The alpha 3 beta 1 integrin is associated with mammary carcinoma cell metastasis, invasion, and gelatinase B (MMP-9) activity

The alpha 3 beta 1 integrin is elevated in several types of metastatic tumor and has been associated with increased migration and invasion. Our analysis of a series of mammary carcinomas of different histotypes and their corresponding metastases demonstrated significantly increased expression of alpha 3 beta 1 in the tumor metastases. We therefore studied alpha 3 beta 1 expression of several human breast carcinoma cell lines and its association with the invasive phenotype. The MDA-MB-231 cell line expressed high levels of the beta1, alpha 2, alpha 3, alpha 5, and alpha 6 integrin subunits along with moderate levels of the alpha v beta 3 integrin. This line was highly migratory and the most invasive using a chemo-invasion assay. In contrast, the other lines tested, MDA-MB-145, MCF-7, and SK-BR-3, showed lower migratory and invasive activity and reduced alpha 3 integrin subunit expression. Metalloproteases capable of degrading collagen IV are necessary for the invasive process. RT-PCR showed that MDA-MB-231 cells expressed MMP-9, but not MMP-2, gelatinase/collagenase IV. Gelatin zymography demonstrated that invading MDA-MB-231 cells released high levels of MMP-9 gelatinase activity. A direct role for this gelatinase in MDA-MB-231 cell invasion was confirmed by inhibition of invasion using the metalloprotease inhibitor Batimastat. Treatment of MDA-MB-231 cells with a function-blocking anti-alpha 3 antibody strongly inhibited migration and invasion. This correlated with a marked reduction in MMP-9 activity produced by MDA-MB-231 cells, suggesting a role for alpha 3 beta 1 ligand binding in cell signaling and regulation of extracellular matrix degradation.     

Phorbol ester TPA modulates chemoresistance in the drug sensitive breast cancer cell line MCF-7 by inducing expression of drug efflux transporter ABCG2

Recent studies have indicated a link between levels of cyclooxygenase-2 (COX-2) and development of the multidrug resistance (MDR) phenotype. The ATP-binding cassette sub-family G member 2 (ABCG2) is a major MDR-related transporter protein that is frequently overexpressed in cancer patients. In this study, we aimed to evaluate any positive correlation between COX-2 and ABCG2 gene expression using the COX-2 inducer 12-O-tetradecanoylphorbol-13-acetate (TPA) in human breast cancer cell lines. ABCG2 mRNA and protein expression was studied using real-time RT-PCR and flow cytometry, respectively. A significant increase of COX-2 mRNA expression (up to 11-fold by 4 h) was induced by TPA in MDA-MB-231 cells, this induction effect being lower in MCF-7 cells. TPA caused a considerable increase up to 9-fold in ABCG2 mRNA expression in parental MCF-7 cells, while it caused a small enhancement in ABCG2 expression up to 67 % by 4 h followed by a time-dependent decrease in ABCG2 mRNA expression in MDA-MB-231 cells. TPA treatment resulted in a slight increase of ABCG2 protein expression in MCF-7 cells, while a time-dependent decrease in ABCG2 protein expression was occurred in MDA-MB-231 cells. In conclusion, based on the observed effects of TPA in MDA-Mb-231 cells, it is proposed that TPA up-regulates ABCG2 expression in the drug sensitive MCF-7 breast cancer cell line through COX-2 unrelated pathways.     

上皮性肿瘤细胞中Snail与E-cadherin的表达及其与体外侵袭性的关系

目的 研究上皮性肿瘤细胞Snail与E-cadherin的表达以及与瘤细胞表型、转移潜能的关系。方法 采用Northern blot、共聚焦激光显微镜研究6株不同组织来源、不同分化程度及转移潜能的上皮性肿瘤细胞、1株正常上皮细胞、1株成纤维细胞中Snail与E-cadherin mRNA和蛋白表达与定位;Boyden小室体外侵袭实验反映细胞的转移潜能。结果 在分化程度较高的癌细胞和对照正常上皮细胞中,E-cadherin mRNA与蛋白表达较强,而Snail表达缺如;在分化程度低、转移潜能高的癌细胞和对照成纤维细胞中,E-cadherin、Snail mRNA及蛋白表达与上述情况相反。E-cadherin多定位于细胞胞浆和胞膜,Snail主要定位于胞核和胞浆。结论 在上皮性肿瘤细胞中E-cadherin和Snail在mRNA与蛋白表达水平上存在逆反关系,且与细胞分化、转移潜能相关。     

