用非损伤微测技术研究肿瘤细胞的耐药性与其胞外H+流变性的相关性

介绍了人乳腺癌细胞(药物敏感株MCF-7/S及耐药株MCF-7/R)在化疗药物阿霉素(adriamycin,ADR)处理下,细胞外pH和H 流动方向和速率的变化.为此,建立了一种基于非损伤微测技术(non-invasive micro-test technique,NMT)的药物抗性研究方法(drug resistance study method,DRSM),该方法可用于研究器官/组织/细胞外离子/分子活性与肿瘤细胞耐药性之间的相互关系.结果显示存在一个持续的并以固有振荡形式出现的胞外H 流现象.此外,耐药株净H 流在加ADR前趋近于零,而敏感株净H 流呈明显内流.敏感株和耐药株加ADR后净H 均呈外流,但耐药株的净H 外流速率要高于敏感株5倍.与净H 流动速率结果相一致的是胞外的pH也产生了相应的变化.因此,实验为胞外H 活性与肿瘤耐药性的相互关联提供了直接证据.     

MCF-7/VD(R): a new vitamin D resistant cell line

Several in vitro and in vivo experiments have demonstrated potent cell regulatory effects of vitamin D compounds in cancer cells. Moreover, a promising phase I study with the vitamin D analogue Seocalcitol (EB 1089) in patients with advanced breast and colon cancer has already been carried out and more clinical trials evaluating the clinical effectiveness of EB 1089 in other cancer types are in progress (M?rk Hansen et al. [2000a]). However, only little is known about the mechanisms underlying the actions of vitamin D or about the possible development of drug resistance in the patients. Therefore, in an attempt to gain more insight into these aspects, we have developed the MCF-7/VD(R) cell line, a stable subclone of the human MCF-7 breast cancer cell line, which is resistant to the growth inhibitory and apoptosis inducing effects of 1alpha,25(OH)(2)D(3). Despite this characteristic, receptor studies on the VDR have clearly demonstrated that the MCF-7/VD(R) cells contain fully functional VDRs, although in a lower number than seen with the parental MCF-7 cells. The regulation of the 24-hydroxylase enzyme appeared to be intact in the MCF-7/VD(R) cells and no differences with regard to growth rate and morphological appearance between the MCF-7/VD(R) cells and the parental MCF-7 cells were observed. Interestingly, however, the sensitivity of the MCF-7/VD(R) cells to the pure anti-estrogen ICI 182,780 was found to be increased. The MCF-7/VD(R) cell line shows characteristics different from those of previously described vitamin D resistant breast cancer cell lines but also some similarities. Together such vitamin D resistant cell lines therefore serve as a useful tool for studying the exact mechanism of action of vitamin D and the development of vitamin D resistance.     

Metabolomic approach to evaluating adriamycin pharmacodynamics and resistance in breast cancer cells

Continuous exposure of breast cancer cells to adriamycin induces high expression of P-gp and multiple drug resistance. However, the biochemical process and the underlying mechanisms for the gradually induced resistance are not clear. To explore the underlying mechanism and evaluate the anti-tumor effect and resistance of adriamycin, the drug-sensitive MCF-7S and the drug-resistant MCF-7Adr breast cancer cells were used and treated with adriamycin, and the intracellular metabolites were profiled using gas chromatography mass spectrometry. Principal components analysis of the data revealed that the two cell lines showed distinctly different metabolic responses to adriamycin. Adriamycin exposure significantly altered metabolic pattern of MCF-7S cells, which gradually became similar to the pattern of MCF-7Adr, indicating that metabolic shifts were involved in adriamycin resistance. Many intracellular metabolites involved in various metabolic pathways were significantly modulated by adriamycin treatment in the drug-sensitive MCF-7S cells, but were much less affected in the drug-resistant MCF-7Adr cells. Adriamycin treatment markedly depressed the biosynthesis of proteins, purines, pyrimidines and glutathione, and glycolysis, while it enhanced glycerol metabolism of MCF-7S cells. The elevated glycerol metabolism and down-regulated glutathione biosynthesis suggested an increased reactive oxygen species (ROS) generation and a weakened ability to balance ROS, respectively. Further studies revealed that adriamycin increased ROS and up-regulated P-gp in MCF-7S cells, which could be reversed by N-acetylcysteine treatment. It is suggested that adriamycin resistance is involved in slowed metabolism and aggravated oxidative stress. Assessment of cellular metabolomics and metabolic markers may be used to evaluate anti-tumor effects and to screen for candidate anti-tumor agents.     

Interactions between normal mammary epithelial cells and mammary tumour cells in a model system

Abstract. Normal mammary epithelial (NME) cells and MCF-7 cells aggregate and grow as spheroids when cultured on extracellular matrix derived from Engelbreth/ Holmes/Swarth (EHS) tumour. NME cells stop dividing and differentiate but MCF-7 cells continue to proliferate, although growth is counterbalanced by cell death. In mixed cultures of NME cells and MCF-7 cells, the two cell types form mixed aggregates but then segregate to form well separated domains, often joined by only a narrow neck of cells. In these mixed cultures the growth of MCF-7 cells is inhibited by a factor secreted by NME cells into the medium.     

