Epidermal growth factor and amphiregulin up-regulate matrix metalloproteinase-9 (MMP-9) in human breast cancer cells

The EGF family of proteins encompasses several polypeptides such as epidermal growth factor (EGF), transforming growth factor alpha (TGFα), amphiregulin (AR) and heregulin (HRG-β1). These polypeptides regulate proliferation in breast cancer cells through interaction with membrane receptors. It has been previously shown that high EGF receptor number correlates with aggressive behavior and increased metastasis in human breast cancer. In the present study, we investigated the association between EGF and EGF-like ligand-induced DNA synthesis and secretion of MMP-9 and MMP-2 in metastatic SKBR-3 cells and non-metastatic MCF-7 breast cancer cells. Exposure of SKBR-3 cells to EGF or Ar induces expression of MMP-9 but has no effect on MMP-2 secretion. In contrast to EGF and AR, HRG had no effect on gelatinase induction. None of the EGF polypeptides had any effect on gelatinase induction in MCF-7 non-metastatic breast cancer cells. While a relatively specific inhibitor of EGF receptor tyrosine kinase, PD 153035, inhibited EGF-, AR- and HRG-induced cell proliferation, it had no effect on MMP-9 induced by EGF and AR. Experimental evidence suggest that signalling mechanisms for cell proliferation and MMP-9 induction are mediated by different pathways down-stream of EGF receptor autophosphorylation or that low levels of EGF- induced signal that escape inhibition are sufficient to induce MMP-9 but unable to support cell proliferation. In addition, our results suggest that EGF and AR may modulate invasion of metastatic breast cancer cells by increasing the expression of MMPs. Int. J. Cancer 70:722–726, 1997. © 1997 Wiley-Liss, Inc.     

Fas抗原在乳腺癌组织中的表达

目的检测乳腺癌组织中Fas基因表达并探讨其临床意义.方法用流式细胞技术对36例乳腺癌组织和癌旁乳腺组织中Fas抗原的表达进行检测.同时检测癌组织中细胞凋亡率.结果乳腺癌组织(阳性细胞数941±846)和癌旁乳腺组织(阳性细胞数560±551)中均有Fas抗原表达,其表达水平有统计学差异(P＜0.05),细胞凋亡率有显著差异(P＜0.05).有腋淋巴结转移的乳腺癌Fas抗原表达(阳性细胞数181±59)明显低于无腋淋巴结转移者(阳性细胞数1320±801),(P＜0.05);乳腺癌组织中雌激素受体的表达与Fas抗原表达及细胞阳性细胞数凋亡率均无关.乳腺癌组织中Fas抗原表达与癌细胞凋亡率有显著相关性(r=0.603,P＜0.01).结论乳腺癌和癌旁乳腺组织均可发生凋亡,Fas基因参与乳腺癌的发生和发展.采用特异性改变凋亡阈限的治疗方案,可能有利于改变乳腺癌的自然进程.     

Increased Fas expression reduces the metastatic potential of human osteosarcoma cells.

PURPOSE: The process of metastasis requires the single tumor cell that seeds the metastatic clone to complete a complex series of steps. Identifying factors responsible for these steps is essential in developing and improving targeted therapy for metastasis. Resistance to receptor-mediated cell death, such as the Fas/Fas ligand pathway, is one mechanism commonly exploited by metastatic cell populations. EXPERIMENTAL DESIGN AND RESULTS: LM7, a subline of the SAOS human osteosarcoma cell line with low Fas expression, was selected for its high metastatic potential in an experimental nude mouse model. When transfected with the full-length Fas gene (LM7-Fas), these cells expressed higher levels of Fas than the parental LM7 cells or LM7-neo control-transfected cells. These cells were also more sensitive to Fas-induced cell death than controls. When injected intravenously into nude mice, the LM7-Fas cell line produced a significantly lower incidence of tumor nodules than control cell lines. Lung weight and tumor nodule size were also decreased in those mice injected with LM7-Fas. Levels of Fas were quantified in osteosarcoma lung nodules from 17 patients. Eight samples were Fas negative, whereas the remaining 9 were only weakly positive compared with normal human liver (positive control). CONCLUSIONS: Our results demonstrate that altering Fas expression can impact the metastatic potential of osteosarcoma cells. We conclude that the increase of Fas on the surface of the LM7 osteosarcoma cells increased their sensitivity to Fas-induced cell death in the microenvironment of the lung, where Fas ligand is constitutively expressed. Thus, loss of Fas expression is one mechanism by which osteosarcoma cells may evade host resistance mechanisms in the lung, increasing metastatic potential. Fas may therefore be a new therapeutic target for osteosarcoma.     

