以及Ki-67(0.57±0.05比0.18±0.02)、MMP-2(0.74±0.07比0.28±0.03)和MMP-9蛋白表达量(0.64±0.04比0.22±0.02)降低,差异有统计学意义(P均<0.001).与miR-NC组比较,miR-1182组HeLa细胞增殖活力(0.88±0.04比0.46±0.04)、迁移数量[(89.74±5.53)个比(46.63±3.79)个]和侵袭数量[(68.03±4.34)个比(34.63±3.37)个]以及Ki-67(0.59±0.04比0.24±0.02)、MMP-2(0.76±0.05比0.33±0.03)和MMP-9蛋白表达量(0.66±0.04比0.29±0.03)降低,差异有统计学意义(P均<0.001)。circ_0000218靶向负调控miR-1182表达。与si-circ_0000218+anti-miR-NC组比较,si-circ_0000218+anti-miR-1182组HeLa细胞增殖活力(0.35±0.03比0.76±0.04)、迁移数量[(35.58±3.11)个比(77.04±4.08)个]和侵袭数量[(25.44±2.29)个比(57.61±3.47)个]以及Ki-67(0.16±0.02比0.46±0.04)、MMP-2(0.26±0.02比0.65±0.04)和MMP-9蛋白表达量(0.20±0.02比0.57±0.04)升高,差异有统计学意义(P均<0.001)。结论circ_0000218通过靶向吸附miR-1182可促进宫颈癌HeLa细胞增殖、迁移和侵袭。     

ROCK signaling mediates the adoption of different modes of migration and invasion in human mammary epithelial tumor cells

For the invasive migration of tumor cells, at least two mechanisms are currently discussed: (1) the mesenchymal mode depending on extracellular proteolysis and (2) the proteolysis-independent amoeboid mode depending on the activity of the Rho kinase ROCK. The ability of tumor cells to switch between different modes of motility has been shown to limit the efficiency of agents aimed to reduce invasion. Here we show by combining 2D and 3D migration assays that human mammary tumor cells exhibited a strongly reduced migration velocity as compared to their normal counterparts indicating that high invasiveness is not necessarily correlated with high migratory capacity in 2D assays. This reduced migration was apparently due to significant differences in actin organization, decreased persistence of lamellipodia by 50% and increased cell substrate adhesion. These differences resulted from a 2.5-fold higher activity of ROCK and were mediated by its downstream effectors myosin light chain kinase and cofilin. Thus, inhibition of ROCK activity caused a marked increase in 2D migration efficiency by 40%, without, however, affecting 3D invasion. A massive reduction of invasion by 60% was achieved by the simultaneous inhibition of the ROCK-dependent amoeboid and the extracellular proteolysis-dependent mesenchymal mode. These results may point to a new efficient strategy for blocking tumor cell invasion in vivo.     

Expression of Nucleophosmin/NPM1 correlates with migration and invasiveness of colon cancer cells

Background

We aimed to examine the expression level of Nucleophosmin (NPM1) protein in colon cancer tissues and to investigate the potential role of NPM1 in the regulation of cell migration and invasiveness.

Methods

Immunohistochemical assay was performed to examine the expression pattern of NPM1 in 31 groups of colonic carcinoma samples, including colon tumors, adjacent normal tissues, and matched metastatic lymph nodes from the same patients. Small interfering RNA technique and exogenous expression of wild type NPM1 methods were used to further verify the function of NPM1.

Results

High-expression of NPM1 correlates with lymph node metastasis (P = 0.0003) and poor survival rate of human colon cancer patients (P = 0.017). SiRNA-mediated reduction of NPM1 was also shown to inhibit the migration and invasiveness of metastatic colon cancer HCT116 cell line. In addition, the exogenous expression of NPM1 in HT29 cells, a NPM1 low expression and low invasive colon cancer cell line, enhanced cell migration and invasiveness along with increased cell proliferation.

Conclusions

The current study uncovered the critical role of NPM1 in the regulation of colon cancer cells migration and invasion, and NPM1 may serve as a potential marker for the prognosis of colon cancer patients.     