A molecular role for lysyl oxidase in breast cancer invasion

We identified previously an up-regulation in lysyl oxidase (LOX) expression,an extracellular matrix remodeling enzyme, in a highly invasive/metastatic human breast cancer cell line, MDA-MB-231, compared with MCF-7, a poorly invasive/nonmetastatic breast cancer cell line. In this study, we demonstrate that the mRNA expression of LOX and other LOX family members [lysyl oxidase-like (LOXL), LOXL2, LOXL3, and LOXL4] was observed only in breast cancer cells with a highly invasive/metastatic phenotype but not in poorly invasive/nonmetastatic breast cancer cells. LOX and LOXL2 showed the strongest association with invasive potential in both highly invasive/metastatic breast cancer cell lines tested (MDA-MB-231 and Hs578T). To determine whether LOX is directly involved in breast cancer invasion, LOX antisense oligonucleotides were transfected into MDA-MB-231 and Hs578T cells, and found to inhibit invasion through a collagen IV/laminin/gelatin matrix in vitro compared with LOX sense oligonucleotide-treated and untreated controls. In addition, treatment of MDA-MB-231 and Hs578T cells with beta-aminopropionitrile (an irreversible inhibitor of LOX enzymatic activity) decreased invasive activity. Conversely, MCF-7 cells transfected with the murine LOX gene demonstrated a 2-fold increase in invasiveness that was reversible by the addition of beta-aminopropionitrile in a dose-dependent manner. In addition, endogenous LOX mRNA expression was induced when MCF-7 cells were cultured in the presence of fibroblast conditioned medium or conditioned matrix, suggesting a role for stromal fibroblasts in LOX regulation in breast cancer cells. Moreover, the correlation of LOX up-regulation and invasive/metastatic potential was additionally demonstrated in rat prostatic tumor cell lines, and human cutaneous and uveal melanoma cell lines. These results provide substantial new evidence that LOX is involved in cancer cell invasion.     

Histone deacetylases 1, 6 and 8 are critical for invasion in breast cancer

Histone deacetylases (HDACs) are associated with the development and progression of cancer, but it is not known which of the HDAC isoforms play important roles in breast cancer metastasis. This study identified the specific HDAC isoforms that are necessary for invasion and/or migration in human breast cancer cell lines. MDA-MB-231 cells were significantly more invasive and expressed higher levels of matrix metalloproteinase-9 (MMP-9) compared to MCF-7 cells. We compared the expression of HDAC isoforms between MCF-7 and MDA-MB-231 cells and found greater expression of HDAC4, 6 and 8 in MDA-MB-231 cells by RT-PCR and Western blot analyses. In addition, apicidin, a histone deacetylase inhibitor, was shown to attenuate the invasion, migration and MMP-9 expression in MDA-MB-231 cells. Using specific siRNAs directed against HDAC1, 4, 6 and 8, we show that inhibition of HDAC1, 6 and 8, but not HDAC4, are responsible for invasion and MMP-9 expression in MDA-MB-231 cells. We analyzed the invasiveness of MCF-7 cells overexpressing HDAC1, 4, 6 or 8 and found that overexpression of HDAC1, 6 or 8 increased invasion and MMP-9 expression. By developing HDAC isoforms as potential biomarkers for breast cancer metastasis, the present study can be extended to developing therapies for breast cancer invasion.     

High Expression of the RECK Gene in Breast Cancer Cells is Related to Low Invasive Capacity

OBJECTIVE To investigate the expression of the RECK gene in human breast (cancer) cell lines, and to determine the relationship between RECK gene expression and the invasive capacity of the breast cancer cell lines. METHODS The invasive capacity of breast (cancer) cell lines including HBL-100, MCF-7 and MDA-MB-435S were determined by the Tran-swell method. The protein expression levels of RECK, MMP-2 and MMP-9 genes in these three cell lines were measured by immunocytochemical methods. The expressions of the RECK gene and protein level were measured by RT-PCR and Western blots in the cell lines respectively. RESULTS The order of the invasive capacity of the breast (cancer) cell lines was MDA-MB-435S, being the highest, and HBL-100, being the lowest. The invasive capacity difference between any two groups among the three groups was significant (P<0.01). The protein expression level of the RECK gene in the HBL-100 cell line was highest, and no expression was detected in MDA-MB-435S cells. Moreover, the expression of the RECK gene was negatively correlated with the expression of the MMP-2 and MMP-9 genes. The mRNA level of the RECK gene in HBL-100 cells was the highest, but no expression was found in the MDA-MB-435S cells (P<0.001). CONCLUSION There was a significant negative correlation between the expression level of the RECK gene and invasive capacity in vitro, and the RECK gene expression showed an inverse proportion to that of the MMP-2, MMP-9 genes.     

