MDM2反义寡核苷酸联合紫杉醇对乳腺癌MCF-7细胞株的作用

目的:探讨靶向MDM2反义寡核苷酸(ASON)联合紫杉醇对乳腺癌MCF-7细胞株的影响。方法:合成一段与MDM2 mRNA特异性结合的反义寡核苷酸和与反义寡核苷酸有4个碱基不同的的错义寡核苷酸(MON),脂质体2000介导不同浓度的MDM2ASON转染MCF-7乳腺癌细胞系,转染的乳腺癌细胞通过1μmol/L紫杉醇药物处理后,采用RT-PCR和Western Blot方法检测MDM2 ASON联合紫杉醇的协同作用及对乳腺癌MCF-7细胞株的抑制效率,MTT观察给药后MCF-7细胞的增殖能力和药物敏感性。结果:MDM2反义寡核苷酸联合紫杉醇明显下调MDM2 mRNA及MDM2蛋白表达水平,抑制MCF-7细胞的生长,随着MDM2 ASON浓度的增加,MDM2表达越来越低,协同作用越来越强,呈剂量依赖关系,A500联合紫杉醇的协同作用最明显,MTT显示紫杉醇处理的转染MCF-7细胞增殖抑制率明显增高,A500抑制增殖作用最明显,抑制率达(13.0±0.84)%。结论:不同浓度MDM2 ASON转染后的乳腺癌MCF-7细胞,等浓度紫杉醇处理后,乳腺癌MCF-7细胞MDM2表达明显降低,细胞凋亡增加,,MDM2 ASON联合紫杉醇对MCF-7细胞有协同作用,提高了乳腺癌MCF-7细胞对紫杉醇的药物敏感性。     

Characterization of insulin-like growth factor binding proteins from human breast cancer cells

The insulin-like growth factor binding proteins (IGF-BPs) are structurally and immunologically distinct from the IGF type 1 or type 2 receptors and are characterized by two major forms: a large, GH-dependent BP found in human plasma (Mr = 150 k) and a small GH-independent BP (Mr = 28-42 k) present in human plasma, amniotic fluid, and HEP G2 cells. Using affinity cross-linking techniques, we have identified several binding proteins secreted by human breast cancer cell lines (Hs578T, MDA-231, T-47D, and MCF-7). Under nonreducing conditions these proteins migrated at an apparent Mr = 35, 28, 27, and 24 k, while reducing conditions revealed bands of apparent Mr = 35, 32, 27, and 24 k. Competitive binding studies in T-47D-conditioned media demonstrated that these BPs bound more IGF-II than IGF-I, and that IGF-II potently inhibited binding of either IGF-I or -II. Immunological studies using a polyclonal antibody against the HEP G2 small BP revealed no immunoreactive BP in conditioned media from MCF-7 and T-47D and only slight immunoreactivity in conditioned media from Hs578T and MDA 231. Analysis by Northern blot, using a probe from the cDNA sequence of the HEP G2 BP, demonstrated that Hs578T and MDA-231 cell lines contained small amounts of the 1.65 kilobase mRNA characteristic of the HEP G2 BP, while MCF-7 and T-47D tested negative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secretion of prosaposin, a multifunctional protein, by breast cancer cells.

Western blotting and immunodetection with three antibodies were used to probe conditioned media of breast cancer cells (MDA231, MDA435, MCF-7) for prosaposin, a lysosomal protein that occurs in milk. It was readily detected in media from these cells, and from that of an sv40-transformed mammary epithelial cell, HBL100, but not from medium of human neural tumor cells (SK-N-MC). In cultures of MCF-7 cells, the prosaposin pattern of secretion over time closely resembled that of procathepsin D, another lysosomal protein occurring in milk. Supplementing medium with 17beta-estradiol (0. 1-100 nM) dose dependently increased secretion of both proteins after 48 h without changes in cell viability. The influence of 17beta-estradiol on secretion could play a role in the trophic activity of prosaposin in cellular differentiation and cell death protection. In concert with other lysosomal proteins in the tumor environment, such as procathepsin D, prosaposin may be a factor in eliminating barriers to tumor metastasis by facilitating hydrolysis of membrane glycolipids. The number of milk proteins known to be secreted by breast cancer cells is growing. There is evidence that at least some of these may be secreted in an endocrine manner in the normal, non-lactating breast.     

