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.     

Oestrogen sulphatase activity in hormone-dependent and hormone-independent breast-cancer cells: modulation by steroidal and non-steroidal therapeutic agents.

Oestrogen sulphatase may play an important role in providing intracellular oestrogens from E1S for the growth and maintenance of breast tumours. In this study, we characterized oestrogen sulphatase in the hormone-dependent (ER/PR+) MCF-7 and in the hormone-independent (ER/PR-) MDA-MB-231 breast-cancer cells and, furthermore, examined its modulation by MPA, 4-OH-A4, tamoxifen, danazol, ethinyloestradiol and DHAS in both these cell types. Our detailed study of oestrogen sulphatase activity as a function of incubation time, E1S concentration and numbers of MCF-7 and MDA-MB-231 cells showed that more E1S was hydrolysed by MDA-MB-231 cells than by MCF-7 cells at all time points and all substrate concentrations. Additionally, although the Km values of E1S for oestrogen sulphatase in both MCF-7 and MDA-MB-231 cells were similar, the Vmax values, and therefore the activity, differed greatly. The effect of various steroidal and non-steroidal compounds also suggested differences in these 2 cell lines with respect to oestrogen sulphatase inhibition or stimulation. MPA significantly increased the hydrolysis of [3H]E1S in both cell lines, possibly through its effect on membrane fluidity. Tamoxifen increased E1S hydrolysis in MDA-MB-231 cells but not in MCF-7 cells, whereas 4-OH-A4 inhibited E1S in MCF-7 cells but not in MDA-MB-231 cells. Danazol (an isoxazol derivative of 17 alpha-ethinyltestosterone), 17 alpha-ethinyloestradiol and DHAS all significantly inhibited oestrogen sulphatase activity in both cell lines. Furthermore, danazol had a growth-inhibitory effect on both MCF-7 and MDA-MB-231 cells, although MCF-7 cells appeared to be more sensitive to growth inhibition by danazol.     

Effects of environmental estrogens on tumor necrosis factor alpha-mediated apoptosis in MCF-7 cells.

Environmental estrogens represent a class of compounds which have been shown to mimic the effects or activity of the naturally occurring ovarian hormone 17beta-estradiol. Given the role of 17beta-estradiol in cell survival in a number of systems, we wished to determine if environmental estrogens protect MCF-7 cells from apoptosis. Here we demonstrate that the organochlorine pesticides o, p' DDT and alachlor, like 17beta-estradiol, have the ability to suppress tumor necrosis factor alpha (TNF)-induced apoptosis in estrogen receptor (ER)-positive MCF-7 breast carcinoma cells. These compounds, however, did not affect TNF-induced apoptosis of the ER-negative MDA-MB-231 cell line. The ability of these compounds to suppress apoptosis in MCF-7 cells was correlated with an ER-dependent increase in Bcl-2 expression. Taken together these results demonstrate that estrogenic organochlorine pesticides like o, p' DDT and alachlor may partially mimic the primary endogenous estrogen, 17beta-estradiol, and function to suppress apoptosis in ER-responsive cells.     

Inhibition of colon and breast carcinoma cell growth by interleukin-4.