Delphinidin inhibits BDNF-induced migration and invasion in SKOV3 ovarian cancer cells

Brain-derived neurotrophic factor (BDNF), the TrkB ligand, is associated with aggressive malignant behavior, including migration and invasion, in tumor cells and a poor prognosis in patients with various types of cancer. Delphinidin is a diphenylpropane-based polyphenolic ring structure-harboring compound, which exhibits a wide range of pharmacological activities, anti-tumor, anti-oxidant, anti-inflammatory, anti-angiogenic and anti-mutagenic activity. However, the possible role of delphinidin in the cancer migration and invasion is unclear. We investigated the suppressive effect of delphinidin on the cancer migration and invasion. Thus, we found that BDNF enhanced cancer migration and invasion in SKOV3 ovarian cancer cell. To exam the inhibitory role of delphinidin in SKOV3 ovarian cancer migration and invasion, we investigated the use of delphinidin as inhibitors of BDNF-induced motility and invasiveness in SKOV3 ovarian cancer cells in vitro. Here, we found that delphinidin prominently inhibited the BDNF-induced increase in cell migration and invasion of SKOV3 ovarian cancer cells. Furthermore, delphinidin remarkably inhibited BDNF-stimulated expression of MMP-2 and MMP-9. Also, delphinidin antagonized the phosphorylation of Akt and nuclear translocation of NF-κB permitted by the BDNF in SKOV3 ovarian cancer cells. Taken together, our findings provide new evidence that delphinidin suppressed the BDNF-induced ovarian cancer migration and invasion through decreasing of Akt activation.     

虫草素对肺癌细胞生长及迁移的影响

探讨虫草素对非小细胞肺癌细胞株H1781细胞凋亡及迁移的影响及作用机制。培养H1781细胞并分组,对照组用不含药物的培养基处理,虫草素组用含有10、20、30和40 μmol/L虫草素处理,处理24 h后测定细胞活力,通过显微镜观察细胞形态学,HE染色观察虫草素对细胞整体的影响,细胞免疫荧光技术检测细胞中MMP-9和DAPI核染色情况观察细胞凋亡,Western blotting检测凋亡等相关蛋白表达。与对照组相比,虫草素处理24 h后,H1781细胞系活力显著降低;细胞数量明显减少;HE染色观察发现随着虫草素浓度增加,细胞数量及细胞集团明显变少,免疫荧光技术检测发现药物处理后细胞凋亡明显促进;划痕实验发现虫草素明显降低细胞迁移能力;Western blotting实验中Bax、cleaved caspase-3蛋白表达明显上调,MMP-9、Bcl-2蛋白表达明显下调。虫草素对肺癌H1781细胞迁移有抑制作用、对凋亡有促进作用,推测其作用机制为上调促凋亡蛋白表达、下调抗凋亡蛋白表达。     

Migration-stimulating factor (MSF) is over-expressed in non-small cell lung cancer and promotes cell migration and invasion in A549 cells over-expressing MSF

Migration-stimulating factor (MSF), an oncofetal truncated isoform of fibronectin, is a potent stimulator of cell invasion. However, its distribution and motogenic role in non-small cell lung cancer (NSCLC) have never been identified. In this study, real-time PCR and immunohistochemical staining (IHC) were performed to detect MSF mRNA and protein levels in tumor tissues and matched adjacent tumor-free tissues. Furthermore, to examine the effect of MSF on invasiveness, MSF was upregulated in A549 cells. The invasiveness and viability of A549 cells were then determined using a transwell migration assay and the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assays, respectively. The expression level of MSF in NSCLC tissue was markedly higher than in matched adjacent tumor-free tissue. Additionally, the level of MSF protein expression in stage III and IV NSCLC samples was higher than in stage I and II NSCLC samples. More importantly, we also demonstrated that migration and invasion of A549 cells increased substantially after upregulating MSF, although proliferation remained unchanged. Meanwhile, we found no correlation between increasing motility and invasiveness of MSF-overexpressing cells and expression levels and activities of matrix metalloprotease MMP-2 and MMP-9. Our current study shows that MSF plays a role in migration and invasion of A549 cells and suggests that MSF may be a potential biomarker of NSCLC progression.     