Regulation of breast cancer growth by insulin-like growth factors

The IGFs may be important autocrine, paracrine or endocrine growth factors for human breast cancer. IGF-I and II stimulate growth of cultured human breast cancer cells. IGF-I is slightly more potent, paralleling its higher affinity for the IGF-I receptor. Antibody blockade of the IGF-I receptor inhibits growth stimulation induced by both IGFs, suggesting that this receptor mediates the growth effects of both peptides. However, IGF-I receptor blockade does not inhibit estrogen (E₂)-induced growth suggesting that secreted IGFs are not the major mediators of E₂ action. Several breast cancer cell lines express IGF-II mRNA by both Northern analysis and RNase protection assay. IGF-II activity is found in conditioned medium by radioimmuno and radioreceptor assay, after removal of somatomedin binding proteins (BP) which are secreted in abundance. IGF-I is undetectable. BPs of 25 and 40 K predominate in ER-negative cell lines while BPs of 36 K predominate in ER-positive cells. Blockade of the IGF-I receptor inhibits anchorage-independent and monolayer growth in serum of a panel of breast cancer cell lines. Growth of one line (MDA-231) was also inhibited in vivo by receptor antibody treatment of nude mice. The antibody had no effect on growth of MCF-7 tumors. These data suggest the IGFs are important regulators of breast cancer cell proliferation and that antagonism of this pathway may offer a new treatment strategy.     

Increased autocrine production of insulin-like growth factor II (IGF-II) alters serum sensitivity of MCF-7 human breast cancer cell proliferation

Abstract. Regulation of the growth of breast cancer cells is the result of a complex interaction between steroid hormones and growth factors, and in particular of oestrogen and insulin-like growth factors (IGF). Alteration of any one mitogenic component can affect the cell response to other pathways. Previous work has shown that increased autocrine production of IGF-II from a transfected inducible expression vector can result in reduced oestrogen sensitivity of growth of MCF-7 human breast cancer cells. This report describes alterations to non-oestrogen regulated pathways of cell growth following enhanced IGF-II expression in these transfected MI7 cells. Serum sensitivity of cell growth in the absence of oestrogen was found to differ between MI7 and untransfected MCF-7 cells, in that growth of MI7 but not MCF-7 cells was strongly inhibited by high serum levels. Increased serum had no effect on levels of IGF-II mRNA, IGFIR, IGFBP4 mRNA, or IGFBP secreted in MI7 cells. However, growth inhibition by serum in MI7 cells could be overcome by increasing levels of IGF-II in the serum or by removal of IGFBP onto polycarbonate membranes. Thus, the growth inhibition by serum in MI7 cells is concluded to result from the increased levels of IGFBP added with higher serum. This would support an inhibitory role for IGFBP on growth of breast cancer cells when cell growth is being driven by IGF pathways in the absence of oestrogen, and would suggest that cellular sensitivity to such factors can depend on levels of endogenous IGF production.     

Altered cathepsin D metabolism in PHEX antisense human osteoblast cells

X-linked hypophosphatemia (XLH), the most common form of hereditary rickets, is caused by loss-of-function mutations of PHEX gene in osteoblast cells, leading to rachitic bone disease and hypophosphatemia. Available evidence today indicates that the bone defect in XLH is caused not only by hypophosphatemia and altered vitamin D metabolism, but also by locally released osteoblastic mineralization inhibitory factor(s), referred to as minhibin. In our present study, we found that suppression of PHEX expression by PHEX antisense in human osteoblast cells caused an increase in cathepsin D expression at protein, but not mRNA, levels. This was associated with a decrease in cathepsin D degradation and an increased cathepsin D release into culture media. Our results also showed that lowering cathepsin D activity in antisense cell conditioned media abolished their inhibitory effect on osteoblast cell calcification, suggesting the involvement of cathepsin D in mediating the minhibin activity of the antisense cell conditioned media.     