Within human carcinomas, there is often an infiltration of lymphocytes and other cells of the immune system. A variety of cytokines are produced by such cells that could have a paracrine influence on the growth of tumor epithelium. The effect of one of these cytokines, interleukin-4 (IL-4), on human breast and colon cancer cell lines was therefore examined. IL-4 inhibited the growth of human colon (HT 29) and breast [MCF-7 wild type (MCF-7 WT), MCF-7 Adriamycin-resistant (MCF-7r), MDA-MB-231, and MDA-MB-468] carcinoma cells in culture. Competitive binding of 125I-IL-4 demonstrated the presence of 2000 high affinity IL-4-binding sites on HT 29 cells. The Kd for specific binding of 125I-IL-4 to HT 29 cells was 77 pM. Further studies were conducted on the estrogen-dependent MCF-7 WT and estrogen-independent MDA-MB-231 breast carcinoma lines. Concentrations of IL-4 of 10-100 nM were required to significantly inhibit growth of these carcinoma cell lines; e.g., with MCF-7 WT cells, half-maximal inhibition of growth occurred at 20 nM IL-4. Specific binding of 125I-IL-4 was detected to MCF-7 WT and MDA-MB-231 cells, but the low level of binding precluded Scatchard analysis. IL-4 inhibited 90% of the 17 beta-estradiol-stimulated growth of MCF-7 WT cells in a dose-dependent manner but without a change in estrogen receptor expression. Inhibition of growth by IL-4 was less in the absence of estrogens. Combined treatment with IL-4 and other known inhibitors of breast carcinoma cell growth [transforming growth factor-beta 1 (TGF-beta 1) and the antiestrogen tamoxifen] showed additive inhibition. The hormone-independent cell lines MCF-7r and MDA-MB-231 were additively inhibited by IL-4 and TGF-beta 1. This was not the case with MDA-MB-468 cells in which inhibition by IL-4 and TGF-beta 1 was of similar magnitude but no significantly greater effect was observed on combined treatment. No secretion of IL-4 was detected from these cell lines either basally or on treatment with TGF-beta 1 or tamoxifen, and we conclude that IL-4 is a nonautocrine inhibitor of breast carcinoma cell growth.     

miRNA-34a抑制乳腺癌细胞MCF-7、MDA-MB-231的增殖与侵袭

目的:研究miRNA-34a(miR-34a)对乳腺癌细胞MCF-7、MDA-MB-231的生物调控作用。方法:采用定量PCR检测人乳腺上皮细胞MCF-10A,乳腺癌细胞株MCF-7、T47D、MDA-MB-231、MDA-MB-453、Hs578T中miR-34a的表达水平。通过miR-34a mimics分别上调MCF-7、MDA-MB-231细胞中miR-34a的表达水平,MTT和Transwell检测肿瘤细胞增殖能力、侵袭力等生物学行为的变化。结果:乳腺癌细胞MCF-7、T47D、MDA-MB-231、MDA-MB-453、Hs578T中miR-34a处于低表达水平。通过miR-34a mimics上调MCF-7、MDA-MB-231细胞中miR-34a的表达后,细胞的增殖能力被miR-34a抑制（P＜0.05）,miR-34a对细胞侵袭有显著抑制作用（P＜0.05）。结论:miR－34a在乳腺癌细胞MCF-7、T47D、MDA-MB-231、MDA-MB-453及Hs578T中低表达,miR-34a抑制乳腺癌细胞MCF-7、MDA-MB-231的细胞增殖和侵袭能力。     

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.     

Cytokines facilitate chemotactic motility of breast carcinoma cells

BACKGROUND: Both growth and motility of various tumor cells have been shown to be influenced by surrounding cells such as lymphocytes, histiocytes and fibroblasts through various cytokines, growth factors and extracellular matrices. The role of cytokines and extracellular matrices produced by lymphocytes, histiocytes and fibroblasts on migration and invasion of breast carcinoma cells has not been fully investigated METHODS: We investigated the effect of hepatocyte growth factor (HGF), interleukin (IL)-6, IL-8, IL-11, soluble type IV collagen and soluble laminin on the migration of 3 human breast carcinoma cell lines, MDA-MB-231, MCF-7 and T-47D, using a cell culture insert and a biocoat matrigel invasion chamber to assess migration across a matrigel-coated polyethylene telephtalate membrane. RESULTS: HGF, IL-6, IL-11 and IL-8 induced significant migration of MDA-MB-231 cells depending on the dose of each cytokine. However, type IV collagen and laminin inhibited migration of MDA-MB-231 cells. In contrast, IL-8 inhibited migration of MCF-7 cells and IL-6 induced significant migration of T-47D cells, while no other cytokine or extracellular matrix induced significant migration of MCF-7 and T-47D cells. Only HGF induced significant invasion of MDA-MB-231 cells depending on the dose. MCF-7 and T-47D cells did not invade in response to any of the cytokines and extracellular matrices tested. CONCLUSIONS: These results suggest the possibility that the potency of chemotaxis or chemoinvasion differs according to the breast carcinoma cell line and that various cytokines and extracellular matrices secreted by lymphocytes, histiocytes and fibroblasts in the stroma of breast carcinoma can affect the invasion of breast carcinoma cells.     