Effect of phorbol ester and cytokines on matrix metalloproteinase and tissue inhibitor of metalloproteinase expression in tumor and normal cell lines.

The effect of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the cytokines interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) on matrix metalloproteinases (MMP) and metalloproteinase inhibitors was studied in a variety of human cell lines. Expression of the mammalian collagenase (MMP-1), 72-kD gelatinase/type IV collagenase (MMP-2), stromelysin (MMP-3), 92-kD gelatinase/type IV collagenase (MMP-9), and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) was assessed by zymography and Northern blot analysis. MMP-2 and TIMP-2 activities were refractory to TPA, IL-1 and TNF-alpha treatment in most of the cell lines. In contrast, MMP-3, MMP-9 and TIMP-1 activities were markedly stimulated by TPA in most of the tumor cell lines and human umbilical vein endothelial cells (HUVEC), whereas the fibroblast lines were minimally stimulated or unresponsive to TPA. The MMP-3, MMP-9 and TIMP-1 stimulation in response to IL-1 and TNF-alpha treatment was detected in some of the tumor cell lines and HUVEC. The increase in activity was less marked than in TPA. A breast carcinoma cell line, MDA-MB-231, which did not express MMP-2, had high expression of MMP-3 and MMP-9 which were unaffected by TPA and cytokine treatment. Northern blot analysis of MMP and TIMP mRNA expression reflected the zymogram findings for most of the cell lines. TPA-mediated stimulation of MMP-1 was similar to that of MMP-3 and MMP-9. Exceptions were the fibroblast cell lines which showed either a much more marked mRNA response of MMP-9 to TPA than observed at protein level, or a high constitutive MMP-9 mRNA when MMP-9 activity was not detectable by zymography. TPA-mediated stimulation of MMP-9 and TIMP-1 activity was blocked by staurosporine, an inhibitor of protein kinase C (PKC). A non-PKC-activating phorbol ester, 4 alpha-phorbol-12,13-didecanoate, did not stimulate MMP-9 and TIMP-1 activity. TPA treatment caused the increased expression of c-fos containing AP-1-specific binding activity in selected tumor cell lines. This activity was maximal at 6 h. An association was observed between AP-1 binding activity and increased expression of MMP-1, MMP-3 and MMP-9, which possess TPA-responsive elements (TRE). TPA-sensitive MMPs and TIMP-1 were variably stimulated by biologically relevant cytokines, such as IL-1 and TNF-alpha.(ABSTRACT TRUNCATED AT 400 WORDS)     