Breast cancer cells express cathepsins B and L but not cathepsins K or H

Lysosomal cysteine proteinases (cathepsins) are considered to play a role in bone degradation mediated by metastatic breast cancers. To evaluate which cathepsin contributes to the osteolysis, we quantitatively determined the expression levels of four cathepsins in two breast cancer cell lines, MCF-7 and MDA-MB-231, by competitive RT-PCR. Cathepsin K, which is the most abundant cathepsin in osteoclasts, was not detected in either cell lines. We also failed to detect cathepsin H mRNA. By contrast, we found significant expression of cathepsins B and L in both cell lines. By Northern blot analysis cathepsin B mRNA was detected in a single form in these cells, whereas osteoclasts contained multiple forms of the mRNA. Cathepsin B protein was also detected by Western blotting as a single immunoreactive band corresponding to its mature enzyme. These findings suggest that osteolysis associated with metastatic breast cancers takes place in a different way from osteoclast-mediated bone resorption.     

Expression of transforming growth factor alpha and its messenger ribonucleic acid in human breast cancer: its regulation by estrogen and its possible functional significance

We have studied the estrogenic regulation and the potential autocrine role of transforming growth factor alpha (TGF alpha) in the human breast cancer cell line MCF-7. A biologically active apparent mol wt 30 k TGF alpha was identified by gel filtration chromatography in medium conditioned by MCF-7 breast cancer cells. We previously reported induction of TGF alpha levels in medium by 17 beta-estradiol. We now report correlated increases in TGF alpha mRNA, by Northern and slot blot analysis, after estrogen treatment of MCF-7 cells in vitro. In vivo experiments confirmed these data: estrogen withdrawal from MCF-7 tumor-bearing nude mice resulted in a decline in tumor size and TGF alpha mRNA levels. To explore the functional significance of TGF alpha in MCF-7 cells, anti-TGF alpha antibody was added to MCF-7 soft agar cloning assays. Inhibition of MCF-7 growth resulted, supporting an autocrine role for TGF alpha. Further experiments using an anti-EGF receptor antibody expanded this data, demonstrating inhibition of estrogen-stimulated monolayer MCF-7 cell growth. Examining the generality of TGF alpha expression, 4.8 kilobase TGF alpha mRNAs were seen in three other human breast cancer cell lines, MDA-MB-231, ZR 75B, and T47D. Expression of TGF alpha mRNA was detected in 70% of estrogen receptor positive and negative primary human breast tumors from 40 patients when examined by slot blot and Northern analysis. Thus, we have demonstrated broad expression of TGF alpha in human breast cancer, its hormonal regulation in an estrogen-responsive cell line, and its possible functional significance in MCF-7 cell growth.     

Autocrine production of insulin-like growth factor II using an inducible expression system results in reduced estrogen sensitivity of MCF-7 human breast cancer cells.

An inducible expression vector, utilizing the metal response elements from the human metallothionein IIA promoter and encoding human prepro-insulin-like growth factor II (IGF-II), was transfected into MCF-7 McG cells, an MCF-7 subline which exhibits an estrogen dependent phenotype in vitro and does not express detectable levels of IGF-II mRNA. Two stably transfected clones, designated MI5 and MI7, which expressed IGF-II mRNA in a Zn2+ regulated manner, were isolated. Clone MI5 did not secrete detectable levels of IGF-II activity, as determined by radioimmunoassay of conditioned medium, but clone MI7 secreted high levels of IGF-II activity in a Zn2+ inducible fashion. Clone MI5 and control clones transfected with the selection plasmid pSV2 neo and the control plasmid pSP65 continued to display an estrogen dependent phenotype in vitro. However, under both anchorage dependent and anchorage independent growth conditions, clone MI7 cells exhibited an estrogen responsive, rather than dependent, phenotype. Moreover, when grown in the presence of inducing concentrations of Zn2+, MI7 cells were either virtually (for anchorage dependent growth) or completely (anchorage independent growth) unresponsive to estradiol. Both the basal growth rate in the absence of metal ions and the Zn2+ induced increases in cell proliferation could be inhibited by the monoclonal antibody alpha-IR3, which blocks the binding site of the IGF-I receptor. The antiestrogens tamoxifen and 4-hydroxytamoxifen were found to enhance the growth stimulation resulting from Zn2+ induced IGF-II production in MI7 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estradiol down-regulates the mannose-6-phosphate/insulin-like growth factor-II receptor gene and induces cathepsin-D in breast cancer cells: a receptor saturation mechanism to increase the secretion of lysosomal proenzymes.