Laminin and estradiol regulation of the plasminogen-activator system in MCF-7 breast-carcinoma cells

We have investigated the effects of laminin, on the plasminogen-activator system of MCF-7 breast-carcinoma cells. MCF-7 cells were incubated on plastic or laminin-coated wells, and medium and cell lysate aliquots were assayed for tissue-type (tPA) and urokinase-type plasminogen activator (uPA) by a chromogenic assay in combination with anti-uPA antibodies. Cells cultured on laminin displayed a 5-fold increase in tPA activity and a 2-fold decrease in uPA activity relative to cells on plastic. These effects could be mimicked by laminin fragment P1 but not by collagen I or fibronectin. tPA activity of cells treated with estradiol (10 nM) was 3-fold higher, that of cells on laminin treated with estradiol was 15-fold higher, than that of control. Northern-blot analysis showed that tPA mRNA levels were up-regulated by estradiol and laminin, whereas PAI-1 mRNA levels were down-regulated by laminin and not affected by E2. Concomitant treatment with laminin and estradiol, decreased PAI-1 mRNA and increased tPA mRNA levels, accounting for the synergistic increase in tPA activity. Laminin exerted only a modest (approx. 2-fold) inhibitory effect on uPA mRNA levels. In the breast-carcinoma cell line MDA-MB-231, down-regulation of PAI-1 and uPA mRNA by laminin was not observed. Adhesion assays indicated that α2β1 is the predominant receptor for laminin in MCF-7 cells. MDA-MB-231 cells expressed α2 (54%) but this integrin is not used as a laminin receptor. These results support a role for α2β1 in mediating interactions of MCF-7 with LN. Int. J. Cancer 76:77–85, 1998.© 1998 Wiley-Liss, Inc.     

Inhibition of estrogen-dependent breast cell responses with phenylacetate

The aromatic fatty acid phenylacetate (PA) and its analogs have come under intense investigation due to their ability to cause the growth arrest of a variety of neoplasia, including human breast cancer. We have determined that PA and its halide derivative 4-chlorophenylacetate (4-CPA) showed marked antiproliferative activity on 3 of 6 human breast cancer cell lines tested. Interestingly, the 3 cell lines that were growth inhibited by PA and 4-CPA were estrogen receptor (ER) positive (T47-D, MCF-7 and ZR-75-1) whereas those that were little affected by these compounds were ER-negative (MDA-MB-157, MDA-MB-231 and SK-Br-3). Dose response studies indicated that 4-CPA inhibited the growth of the sensitive (ER+) cell lines with a potency 3-4 times that of PA. These findings suggest that there is "cross-talk" between the PA and estrogen signaling pathways such that PA can directly inhibit estrogen-dependent events. This hypothesis was directly tested in vitro using ER+ MCF-7 cells that were stably transfected with a luciferase reporter construct driven by the full length (1745 bp) cyclin D1 promoter (MCF-7-D1). Our experiments with MCF-7-D1 cells indicated that PA and 4-CPA inhibited basal and estrogen-induced reporter gene activity by up to 90%, resulting in almost complete elimination of estrogen-dependent cyclin D1 gene activation. Using a reporter gene construct (ERE(V)-tk-Luc) containing a canonical estrogen response element that was transiently transfected into MCF-7 and MDA-MB-231 cells, we have also demonstrated inhibition of promoter activity by PA and 4-CPA that was directly mediated by blockage of activity through the ERE. Taken together, these findings indicate that PA analogs possess potent antiestrogen properties that may, at least partly, account for their antiproliferative effects on ER+ breast cancer cells. The data suggests a novel mechanism of action that might bypass some of the limitations of conventional antiestrogen therapy.     