Effect of interleukin (IL)-4 cytotoxin on breast tumor growth after in vivo gene transfer of IL-4 receptor alpha chain.

Although human breast cancer cells express interleukin-4 receptors (IL-4Rs), a recombinant fusion protein, IL-4 cytotoxin, did not mediate desirable antitumor activity in tumor models of breast cancer. Recent studies have identified that a primary IL-4 binding protein, IL-4Ralpha chain, is internalized after binding to IL-4 in cancer cells. The consequent expression of high-level IL-4Ralpha in tumor cells sensitizes them to the cytotoxic effect of IL-4 cytotoxin in vitro. To assess whether overexpression of IL-4Ralpha chain in vivo by plasmid-mediated gene transfer can enhance antitumor activity of IL-4 cytotoxin in mouse models of breast tumor, we injected MDA-MB-231 human breast cancer cells in both flanks of athymic nude mice. Animals then received three intratumoral (i.t.) injections of either IL-4Ralpha encoding vector (left flank) or vector only (right flank) mixed with liposome followed by IL-4 cytotoxin administration. Both i.p. and i.t. administration of IL-4 cytotoxin profoundly reduced the growth of IL-4Ralpha plasmid-injected MDA-MB-231 tumors, compared with control. Innate immune cells, including macrophages and neutrophils, were found to infiltrate at the regressing tumor site. This study provides proof of principle that i.t. IL-4Ralpha plasmid injection followed by systemic or i.t. IL-4 cytotoxin administration may be a useful strategy for the treatment of breast cancer.     

Fas expression inversely correlates with metastatic potential in osteosarcoma cells

A complex series of steps must take place to allow for a single cell to metastasize. Identifying factors responsible for these steps is essential in developing targeted therapy. We developed series of osteosarcoma cell lines with differing metastatic potentials. We used them to investigate mechanisms of metastasis and possible therapeutic targets for osteosarcoma metastasis to the lung in a nude mouse model. No correlation was found between epidermal growth factor receptor (EGFR), insulin-like growth factor receptor inhibitor (IGF-I-R), gelatinase, p53, metalloproteinase 9 (MMP 9), platelet derived growth factor receptor (PDGF-R), vascular endothelial growth factor (VEGF) and c-met expression and metastatic potential as measured by Northern analysis. By contrast, Fas expression inversely correlated with metastatic potential, and manipulation of Fas expression altered the metastatic phenotype of the cell. Our data indicate that fas gene expression may offer a new therapeutic target for the treatment of metastatic osteosarcoma in the lung.     

Serum IL-8 and IL-12 levels in breast cancer

Interleukins (ILs) are known to play a fundamental role in cancer. We investigated the serum levels of IL-8 and IL-12, in breast cancer patients, and their relationship with the prognostic parameters and therapy. Fortyeight patients with pathologically verified breast carcinoma and 21 healthy controls were enrolled into the study. Serum samples were obtained at baseline and after two cycles of chemotherapy. Serum IL-8 and IL-12 levels were determined using enzyme-linked immunosorbent assay (ELISA). There was no significant difference in the baseline serum IL-8 and IL-12 levels between breast cancer patients and healthy controls (p = 0.365 andp = 0.871, respectively), no significant correlation between the prognostic parameters and the serum IL-8, IL-12 levels. However, in the subgroup consisting of metastatic breast cancer patients, baseline serum IL-8 levels were significantly higher compared with non-metastatic disease (p = 0.047). Anthracycline-based chemotherapy and the addition of taxane did not change the levels of both serum IL-8 and IL-12. Serum IL-8 level may be useful in determining metastatic breast cancer. Larger studies are needed to confirm this finding.     