We have studied the regulation by estradiol of the mannose-6-phosphate (Man-6-P)/insulin-like growth factor-II (IGF-II) receptor concentration in different breast cancer cell lines. The mRNA level was assayed by Northern blot using the H5.1 cDNA probe. The protein level was assayed by Western ligand blot, by binding saturation with [125I]procathepsin-D on total membrane preparations, and by immunoprecipitation of 35S-labeled proteins. In three estrogen receptor-positive cell lines (MCF7, T47D, and ZR75-1), estradiol specifically decreased the steady state level of the Man-6-P/IGF-II receptor protein and mRNA. Moreover, in different cell lines and in primary culture of normal mammary cells, the secretion of procathepsin-D was inversely correlated with the level of Man-6-P/IGF-II receptor protein and mRNA. We conclude that estradiol down-regulates the Man-6-P/IGF-II receptor in breast cancer cells. Since two of its ligands, procathepsin-D and IGF-II, are induced by estrogen, we propose that the Man-6-P/IGF-II receptor becomes saturated after estrogen treatment. This model might explain the previously described estrogen-induced secretion of procathepsin-D and other lysosomal proenzymes routed by the same transport system.     

Tamoxifen induces TGF-β1 activity and apoptosis of human MCF-7 breast cancer cells in vitro

We report here that the antiestrogen tamoxifen (TAM) induces cell death in human breast cancer cell line MCF-7. We assessed the type of cell death induced by TAM in this breast cancer cell line on the basis of morphological and biochemical characteristics. Dying cells showed morphological characteristics of apoptosis, such as chromatin condensation and nuclear disintegration. DNA isolated from these cells revealed a pattern of distinctive DNA bands on agarose gel. The DNA fragmentation in MCF-7 cells induced by TAM could also be detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling. Northern blot hybridization revealed a substantial increase in the amounts of TRPM-2 and TGF-β1 mRNAs in MCF-7 cells after treatment with TAM. In contrast, the mRNA level of the estrogen-induced pS2 gene was strongly suppressed. The biological activity of TGF-β was increased at least fourfold in the media from MCF-7 cells treated with TAM. The results presented in this study suggest that TAM induces apoptosis of MCF-7 cells and it may be mediated by the secretion of active TGF-β. © 1996 Wiley-Liss, Inc.     

Expression of insulin-like growth factor-II and insulin-like growth factor binding proteins during Caco-2 cell proliferation and differentiation

The components of the insulin-like growth factor (IGF) axis and their roles in regulating proliferation and differentiation of the human colon adenocarcinoma cell line, Caco-2, have been investigated. Caco-2 cells proliferated in serum-free medium at 75% the rate observed in medium containing 10% fetal bovine serum. IGF-I (10 nM) increased Caco-2 cell growth in serum-free medium, but not to the rate seen with serum. Multiple IGF-II mRNA species were produced by Caco-2 cells, but IGF-I mRNA was undetectable. Secretion of radioimmunoassayable IGF-II corresponded with steady-state levels of IGF-II mRNA, neither of which was observed to change markedly over the course of 16 days of Caco-2 cell differentiation. Levels of sucrase-isomaltase mRNA, a marker for enterocytic differentiation, increased 12-fold between days 5 and 16 of culture. Northern blotting of total RNA and ligand blot and immunoblot analyses of serum-free conditioned medium revealed that Caco-2 cells produce several IGF binding proteins (IGFBPs), including IGFBP-2, -3, and -4, as well as a 31,000 M, species that was not identified. The pattern of IGFBP secretion changed dramatically during Caco-2 cell differentiation: IGFBP-3 and IGFBP-2 increased 8.5-fold and 5-fold, respectively, whereas IGFBP-4 and the 31,000 M, species decreased 43% and 90%. Caco-2 cell clones stably transfected with a human IGFBP-4 cDNA construct exhibited a 60% increase in steady-state level of IGFBP-4 mRNA, and secreted twice as much IGFBP-4 protein as controls. Moreover, IGFBP-4-overexpressing cells proliferated at only 25% the rate of control cells in serum-free medium, in conjunction with a 70% increase in expression of sucrase-isomaltase. In summary, these studies indicate that a complex IGF axis is involved in autocrine regulation of Caco-2 cell proliferation and differentiation. © 1996 Wiley-Liss, Inc.     