Rhodanine-based PRL-3 inhibitors blocked the migration and invasion of metastatic cancer cells

PRL-3, phosphatase of regenerating liver-3, plays a role in cancer progression through its involvement in invasion, migration, metastasis, and angiogenesis. We synthesized rhodanine derivatives, CG-707 and BR-1, which inhibited PRL-3 enzymatic activity with IC₅₀ values of 0.8 μM and 1.1 μM, respectively. CG-707 and BR-1 strongly inhibited the migration and invasion of PRL-3 overexpressing colon cancer cells without exhibiting cytotoxicity. The specificity of the inhibitors on PRL-3 phosphatase activity was confirmed by the phosphorylation recovery of known PRL-3 substrates such as ezrin and cytokeratin 8. The compounds selectively inhibited PRL-3 in comparison with other phosphatases, and CG-707 regulated epithelial-to-mesenchymal transition (EMT) marker proteins. The results of the present study reveal that rhodanine is a specific PRL-3 inhibitor and a good lead molecule for obtaining a selective PRL-3 inhibitor.     

lncRNA MAGI2-AS3下调miR-31-5p抑制肺腺癌细胞的增殖、迁移、侵袭并促进凋亡

目的研究lncRNA MAGI2-AS3对肺癌A549细胞增殖、迁移、侵袭和凋亡的影响和潜在的分子机制。方法根据转染载体不同将A549细胞分为pcDNA3.1组(转染pcDNA3.1)、pcDNA3.1-MAGI2-AS3组(转染pcDNA3.1-MAGI2-AS3)、anti-miR-NC组(转染anti-miR-NC)、anti-miR-31-5p组(转染anti-miR-31-5p)、pcDNA3.1-MAGI2-AS3+miR-NC组(共转染pcDNA3.1-MAGI2-AS3和miR-NC)、pcDNA3.1-MAGI2-AS3+miR-31-5p组(共转染pcDNA3.1-MAGI2-AS3和miR-31-5p mimics)。实时荧光定量PCR(qRT-PCR)检测miR-31-5p和MAGI2-AS3 RNA的表达,四氮唑蓝(MTT)法测定A549细胞增殖活性,Transwell实验检测细胞迁移和侵袭能力,双荧光素酶报告系统验证MAGI2-AS3与miR-31-5p的调控关系,流式细胞术检测细胞凋亡与周期。两组间比较采用独立样本t检验进行分析;多组间比较采用单因素方差分析,组内多重比较采用SNK-q检验。结果与人正常肺细胞HBE相比,肺癌细胞A549中的MAGI2-AS3表达量(0.48±0.03比1.29±0.06)降低,miR-31-5p表达量(1.01±0.05比0.25±0.02)升高;与pcDNA3.1组比较,pcDNA3.1-MAGI2-AS3组A549细胞活力(0.48±0.04比0.77±0.06)、迁移[(81.33±2.87)个比(124.33±3.09)个]和侵袭[(32.00±2.83)个比(53.00±3.27)个]细胞数、S期细胞所占比例(23.01﹪±1.00﹪比32.95﹪±1.06﹪)均降低,凋亡率(19.95﹪±1.25﹪比7.23﹪±0.51﹪)、G0-G1期细胞所占比例(43.58﹪±2.15﹪比33.56﹪±1.23﹪)均升高;与anti-miR-NC组比较,anti-miR-31-5p组A549细胞活力(0.53±0.04比0.78±0.06)、迁移[(76.00±3.74)个比(108.33±2.87)个]和侵袭[(30.00±1.63)个比(42.33±2.05)个]细胞数、S期细胞所占比例(24.43﹪±1.13﹪比32.91﹪±1.08﹪)降低,凋亡率(18.21﹪±1.24﹪比7.29﹪±0.51﹪)、G0-G1期细胞所占比例(41.56﹪±2.19﹪比33.53﹪±1.27﹪)升高,差异有统计学意义(P均<0.05);双荧光素酶报告系统结果显示,MAGI2-AS3靶向负调控miR-31-5p的表达。与pcDNA3.1-MAGI2-AS3+miR-NC组比较,pcDNA3.1-MAGI2-AS3+miR-31-5p组A549细胞活力(0.68±0.06比0.50±0.04)、迁移[(91.00±1.63)个比(52.67±2.62)个]和侵袭[(62.67±2.49)个比(31.67±4.03个)]细胞数升高,凋亡率(10.59﹪±1.0﹪比21.11﹪±1.14﹪)降低,差异有统计学意义(P均<0.05)。结论lncRNA MAGI2-AS3通过靶向miR-31-5p抑制A549细胞的增殖、迁移和侵袭,促进细胞凋亡。lncRNA MAGI2-AS3是肺癌潜在分子治疗靶点。     

Profilin 2 promotes migration,invasion, and stemness of HT29 human colorectal cancer stem cells