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。     

Glucocorticoid modulation of plasminogen activators and of one of their inhibitors in the human mammary carcinoma cell line MDA-MB-231

In cultures of the human mammary carcinoma-derived cell line MDA-MB-231, plasminogen activator (PA) activity was reduced substantially following treatment with the glucocorticoid dexamethasone. These cells produced urokinase-type PA (u-PA) and tissue-type PA (t-PA), and both enzymes were decreased in dexamethasone-treated cultures. The drop in u-PA activity was associated with a decrease in the synthesis of single-chain pro-u-PA and in the concentration of u-PA messenger RNA; however, the decrease in u-PA activity was more extensive than could be accounted for by inhibition of enzyme synthesis only, suggesting that postsynthetic events were also involved. The comparatively small dexamethasone-induced decrease in t-PA activity was not associated with a change in the concentration of t-PA messenger RNA. Hence, the two PA genes are differentially regulated by the same hormone. MDA-MB-231 cells also produced a PA-specific inhibitor related to that produced by bovine aortic endothelial cells (PAI-1). This inhibitor was present in two forms: one functionally active, and the other which required activation by sodium dodecyl sulfate; both forms were increased in cultures exposed to dexamethasone. Thus, glucocorticoid-induced inhibition of PA activity in these cells results from a decrease in u-PA synthesis and a concomitant increase in the production of a PA inhibitor.     

乳腺癌干细胞富集与鉴定的初步研究

目的:通过从MCF-7、ZR-75-1、MDA-MB-231乳腺癌细胞系中培养富集及鉴定乳腺癌干细胞(breast cancer stem cell,BCSC),寻找培养与富集乳腺癌干细胞的方法。方法:贴壁培养MCF-7、ZR-75-1、MDA-MB-231细胞系,倒置显微镜观察各细胞形态;流式细胞仪分别分选收集CD44-CD24-、CD44-CD24+、CD44+CD24-及 CD44+CD24+ 细胞,其中CD44+CD24-为乳腺癌干细胞,其余三类为对照组;MTT法计数细胞,绘制MCF-7、ZR-75-1、MDA-MB-231细胞系生长曲线;MCF-7细胞系进行无血清悬浮培养1个周期,流式细胞仪检测分子表面标记物CD44+CD24-含量,贴壁培养的CD44+CD24-乳腺癌干细胞为对照组;将分选的MCF-7（CD44+CD24-）和分选的其余MCF-7细胞（非CD44+CD24-）进行干性成球实验,鉴定CD44+CD24-干性表达。结果:MCF-7、MDA-MB-231细胞系富含表面标志物CD44-CD24-的乳腺癌细胞;ZR-75-1细胞系富含分子表面标志物CD44+CD24+的乳腺癌细胞;生长曲线显示MCF-7、ZR-75-1、MDA-MB-231均呈持续增长,MDA-MB-231细胞生长较MCF-7、ZR-75-1细胞快;通过无血清悬浮培养CD44+CD24-乳腺癌干细胞由19.4%富集到88.9%;成球实验中CD44+CD24-表型细胞成球数量较分选的其余MCF-7细胞（非CD44+CD24-表型）明显增多,成球率分别为（36.5±1.7）%,（1.1±0.5）%。结论:流式细胞仪可成功分选出分子表面标志物为CD44+CD24-的乳腺癌干细胞;CD44+CD24-可能不是乳腺癌干细胞唯一的表面标志物;MDA-MB-231细胞系较MCF-7、ZR-75-1细胞系生长快;无血清悬浮培养法可简便、高效地富集乳腺癌干细胞;CD44+CD24-乳腺癌干细胞干性表达较强。     