Modulation of glutathione peroxidase expression by selenium: effect on human MCF-7 breast cancer cell transfectants expressing a cellular glutathione peroxidase cDNA and doxorubicin-resistant MCF-7 cells.

We have studied the effect of selenium on the expression of a cellular glutathione peroxidase, GSHPx-1, in transfected MCF-7 cells and in doxorubicin-resistant (Adrr) MCF-7 cells. A GSHPx-1 cDNA with a Rous Sarcoma virus promoter was transfected into a human mammary carcinoma cell line, MCF-7, which has very low endogenous cytosolic glutathione (GSH) peroxidase activity and no detectable message. The transfectant with the highest GSH peroxidase activity among the isolates, MCF-7H6, was characterized. Adrr MCF-7 cells, a subline of MCF-7 cells, also has elevated GSH peroxidase activity. GSH peroxidase expressed by MCF-7H6 and Adrr MCF-7 cells is similar to the endogenous GSHPx-1 based on molecular weight, immunoreactivity, and metabolic labeling with 75Se. MCF-7H6 and Adrr MCF-7 cells grown in Se-deficient media had 2.6 +/- 2.4 (mean +/- S.D.) and 4.2 +/- 3.6 units/mg protein of GSH peroxidase specific activity, respectively. Se supplementation increased GSH peroxidase activity in a concentration- and time-dependent fashion. Enzymatic activity reached a level of 164 +/- 62 in MCF-7H6 cells and 114 +/- 27 in Adrr MCF-7 cells within 5 days of growth in media supplemented with 30 nM Se. Northern analysis revealed that Se-deficient MCF-7H6 cells expressed 2.1 +/- 0.4-fold less GSHPx-1 mRNA than their Se-sufficient counterparts. Similarly, Se-deficient Adrr MCF-7 cells expressed 3.3 +/- 1.8-fold less GSHPx-1 mRNA than their Se-supplemented counterparts after the quantity of mRNA was normalized with beta-actin. These studies suggest that modulation of GSH peroxidase activity by Se in both MCF-7H6 transfectants expressing pRSV-GSHPx-1 and Adrr MCF-7 cells expressing endogenous GSHPx-1 occurs largely at the translational level, and to a lesser degree at the level of mRNA, possibly by stabilizing GSHPx-1 mRNA since the transfected cDNA in MCF-7H6 cells has only 5 nucleotides 5' to the AUG initiation codon.     

siRNA-cyclin D1对乳腺癌MCF-7细胞增殖的抑制作用

研究小干扰RNA(small interfering RNA,siRNA)对乳腺癌MCF-7细胞株cyclin D1表达的抑制及对细胞增殖的影响。化学合成针对cyclin D1基因的siRNA,转染MCF-7细胞株;分别应用荧光定量PCR和免疫印迹测定cyclin D1 mRNA和蛋白的表达,CCK-8测定细胞的增殖活性,流式细胞仪检测细胞周期,软琼脂培养检测细胞克隆形成能力。在实验中,10、50、100 nmol/L siRNA-cyclin D1分别使MCF-7细胞cyclin D1 mRNA表达降低了57．85%、63．22%和68．02%,蛋白表达降低了51．13%、62．09%、77．68%。转染siRNA-cyclin D1后,细胞增殖受到抑制,细胞周期阻滞于G1期,软琼脂克隆形成率降低。结果提示siRNA可以有效抑制MCF-7细胞株中cyclin D1的表达,使细胞周期阻滞于G1期,从而抑制细胞增殖。     