过表达lncRNA LINC00886靶向调控miR-451a影响乳腺癌MDA-MB-231细胞生物学行为

目的:探究过表达长链非编码RNA（lncRNA）LINC00886通过调控微小RNA-451a（miR-451a）对乳腺癌（BC）MDA-MB-231细胞生物学行为的影响。方法:荧光定量PCR（qRT-PCR）检测人乳腺上皮细胞系（MCF-12A）和4种BC细胞系（HCC1937、SUM159、SK-BR-3和MDA-MB-231）中lncRNA LINC00886、miR-451a表达。将MDA-MB-231细胞分为对照组、LINC00886-NC组、LINC00886组、LINC00886+miR-NC组、LINC00886+miR-451a组。qRT-PCR法检测MDA-MB-231细胞中lncRNA LINC00886、miR-451a表达水平;克隆形成实验和EdU染色检测MDA-MB-231细胞增殖能力;流式细胞术、Transwell小室实验、划痕愈合实验分别用来检测MDA-MB-231细胞凋亡、侵袭、迁移;蛋白印迹法检测MDA-MB-231细胞中增殖细胞核抗原（PCNA）、Bcl-2相关X蛋白（Bax）、B淋巴细胞瘤-2（Bcl-2）、活化的半胱氨酸天冬氨酸蛋白酶3（Cleaved caspase-3）、基质金属蛋白酶（MMP）-2、MMP-9蛋白表达;双荧光素酶报告基因检测lncRNA LINC00886与miR-451a的靶向关系。结果:与人乳腺上皮细胞MCF-12A相比,BC细胞系中lncRNA LINC00886表达水平降低,miR-451a表达水平升高（P＜0.05）;过表达lncRNA LINC00886可升高MDA-MB-231细胞凋亡率、Bax和Cleaved caspase-3蛋白表达,降低miR-451a表达、集落形成数、EdU阳性细胞百分比、侵袭细胞数、迁移率以及PCNA、Bcl-2、MMP-2、MMP-9蛋白表达（P＜0.05）;上调miR-451a表达可减弱lncRNA LINC00886过表达对MDA-MB-231细胞的影响（P＜0.05）;lncRNA LINC00886可靶向调控miR-451a表达。结论:过表达lncRNA LINC00886可通过靶向抑制miR-451a表达促进MDA-MB-231细胞凋亡并抑制MDA-MB-231细胞增殖、侵袭和迁移。     

Positive regulation of migration and invasion by vasodilator-stimulated phosphoprotein via Rac1 pathway in human breast cancer cells

This study aimed to investigate the role of the cytoskeleton-associated protein vasodilator-stimulated phosphoprotein (VASP) on the migration and invasion of human breast cancer cells and its relationship to Rac1 which is a member of the Rho family and has been found to be implicated in tumorigenesis, invasion and metastasis. We detected the mRNA and protein expression levels of VASP and Rac1 of the non-invasive breast cancer cell line MCF-7 as well as the invasive cell line MDA-MB-231 by RT-PCR and Western blotting. GST pull-down assay was used to examine the activitiy of Rac1. Accordingly, the cell invasive migration ability was analyzed in a wound-healing assay (2D) and transwell assays (3D migration and invasion). We then used VASP-siRNA to inhibit the expression of VASP in breast cancer cells in order to study the relationship between the VASP expression level and the invasive migration ability of breast cancer cells. Rac1-siRNA was used to decrease the expression of Rac1, and observe its effect on the VASP expression level together with the migration and invasion ability of MCF-7 and MDA-MB-231 cells. Our results revealed that the invasive breast cancer cell line MDA-MB-231 showed a higher Rac1 activity and VASP expression level compared with the non-invasive MCF-7. Inhibition of Rac1 or VASP by siRNA, respectively, decreased the migration and invasion ability of breast cancer cells and the transfection of Rac1 siRNA-mediated reduction of VASP expression at mRNA and protein levels. Collectively, our data showed that the higher expression level of VASP contributed to a higher invasive migration capacity of human breast cancer cells which was controlled by the Rac1 pathway.     

人乳腺癌细胞株WAF1／GIP1／p21基因的表达及其与细胞生物学特性的关系

OBJECTIVE: To study the WAF1/CIP1 gene DNA status, mRNA and protein expressions in human breast cancer cell line and its significance. METHODS: Using cell culture, molecular biological techniques such as Southern blot, Northern blot and immunocytochemical methods, the WAF1/CIP1 gene DNA status, mRNA and protein expression levels in MCF-7 expressing wild type p53(wtp53) and MDA-MB-231 expressing mutant p53 (mtp53) human breast cancer cell lines were detected respectively. The p53 and mdm-2 protein expression levels and cytobiological features of the 2 cell lines were compared and correlated to their WAF1/CIP1 gene expression levels. RESULTS: (1) There was no difference in WAF1/CIP1 gene DNA status in the two breast cancer cell lines. Neither of them showed gene amplificatian or deletion. However, the WAF1/CIP1 mRNA and p21WAF1/CIP1 protein expression levels of MCF-7 cells were higer than those of MDA-MB-231 cells (P < 0.05). (2) The character and cellular distribution of p53 protein in the two cell lines were clearly different. The expression level of mdm-2 proteion was significantly higher in MCE-7 than in MDA-MB-231 cells (P < 0.05). (3) Compered to the other breast cancer cell line, MCF-7 cells were better differentiated, grew more slowly and adhered more closely with each other. CONCLUSION: The WAF1/CIP1 gene expression at mRNA and protein levels is associated with p53 phenotype and some cytobiological features of human breast cancer.     