We investigated the role of profilin 2 in the stemness, migration, and invasion of HT29 cancer stem cells (CSCs). Increased and decreased levels of profilin 2 significantly enhanced and suppressed the self-renewal, migration, and invasion ability of HT29 CSCs, respectively. Moreover, profilin 2 directly regulated the expression of stemness markers (CD133, SOX2, and β-catenin) and epithelial mesenchymal transition (EMT) markers (E-cadherin and snail). CD133 and β-catenin were up-regulated by overexpression of profilin 2 and down-regulated by depletion of profilin 2. SOX2 was decreased by profilin 2 depletion. E-cadherin was not influenced by profilin 2- overexpression but increased by profilin 2- knockdown. The expression of snail was suppressed by profilin 2- knockdown. We speculated that stemness and the EMT are closely linked through profilin 2-related pathways. Therefore, this study indicates that profilin 2 affects the metastatic potential and stemness of colorectal CSCs by regulating EMT- and stemness-related proteins.     

Arachidonic acid promotes migration and invasion through a PI3K/Akt-dependent pathway in MDA-MB-231 breast cancer cells

Arachidonic acid (AA) is a common dietary n−6 cis polyunsaturated fatty acid that under physiological conditions is present in an esterified form in cell membrane phospholipids, however it might be present in the extracellular microenvironment. AA and its metabolites mediate FAK activation, adhesion and migration in MDA-MB-231 breast cancer cells. However, it remains to be investigated whether AA promotes invasion and the signal transduction pathways involved in migration and invasion. Here, we demonstrate that AA induces Akt2 activation and invasion in MDA-MB-231 cells. Akt2 activation requires the activity of Src, EGFR, and PIK3, whereas migration and invasion require Akt, PI3K, EGFR and metalloproteinases activity. Moreover, AA also induces NFκB-DNA binding activity through a PI3K and Akt-dependent pathway. Our findings demonstrate, for the first time, that Akt/PI3K and EGFR pathways mediate migration and invasion induced by AA in MDA-MB-231 breast cancer cells.     

Claudin-7 inhibits human lung cancer cell migration and invasion through ERK/MAPK signaling pathway

Tight junctions are the most apical component of the junctional complex critical for epithelial cell barrier and polarity functions. Although its disruption is well documented during cancer progression such as epithelial–mesenchymal transition, molecular mechanisms by which tight junction integral membrane protein claudins affect this process remain largely unknown. In this report, we found that claudin-7 was normally expressed in bronchial epithelial cells of human lungs but was either downregulated or disrupted in its distribution pattern in lung cancer. To investigate the function of claudin-7 in lung cancer cells, we transfected claudin-7 cDNA into NCI-H1299, a human lung carcinoma cell line that has no detectable claudin-7 expression. We found that claudin-7 expressing cells showed a reduced response to hepatocyte growth factor (HGF) treatment, were less motile, and formed fewer foot processes than the control cells did. In addition, cells transfected with claudin-7 dramatically decreased their invasive ability after HGF treatment. These effects were mediated through the MAPK signaling pathway since the phosphorylation level of ERK1/2 was significantly lower in claudin-7 transfected cells than in control cells. PD98059, a selective inhibitor of ERK/MAPK pathway, was able to block the motile effect. Claudin-7 formed stable complexes with claudin-1 and -3 and was able to recruit them to the cell-cell junction area in claudin-7 transfected cells. When control and claudin-7 transfected cells were inoculated into nude mice, claudin-7 expressing cells produced smaller tumors than the control cells. Taken together, our study demonstrates that claudin-7 inhibits cell migration and invasion through ERK/MAPK signaling pathway in response to growth factor stimulation in human lung cancer cells.     