P-glycoprotein expression in human breast cancer cells

Multidrug resistance in Chinese hamster ovary cells is associated with the Mr 170,000 surface glycoprotein. Using our monoclonal antibody to this protein, we have isolated a complementary DNA clone from an expression vector library. This complementary DNA recognizes a 4.5-kilobase mRNA in drug-resistant but not-sensitive Chinese hamster ovary cells; it also recognizes a 5.0-kilobase mRNA in our Adriamycin-resistant subline of the MDA-231 human breast cancer cell line which is not expressed in the drug-sensitive parent line. Southern blot analysis shows that the P-glycoprotein sequences are greatly amplified in resistant Chinese hamster ovary cells but not in the resistant human breast cancer cells, indicating that amplification and expression of the Mr 170,000 P-glycoprotein gene are not necessarily coordinate events. Amplification of this gene may not be required for multidrug resistance in human cells.     

Concurrent induction of T-cell activation and apoptosis of osteosarcoma cells by adenovirus-mediated B7-1/Fas chimeric gene transfer

To establish an effective B7-based gene therapy against osteosarcoma, we transferred B7-1/Fas chimeric gene adenovirally into poorly immunogenic osteosarcoma cells. We found that adenovirus-mediated rat B7-1/Fas gene transfer induced (i) expression of rat B7-1/Fas chimeric molecules in osteosarcoma cells, (ii) activation of murine T cells, (iii) apoptosis of murine osteosarcoma cells in the presence of anti-rat B7-1 mAb in vitro, and (iv) therapeutic effects more prominently than B7-1 gene transfer on the development of pulmonary metastasis and survival of mice. These findings collectively support the therapeutic value of adenovirus-mediated B7-1/Fas gene transfer on poorly immunogenic osteosarcoma, which is resistant to a treatment protocol using transduction of B7-1 alone.     

Cyclooxygenase-2 gene expression in human breast cancer

Expression of the inducible isoform of the cyclooxygenase gene (PGHS-2, COX-2) which codes for the enzyme that catalyzes formation of prostaglandins, was detected in 13/13 human breast tumors of high grade but not in samples of normal breast tissue. There was a statistically significant linear association between COX-2 gene expression and high (>50%) tumor cell density (p<0.01), with COX-2 protein localized to tumor cells. These results indicate that COX-2 gene expression may be useful as a molecular biomarker for human breast tumors and may also predict sensitivity to treatment with nonsteroidal anti-inflammatory drugs.     

Interleukin (IL)-1A and IL-6: applications to the predictive diagnostic testing of radiation pneumonitis

PURPOSE: To explore the application of interleukin (IL)-1alpha and IL-6 measurements in the predictive diagnostic testing for symptomatic radiation pneumonitis (RP). METHODS AND MATERIALS: In a prospective protocol investigating RP and cytokines, IL-1alpha and IL-6 values were analyzed by enzyme-linked immunosorbent assay from serial weekly blood samples of patients receiving chest radiation. We analyzed sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) over selected threshold values for both cytokines in the application to diagnostic testing. RESULTS: The average coefficient of variation was 51% of the weekly mean IL-1alpha level and 39% of the weekly mean IL-6 value. Interleukin 1alpha and IL-6 became positively correlated with time. Specificity for both cytokines was better than sensitivity. IL-6 globally outperformed IL-1alpha in predicting RP, with higher PPV and NPV. CONCLUSIONS: Our data demonstrate the feasibility of applying IL-1alpha and IL-6 measurements of blood specimens to predict RP. Interleukin-6 measurements offer stronger positive predictive value than IL-1alpha. This application might be further explored in a larger sample of patients.     

Vitamin E succinate (VES) induces Fas sensitivity in human breast cancer cells: role for Mr 43,000 Fas in VES-triggered apoptosis