Androgens increase osteoblast-stimulating activity of human breast cancer cells in vitro

Bone metastases of breast cancers produce not only osteolytic but also osteosclerotic lesions. The latter are often observed after androgenic treatment of the tumor. Potential production of osteoblast stimulating activity (ObSA) in breast cancer cell lines, and possible androgen control of this activity have been investigated. Conditioned media (CM) collected from 4 breast cancer cell lines (MCF-7, ZR75, MDA-MB 231, BT20) was tested in vitro on ROS 17/2,8 osteoblast-like cells and on osteoblasts derived from human bone biopsies. The parameters monitored in osteoblasts were [3H]thymidine incorporation, alkaline phosphatase activity, and osteocalcin secretion. Serum-free media conditioned during 24 h by MCF-7 cells presented the highest ObSA. CM decreased thymidine incorporation in DNA and increased alkaline phosphatase activity in a dose-dependent manner. Bone GLA protein (osteocalcin) secretion by human osteoblasts was not increased however in the presence of CM. MCF-7 cells were cultured in the presence of dihydrotestosterone (DHT) [1-100 nM] for 5 days. Serum-free, DHT-free CM collected after an additional 24 h, contained alkaline-phosphatase stimulating activity which was DHT dose-dependent. Estradiol and 1,25(OH)2D3 failed to elicit a comparable increase of the ObSA in the CM. In conclusion, MCF-7 cells product factor(s) that interfere with bone remodeling. The DHT modulation of ObSA parallels the estradiol control of MCF-7 cells osteolytic lesions in relation with Prostaglandin E secretion. Sex hormones at physiological and pharmacological levels might thus control both osteosclerotic and osteolytic lesions observed in bone deposits of hormone dependent cancers.     

Insulin-like growth factor-binding protein enhancement of insulin-like growth factor-i (IGF-I)–mediated DNA synthesis and IGF-I binding in a human breast carcinoma cell line

The insulin-like growth factors (IGFs) are potent mitogens for malignant cell proliferation. The majority of secreted IGFs are bound to specific IGF-binding proteins (IGFBPs) that are secreted by a large number of cells. These proteins may either inhibit or enhance IGF actions. Breast carcinoma cells secrete a variety of IGFBPs. We have previously demonstrated that retinoic acid (RA) inhibition of IGF-l– stimulated MCF-7 cell proliferation is associated with increased IGFBP-3 levels in the conditioned media. We therefore investigated the effect of recombinant IGFBP-3 as well as IGFBP-2, -4 and -5 on IGF-l stimulation of DNA synthesis and IGF-I binding in the MCF-7 human breast carcinoma cell line. IGFBP-2 and -3 enhanced IGF-l stimulation of DNA synthesis in MCF-7 cells while IGFBP-4 and -5 had no effect. Transfection of MCF-7 cells with an IGFBP-3 expression vector resulted in the enhanced secretion of IGFBP-3 with an accompanying increase in IGF-l binding as well as increased cell proliferation upon treatment of the cells with IGF-l. IGF-l preincubation of MCF-7 cells transfected with control pSVneo plasmids results in cells refractory to further IGF-l stimulation of thymidine incorporation while IGF-l continues to stimulate [³H]-thymidine incorporation in IGFBP-3–transfected MCF-7 cells, suggesting that IGFBP-3 protects the cells from IGF-l–mediated down regulation of its receptor. Therefore, IGFBP-3 secreted by MCF-7 cells can enhance IGF-l stimulation of DNA synthesis, increase IGF-l binding to these cells, and prevent IGF-l–induced desensitization of its own receptor, suggesting that IGFBP-3 plays a significant role in IGF-l–mediated breast carcinoma proliferation. © 1994 Wiley-Liss, Inc.