miR-363-3p靶向 TWIST1调控上皮间质转化抑制非小细胞肺癌A549细胞迁移和侵袭

目的探讨miR-363-3p靶向TWIST1调控非小细胞肺癌A549细胞迁移和侵袭的分子机制。 方法将体外培养的A549细胞分为空白对照组（细胞未做任何处理）;模拟物对照组（转染miR-363-3p模拟物阴性对照）;模拟物组（转染miR-363-3p模拟物）;后期实验另设：模拟物+ pcDNA组（转染miR-363-3p模拟物和pcDNA3.1空载体质粒）;模拟物+ TWIST1组（转染miR-363-3p模拟物和pcDNA3.1-TWIST1过表达质粒）。采用RT-PCR检测细胞中miR-363-3p的表达,Western blot检测细胞中TWIST1蛋白和上皮间质转化（EMT）相关蛋白Vimentin、E-cadherin的表达,Transwell小室实验检测细胞的迁移和侵袭。双荧光素酶报告基因实验检测miR-363-3p和TWIST1的靶向关系。两组间比较采用独立样本t检验,多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。 结果与空白对照组和模拟物对照组比较,模拟物组A549细胞中miR-363-3p表达水平（1.00±0.08、0.97±0.05比3.82±0.45）升高,TWIST1 mRNA （1.00±0.06、0.98±0.06比0.39±0.02）和蛋白的表达水平（0.81±0.05、0.78±0.06比0.42±0.02）降低,差异具有统计学意义（P均< 0.05）。与空白对照组和模拟物对照组比较,模拟物组A549细胞Vimentin蛋白表达水平（0.58±0.04、0.55±0.05比0.36±0.03）降低,E-cadherin蛋白的表达水平（0.22±0.02、0.25±0.03比0.47±0.03）增高,迁移细胞数（85.75±5.45、83.52±6.85比53.05±4.50）和侵袭细胞数（128.26±6.15、125.95±8.05比71.64±5.75）降低,差异有统计学意义（P均< 0.05）。与模拟物对照组比较,模拟物组细胞中Vimentin蛋白的表达水平（0.55±0.05比0.36±0.03）降低,而E-cadherin蛋白表达水平（0.25±0.03比0.47±0.03）升高,迁移细胞数（83.52±6.85比53.05±4.50）和侵袭细胞数（125.95±8.05比71.64±5.75）均减少（P均< 0.05）。TWIST1是miR-363-3p潜在的靶基因。与模拟物+ pcDNA3.1组比较,模拟物+ TWIST1组A549细胞中Vimentin蛋白的表达水平（0.32±0.02比0.42±0.03）升高,而E-cadherin蛋白表达水平（0.56±0.04比0.38±0.03）降低,A549细胞的迁移数（49.45±4.22比67.52±5.05）和侵袭数（72.45±5.73比108.35±6.56）增加（P均< 0.05）。 结论miR-363-3p可通过靶向TWIST1调控EMT抑制A549细胞迁移和侵袭。     

二氯乙酸对人肾癌细胞株A498侵袭及迁移的抑制作用研究

目的探讨二氯乙酸(DCA)对人肾癌细胞株A498侵袭及迁移的影响,并分析其机制。方法取人肾癌细胞株A498培养,分为阴性对照(等容积无菌生理盐水)组、DCA低、中、高(5、10、20 mmol/L)剂量组,培养48 h。Transwell小室实验检测侵袭能力,细胞划痕实验检测迁移能力,实时荧光定量聚合酶链反应(qRT-PCR)检测c-Jun氨基末端激酶(JNK)、钙粘附蛋白E(E-cadherin)、N-钙粘蛋白(N-cadherin)基因表达,Western blot检测蛋白表达及磷酸化JNK(p-JNK)。多组间比较采用单因素方差分析,组间两两比较采用SNK-q检验。结果与DCA高剂量组相比,DCA中、低剂量组、阴性对照组侵袭活性[(75.33±10.09)比(167.89±47.12)、(372.74±50.06)、(530.26±81.22)个/视野]、E-cadherin mRNA相对表达量(1.52±0.12比1.35±0.11、1.02±0.11、0.85±0.12)、E-cadherin蛋白相对表达量(1.32±0.19比0.89±0.14、0.37±0.08、0.13±0.03)均降低(P均<0.001);而迁移率[(10.05±2.31)﹪比(23.95±4.20)﹪、(36.26±5.01)﹪、(53.59±6.71)﹪]、N-cadherin mRNA蛋白相对表达量(0.42±0.06比0.58±0.07、0.80±0.10、0.95±0.11),N-cadherin蛋白相对表达量(0.15±0.03比0.25±0.04、0.74±0.07、0.95±0.11)、p-JNK水平(0.14±0.03比0.29±0.05、0.68±0.07、0.95±0.10)均升高(P均<0.001)。不同剂量和阴性对照组的JNK mRNA与蛋白表达差异均无统计学意义(P>0.05)。结论DCA可抑制人肾癌细胞株A498侵袭及迁移,且一定范围内呈剂量依赖性,推测与调控侵袭及迁移相关基因与蛋白表达有关。