Modulation of u-PA, MMPs and their inhibitors by a novel nutrient mixture in human female cancer cell lines

Cancers of the breast, cervix, uterus and ovary are the most prevalent cancers in women worldwide. Proteases play a key role in tumor cell invasion and metastasis by digesting the basement membrane and ECM components. Strong clinical and experimental evidence demonstrates association of elevated levels of urokinase plasminogen activators (u-PA) and matrix metalloproteinases (MMPs) with cancer progression, metastasis and shortened patient survival. MMP activities are regulated by specific tissue inhibitors of metalloproteinases (TIMPs). Our main objective was to study the effect of a nutrient mixture (NM) on the activity of u-PA, MMPs and TIMPs in human breast, cervix, uterine and ovarian cancer cell lines. Human breast (MDA-MB-231 and MCF-7), cervical (HeLa), uterine (SK-UT-1) and ovarian (SKOV3) cancer cell lines were cultured in their respective media and treated at confluence with NM at 0, 50, 100, 250, 500 and 1000 μg/ml. Analysis of u-PA activity was carried out by fibrin zymography, MMPs by gelatinase zymography and TIMPs by reverse zymography. Both breast and uterine cancer cell lines expressed u-PA, which was inhibited by NM in a dose-dependent manner. However, no bands corresponding to u-PA were detected for HeLa and SK-OV-3 cell lines. On gelatinase zymography, MDA-MB-231 and MCF-7 showed one band corresponding to MMP-9, HeLa showed two bands, an intense band corresponding to MMP-2 and a faint band corresponding to MMP-9, SK-UT-1 showed PMA-induced MMP-9, and SK-OV-3 showed a band corresponding to MMP-2. NM inhibited their expression in all cell lines. The activity of TIMPs was upregulated in all cancer cell lines in a dose-dependent manner. Analysis revealed a positive correlation between u-PA and MMPs and a negative correlation between u-PA/MMPs and TIMPs. These findings suggest the therapeutic potential of NM in the treatment of female cancers.     

Death receptor-dependent and -independent pathways in anticancer drug-induced apoptosis of breast cancer cells

Apoptosis is a crucial event for anticancer drug efficacy. The signal pathways activated by anticancer drugs are classified as death receptor (DR) -dependent or -independent. The mechanisms by which anticancer drugs induce apoptosis via DR-dependent pathways are not fully understood. In the present study, we assessed differential activation of signal transduction pathways leading to apoptosis by anticancer drugs in breast cancer cell lines. Three breast cancer cell lines, MDA-MB-231, MCF-7, and MCF-7ADM, which is drug-resistant, were used. Drug sensitivity and apoptosis were assessed by MTT assay and TUNEL, respectively. Expression of apoptosis-related protein was assessed by Western blotting and RT-PCR. The sensitivities of MDA-MB-231 and MCF-7 cells to mitomycin C (MMC) and adriamycin (ADM) were similar. In contrast, sensitivity of MDA-MB-231 cells to paclitaxel (TXL) was 30-fold greater than that of MCF-7 cells, 0.03 micro M in MDA-MB-231 and 0.9 micro M in MCF-7 cells, respectively. Treatment with MMC increased expression of DR4 and Fas in a time-dependent manner in MDA-MB-231 cells. Treatment with ADM increased expression of DR4 and DR5 but not Fas, whereas treatment with TXL increased expression of Fas but not DR4 and DR5 in MDA-MB-231 cells. Although treatment of MCF-7 cells with ADM increased expression of DR4 and DR5 but not Fas, expression of DR4, DR5, and Fas by the drug-resistant cells did not change following treatment with ADM. Activation of Fas, DR4, and DR5 in drug-sensitive cells in response to anticancer drugs is dependent on the cytotoxic effect of each drug. In drug-resistant cells, apoptosis is induced via DR-independent pathways mediated by mitochondrial dysfunction.     