Decrease of breast cancer cell invasiveness by sodium phenylacetate (NaPa) is associated with an increased expression of adhesive molecules

Sodium phenylacetate (NaPa), a non-toxic phenylalanine metabolite, has been shown to induce in vivo and in vitro cytostatic and antiproliferative effects on various cell types. In this work, we analysed the effect of NaPa on the invasiveness of breast cancer cell (MDA-MB-231, MCF-7 and MCF-7 ras). Using the highly invasive breast cancer cell line MDA-MB-231, we demonstrated that an 18-hour incubation with NaPa strongly inhibits the cell invasiveness through Matrigel (86% inhibition at 20 mM of NaPa). As cell invasiveness is greatly influenced by the expression of urokinase (u-PA) and its cell surface receptor (u-PAR) as well as the secretion of matrix metalloproteinases (MMP), we tested the effect of NaPa on these parameters. An 18-hour incubation with NaPa did not modify u-PA expression, either on MDA-MB-231 or on MCF-7 and MCF-7 ras cell lines, and induced a small u-PA decrease after 3 days of treatment of MDA-MB-321 with NaPa. In contrast, an 18 h incubation of MDA-MB-231 increased the expression of u-PAR and the secretion of MMP-9. As u-PAR is a ligand for vitronectin, a composant of the extracellular matrix, these data could explain the increased adhesion of MDA-MB-231 to vitronectin, while cell adhesivity of MCF-7 and MCF-7 ras was unmodified by NaPa treatment. NaPa induced also an increased expression of both Lymphocyte Function-Associated-1 (LFA-1) and Intercellular Adhesion Molecule-1 (ICAM-1), which was obvious from 18 hour incubation with NaPa for the MDA-MB-231 cells, but was delayed (3 days) for MCF-7 and MCF-7 ras. Only neutralizing antibodies against LFA-1 reversed the decreased invasiveness of NaPa-treated cells. Therefore we can conclude that the strong inhibition of MDA-MB-231 invasiveness is not due to a decrease in proteases involved in cell migration (u-PA and MMP) but could be related both to the modification of cell structure and an increased expression of adhesion molecules such as u-PAR and LFA-1.     

PPAPDC1A在体内外对乳腺癌细胞增殖、侵袭和转移能力的影响

目的:通过构建稳定过表达和干扰PPAPDC1A的乳腺癌细胞株,探讨PPAPDC1A对乳腺癌细胞增殖、侵袭和转移能力的影响。方法:利用CCK-8和Transwell实验检测PPAPDC1A稳定过表达和干扰后对乳腺癌细胞体外增殖和侵袭能力的影响。采用裸鼠皮下成瘤实验检测PPAPDC1A对乳腺癌细胞体内增殖和裸鼠致瘤性的作用。利用免疫组织化学染色法检测各组肿瘤组织中Ki-67的表达。通过裸鼠尾静脉注射实验检测PPAPDC1A对乳腺癌MCF-7细胞和MDA-MB-231细胞体内转移能力的影响。结果:成功建立稳定过表达PPAPDC1A的乳腺癌MCF-7细胞株和稳定干扰PPAPDC1A的乳腺癌MDA-MB-231细胞株;CCK-8和Transwell实验结果显示,与MCF-7和MCF-7-Vector细胞株相比,MCF-7-PPAPDC1A细胞株的生长速度显著增快,穿膜细胞数量多（P＜0.05）;与此相反,MDA-MB-231-shPPAPDC1A组细胞的生长速度和穿膜细胞数明显少于MDA-MB-231-shNC和MDA-MB-231 细胞株（P＜0.05）。动物实验结果显示,与MCF-7-Vector组相比,MCF-7-PPAPDC1A组的肿瘤生长速度较快,肿瘤的体积较大,Ki-67的阳性率高,肺转移灶的数目增多（P＜0.05）;与此相反,与MDA-MB-231-shNC组相比MDA-MB-231-shPPAPDC1A组的肿瘤生长速度较慢,肿瘤的体积较小,Ki-67的阳性率低,肺转移灶的数目减少（P＜0.05）。结论:PPAPDC1A对乳腺癌细胞的增殖、侵袭和转移有促进作用。