Fas (CD95/APO-1) is an important mediator of apoptosis. We show that Fas-resistant MCF-7, MDA-MB-231, and MDA-MB-435 human breast cancer cells become responsive to anti-Fas (CD95) agonistic antibody-triggered apoptosis after pretreatment or cotreatment with vitamin E succinate (VES; RRR-alpha-tocopheryl succinate). In contrast, no enhancement of anti-Fas agonistic antibody-triggered apoptosis was observed following VES pretreatment or cotreatment with Fas-sensitive primary cultures of human mammary epithelial cells, immortalized MCF-10A cells, or T47D human breast cancer cells. Although VES is itself a potent apoptotic triggering agent, the 6-h pretreatment procedure for Fas sensitization did not initiate VES-mediated apoptosis. The combination of VES plus anti-Fas in pretreatment protocols was synergistic, inducing 2.8-, 3.0-, and 6.3-fold enhanced apoptosis in Fas-resistant MCF-7, MDA-MB-231, and MDA-MB-435 cells, respectively. Likewise, cotreatment of Fas-resistant MCF-7, MDA-MB-231, and MDA-MB-435 cells with VES plus anti-Fas enhanced apoptosis 1.9-, 2.0-, and 2.6-fold, respectively. Functional knockout of Fas-mediated signaling with either Fas-neutralizing antibody (MCF-7-, MDA-MB-231-, and MDA-MB-435-treated cells) or Fas antisense oligomers (MDA-MB-435-treated cells only), reduced VES-triggered apoptosis by approximately 50%. Analyses of whole cell extracts from Fas-sensitive cells revealed high constitutive expression of Mr 43,000 Fas, whereas Fas-resistant cells expressed low levels that were confined to the cytosolic fraction. VES treatment of the Fas-resistant cells caused a depletion of cytosolic Mr 43,000 Fas with a concomitant increase in Mr 43,000 membrane Fas. These data show that VES can convert Fas-resistant human breast cancer cells to a Fas-sensitive phenotype, perhaps by translocation of cytosolic Mr 43,000 Fas to the membrane and show that VES-mediated apoptosis involves Mr 43,000 Fas signaling.     

MicroRNA gene expression deregulation in human breast cancer

MicroRNAs (miRNAs) are a class of small noncoding RNAs that control gene expression by targeting mRNAs and triggering either translation repression or RNA degradation. Their aberrant expression may be involved in human diseases, including cancer. Indeed, miRNA aberrant expression has been previously found in human chronic lymphocytic leukemias, where miRNA signatures were associated with specific clinicobiological features. Here, we show that, compared with normal breast tissue, miRNAs are also aberrantly expressed in human breast cancer. The overall miRNA expression could clearly separate normal versus cancer tissues, with the most significantly deregulated miRNAs being mir-125b, mir-145, mir-21, and mir-155. Results were confirmed by microarray and Northern blot analyses. We could identify miRNAs whose expression was correlated with specific breast cancer biopathologic features, such as estrogen and progesterone receptor expression, tumor stage, vascular invasion, or proliferation index.     

芦荟大黄素对结肠癌细胞株SW620 FasL基因表达和侵袭能力的影响

目的：在体外通过细胞培养，初步研究芦荟大黄素对人结肠癌SW620细胞FasLmRNA表达及对其侵袭能力的影响，为中药抗肿瘤提供实验依据。方法：根据MTT法得到芦荟大黄素对SW620细胞的半数有效抑制浓度（IC50），确定药物作用浓度．SW620细胞分别经芦荟大黄素不同浓度（0．5IC50、IC50）作用后，应用逆转录-聚合酶链反应（RT—PCR）法检测芦荟大黄素作用前后人结肠癌SW620细胞FasL mRNA的变化；应用Transwell细胞侵袭试验检测芦荟大黄素对SW620细胞侵袭能力的影响。结果：芦荟大黄素处理后较处理前SW620细胞FasLmRNA表达水平均明显高于对照组；而且FasLmRNA表达水平随芦荟大黄素作用浓度增加显著上调，芦荟大黄素不同浓度组比较均有显著性差异；随芦荟大黄素作用浓度升高，SW620细胞侵袭能力明显增强，不同浓度组比较均有显著性差异。结论：芦荟大黄素在一定时间内均可上调人结肠癌SW620细胞FasL mRNA的表达，而且这种上调作用在一定范围内呈剂量依赖性，可使结肠癌细胞的侵袭能力增强。     

细胞因子上调大肠癌细胞Fas配体表达

目的：研究白介素１８（ＩＬ－１８）等内源性细胞因子对大肠癌细胞ＦａｓＬ表达水平的调控作用。方法：采用Ｗｅｓｔｅｒｎ蛋白印迹法测定细胞因子处理后大肠癌细胞的ＦａｓＬ表达水平。结果：在检测的６株大肠癌细胞中,全部表达ＦａｓＬ蛋白;ＴＮＦ－α和ＩＦＮ－γ显著上调ＤＬＤ－１ ＦａｓＬ蛋白的表达水平;ＴＮＦ－α和ＩＦＮ－γ以及乙酸肉豆佛波醇（ｐｈｏｔｂｏｌ１２－ｍｙｒｉｓｔａｔｅ １３－ａｃｅｔａｔｅ,ＰＭ