Glycosphingolipid composition of MDA-MB-231 and MCF-7 human breast cancer cell lines

Much evidence has shown that glycosphingolipids are involved in cellular recognition, regulation of cell growth, and metastasis. In the present study, the major glycosphingolipids of two widely studied human breast cancer cell lines were examined. The MCF-7 cell line has functional estrogen and EGF receptors, is dependent on estrogen and EGF for growth, and is uninvasive, while MDA-MB-231 cells are a model for more aggressive, hormone-independent breast cancer. There was twice as much neutral glycolipid in MCF-7 cells as in MDA-MB-231 cells. The major neutral glycolipids in MDA-MB-231 cells were identified as CTH and globoside. MCF-7 cells also contained as the major neutral glycolipids CTH as well as globoside and two other glycolipids which were tentatively identified as galactosylgloboside and fucosylgalactosylgloboside by exoglycosidase treatments. Conversely, the ganglioside content was four fold higher in MDA-MB-231 cells compared to MCF-7 cells. The abundant gangliosides in both cell lines were GM3, GM2, GM1, and GD1a. A minor monosialoganglioside was detected in MDA-MB-231 cells. The striking 18 fold greater amount of GM3 in MDA-MB-231 cells may have important implications because GM3 has been suggested to be involved in regulation of growth factor functions. In agreement, insertion of ganglioside GM3 into the plasma membrane of MCF-7 cells blocked the growth stimulatory effect of EGF.     

Differential regulation of growth and invasiveness of MCF-7 breast cancer cells by antiestrogens

Estrogen increases the ability of the estrogen-dependent MCF-7 human breast cancer cell line to both proliferate and invade through an artificial basement membrane. In studying the response of MCF-7 cells to various antiestrogens, we found that 4-hydroxytamoxifen and tamoxifen inhibited cell proliferation but increased their invasiveness. In contrast, the structurally unrelated benzothiophene antiestrogens, LY117018 and LY156758, were potent antiproliferative agents which did not stimulate invasiveness. The differential effects of these antiestrogenic agents on invasion correlated with changes in production of collagenase IV, while no significant change was seen in the chemotactic activity of the cells. Invasiveness was increased by 17 beta-estradiol or 4-hydroxytamoxifen after a few hours of treatment and was rapidly lost when 17 beta-estradiol was withdrawn. Stimulation of invasiveness with 17 beta-estradiol was blocked by the antiestrogen, LY117018. Cells from the MDA-MB-231 line which lacks estrogen receptors were not affected by estrogen or antiestrogen in terms of proliferation or invasion. These studies indicate that the invasiveness of MCF-7 cells is regulated by antiestrogens through the estrogen receptor and may be mediated by collagenase IV activity. Antiestrogens which reduce both the proliferation and invasiveness of these cells may be interesting new candidates for clinical application.     

Suppression of estrogen-regulated extracellular tissue plasminogen activator activity of MCF-7 cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) suppresses the estrogen enhancement of tissue plasminogen activator (t-PA) by MCF-7 breast cancer cells. 17 beta-estradiol treatment of MCF-7 cells was previously shown to enhance t-PA secretion in a receptor-mediated process dependent on RNA and protein synthesis. The current studies demonstrate that treatment with TCDD, at a concentration as low as 10(-11) M, reduces the 17 beta-estradiol-induced enhancement of t-PA secretion in these cells. Treatment of MCF-7 cells with TCDD alone does not alter t-PA activity nor was inhibition of t-PA activity observed when TCDD was added directly to the enzyme assay. Kinetic studies and the lack of inhibition following in vitro mixing of conditioned media from TCDD-treated and control 17 beta-estradiol stimulated MCF-7 cells argue against TCDD induction of a plasminogen activator inhibitor. The related polychlorinated dibenzofuran, 2,3,7,8,-tetrachlorodibenzofuran, while also active, is less potent that TCDD. Other polychlorinated dibenzodioxins, polychlorinated dibenzofurans, and polychlorinated biphenyls do not suppress 17 beta-estradiol induction of t-PA over the concentrations tested. These results are in agreement with the structure-activity relationships established using these compounds in other assay systems. Treatment with TCDD does not alter the number or affinity of 17 beta-estradiol receptors of MCF-7 cells. TCDD treatment does not suppress constitutive t-PA activity in the estrogen independent breast cancer line MDA-MB-231 nor the t-PA induced by 12-O-tetradecanoylphorbol-13-acetate in HeLa cells. These effects suggest that TCDD is not acting directly on expression of the t-PA genome. Induction of aryl hydrocarbon hydroxylase by TCDD, a cytochrome P-450 regulated metabolic enzyme for which TCDD is the most potent known inducer, was observed in MCF-7 cells but not in MDA-MB-231 or HeLa cells. A plausible mechanism for the antiestrogenic activity of TCDD is based on the metabolic conversion of 17 beta-estradiol to less active derivatives by TCDD induced cytochrome P-450 metabolic enzymes.     

托瑞米芬逆转乳腺癌耐药蛋白介导的多药耐药机制

目的 探讨托瑞米芬逆转乳腺癌耐药蛋白(BCRP)介导的多药耐药机制。方法 通过基因扩增,构建分别由BCRP启动子和巨细胞病毒(CMV)启动子启动表达BCRP的重组质粒pcDNA3-Promoter-BCRP和作为对照的质粒pcDNA3-CMV-BCRP,将其分别转染雌激素受体α（ERα）阳性的MCF-7和ERα阴性的MDA-MB-231乳腺癌细胞系,建立由BCRP启动子和CMV启动子启动表达BCRP的4种耐药细胞系MCF-7/Promoter-BCRP、MCF-7/CMV-BCRP、MDA-MB-231/PromoterBCRP和MDA-MB-231/CMV-BCRP。在耐药细胞培养基中加入托瑞米芬,通过逆转录聚合酶链反应(RT-PCR)、Western blot、外排实验以及细胞毒性实验观察托瑞米芬对不同细胞系的耐药逆转效果。结果与空白对照组（未加药物）相比,托瑞米芬以剂量依赖方式抑制BCRP mRNA的表达,0.1、1和10 μmol/L托瑞米芬处理组MCF-7/Promoter-BCRP细胞中BCRP mRNA的表达水平分别下调29.5％(P＜0.05)、68.1％ (P＜0.01)和97.4％(P＜0.01);MCF-7/Promoter-BCRP细胞经托瑞米芬和17β-雌二醇联合处理后,细胞中BCRP mRNA的相对表达水平为64.2％±1.3％,明显高于托瑞米芬单独处理组(3.8％±0.2％,P＜0.01)。托瑞米芬对各组细胞系中BCRP蛋白表达的调控作用与mRNA相似。经托瑞米芬处理后,MCF-7/Promoter-BCRP细胞内米托蒽醌的荧光强度显著增强,外排米托蒽醌的能力降低了 47.3％ (P ＜0.05);经托瑞米芬和17β-雌二醇联合处理后,MCF-7/Promoter-BCRP细胞内米托蒽醌的荧光强度明显低于托瑞米芬单独处理组,外排米托蒽醌的能力升高了61.5％。托瑞米芬可有效逆转MCF-7/Promoter-BCRP细胞对米托蒽醌的耐药性。上述作用在MCF-7/CMV-BCRP、MDA-MB-231/Promoter-BCRP和MDA-MB-231/CMV-BCRP细胞中未能体现。结论 托瑞米芬可能通过ERot的介导与BCRP启动子上游调控序列中的ERE结合,负性调节BCRP的表达,抑制BCRP蛋白的功能,在体外有效逆转BCRP介导的多药耐药。     

Stimulation by bombesin and inhibition by bombesin/gastrin-releasing peptide antagonist RC-3095 of growth of human breast cancer cell lines.

Recently, it was reported that bombesin/gastrin-releasing peptide (GRP) have mitogenic effects on some human breast cancer cell lines. In this study, we investigated the effects of bombesin/GRP and its receptor antagonist (RC-3095) on the proliferation of three breast cancer cell lines, MDA-MB-231, MCF-7 MIII, and MCF-7. Stimulation by bombesin and inhibition by RC-3095 of cell growth were found in MDA-MB-231 and MCF-7 MIII cells cultured in phenol red-free medium with 5% heat-inactivated and dextran-coated charcoal-treated fetal bovine serum (DCC-FBS). A stimulatory effect by bombesin was not observed in the presence of untreated FBS. [3H]Thymidine incorporation into DNA in these cells was suppressed by RC-3095. MCF-7 cells failed to respond to bombesin and RC-3095 in the presence of either FBS or DCC-FBS. GRP-like immunoreactivity was found in the cell extracts and FBS, but it was undetectable in DCC-FBS. It appears that the stimulatory effect of bombesin on cell proliferation of MCF-7 MIII and MDA-MB-231 cell lines could be obtained because of reduction in the levels of some serum factors in DCC-FBS. These results suggest that bombesin/GRP can act as growth factors through bombesin/GRP receptors in some human breast cancers.     

受体型蛋白酪氨酸磷酸酶O基因甲基化与乳腺癌细胞株化疗敏感性的关系

目的探讨受体型蛋白酪氨酸磷酸酶O（PTPRO）基因不同甲基化状态的乳腺癌细胞株对紫杉醇的敏感性。方法应用甲基化特异性PCR,检测MCF-7、MDA-MB-231和Hs578t乳腺癌细胞株中PTPRO基因启动子Cp G岛甲基化状态,并用RT-PCR法检测PTPRO mRNA表达。采用MTT法检测MCF-7、MDA-MB-231和Hs578t乳腺癌细胞株对0.000 6、0.003 0、0.015 0、0.075 0、0.375 0、1.875 0、9.350 0、46.750 0、234.000 0 mmol/L紫杉醇的敏感性,以及对细胞株进行去甲基化实验后紫杉醇抑制率的改变。两组间均数比较采用配对t检验;在检验方差齐性的前提下,多组间均数比较用单因素方差分析,组间两两比较采用LSD检验。结果乳腺癌MCF-7、MDA-MB-231细胞株中PTPRO基因甲基化,而乳腺癌Hs578t细胞株中PTPRO基因无甲基化。用紫杉醇处理细胞株后再检测PTPRO启动子甲基化情况,结果显示MCF-7、MDA-MB-231细胞株PTPRO基因甲基化率明显降低[（0.861±0.109）比（0.037±0.019）,t=23.326,P=0.000;（0.758±0.114）比（0.086±0.010）,t=16.109,P=0.000]。并且,MCF-7、MDA-MB-231细胞株经5-杂氮脱氧胞嘧啶（DAC）处理后,PTPRO mRNA表达水平明显高于DAC处理前[（0.033±0.006）比（0.166±0.016）,t=28.338,P=0.000;（0.052±0.006）比（0.587±0.087）,t=17.257,P=0.000],其表达水平与启动子Cp G岛甲基化状态有关。紫杉醇对MDA-MB-231、MCF-7及Hs578t细胞株的半数抑制浓度（IC50）分别为8.52、5.87和4.52 mmol/L。不同浓度紫杉醇对Hs578t细胞株的抑制率均比MCF-7和MDA-MB-231细胞株高（F=129.93、182.40、46.26、49.26、33.60、80.31、160.05、69.96、79.98,P均=0.000）。去甲基化实验显示：DAC处理MCF-7和MDA-MB-231乳腺癌细胞株后,不同浓度紫杉醇对细胞株的抑制率与处理前相比,差异均有统计学意义（MCF-7：t=14.195、8.328、7.042、6.385、5.450、8.557、4.788、13.351、11.887,P均〈0.050;MDA-MB-231：t=16.324、30.443、9.177、12.694、9.528、11.912、10.260、18.109?