Positive regulation of normal and tumoral mammary epithelial cell proliferation by fibroblasts in coculture

Summary In the mammary gland, mesenchymal-epithelial interactions are of paramount importance during normal and tumoral developments. We have studied the paracrine growth regulation of a variety of breast epithelial cells in coculture with normal or pathological breast fibroblasts. Two models of coculture were used in which the two cell types were seeded and grown, either together in microchamber slides or separated by a microporous membrane. Under these two conditions, all fibroblasts were shown to stimulate the proliferation of the hormono-responsive breast carcinoma MCF-7 cell line, suggesting that cell contacts were not indispensable for the paracrine stimulation of MCF-7 cell growth by fibroblasts. Moreover, in the Transwell coculture system, the proliferation of a variety of other breast carcinoma cells (MDA-MB231, T47D, and BT-20) was also stimulated by fibroblasts. However, the amplitude of the proliferative response seemed to be dependent on the carcinoma cell line considered. Moreover, the proliferative response of normal mammary epithelial cells to the presence of fibroblasts was shown to be significantly higher than the tumor cell response. The nature of the tissue of fibroblast origin, normal or pathological, did not influence the growth response of the epithelial cells. In this study, we thus demonstrate that fibroblasts are able to stimulate the proliferation of normal and carcinoma cells through paracrine exchange mechanisms. We also conclude that the target epithelial cell phenotype will essentially determine the extent of the proliferative response.     

Novel ceramide analogues display selective cytotoxicity in drug-resistant breast tumor cell lines compared to normal breast epithelial cells.

The sphingolipid ceramide is involved in diverse cell signaling pathways related to proliferation and differentiation. Elevated ceramide also triggers apoptosis. Synthetic ceramide derivatives have been shown to be cytotoxic to tumors, yet few studies have evaluated whether cytotoxicity of synthetic ceramides is selective for tumor cells. We have evaluated the cytotoxic potency of several novel ceramide analogues in the drug-resistant breast tumor cell lines, SKBr3 and MCF-7/Adr, and compared their cytotoxicity in normal breast epithelial cells. Cytotoxicity was assessed using release of lactate dehydrogenase into the culture medium. (2S, 3S)-3-(6'-Dodecylpyridin-2'-yl)-2-butanoylamidopropane-1,3-diol (pyridine-C4-ceramide) produced non-selective cytotoxicity across the three cell types (EC50= 12.8-16.7 microM, at 24 hr). However, 2S,5R-2-(octanoylamido-(3E))-octadecene-1,5-diol (5R-OH-3E-C8-ceramide), (2S,3R)-2-(N-adamantoyl)-(4E)-octadecen-1,3-diol (adamantyl-ceramide), and (2S,3R)-3-(3'-dodecylphenyl)-2-butanoylamidopropane-1,3-diol (benzene-C4-ceramide) exhibited increased cytotoxicity in the tumor cell lines compared to the normal breast epithelial cells. The EC50 values (microM) at 24 hr for these compounds in SKBr3 cells, MCF-7/Adr cells, and normal breast epithelial cells, respectively, were as follows: 5R-OH-3E-C8-ceramide, 18.3, 21.2 and 58.7; adamantyl-ceramide, 10.9, 24.9 and >100; benzene-C4-ceramide, 18.9, 45.5 and >100. At a concentration of 30 microM, the fold increase in cytotoxicity in breast tumor cell lines compared with normal breast epithelial cells was as follows: 5R-OH-3E-C8-ceramide, 23.7 and 19; adamantyl-ceramide, 11.2 and 10.3 and benzene-C4-ceramide, 79.3 and 77.2, for SKBr3 and MCF-7/Adr cells, respectively. Possible mechanisms accounting for selectivity are discussed. Ceramide analogues with relatively selective toxicity against tumor cells may have potential as therapeutic agents. Elucidating the mechanisms of selective cytotoxicity could identify novel targets that may lead to development of anti-neoplastic agents with a higher therapeutic index.     

Inhibition of MCF-7 breast cancer cell proliferation by MCF-10A breast epithelial cells in coculture

A coculture system was developed to investigate the interactions between MCF-10A breast epithelial cells and MCF-7 breast cancer cells stably expressing the green fluorescent protein (MCF-7-GFP). Studies with this MCF-10A/MCF-7-GFP coculture system on microtiter plates and on reconstituted basement membrane (Matrigel), revealed paracrine inhibition of MCF-7-GFP cell proliferation. Epidermal growth factor, which in monocultures modestly enhanced MCF-7-GFP and markedly increased MCF-10A cell proliferation, greatly inhibited MCF-7-GFP cell proliferation in MCF-10A/MCF-7-GFP cocultures. 17beta-Estradiol, which stimulated MCF-7-GFP but not MCF-10A cell proliferation in monoculture, inhibited MCF-7-GFP cell proliferation in MCF-10A/MCF-7-GFP cocultures, an effect that was blocked by the antiestrogen, ICI 182,780. On Matrigel, complex MCF-10A/MCF-7-GFP cellular interactions were observed in real time that resulted in the formation of acinus-like structures. These results indicate a role of normal epithelial cells in inhibiting tumor-cell proliferation and demonstrate the utility of this coculture system as a model of early paracrine control of breast cancer.     

Tumor cell apoptosis polarizes macrophages role of sphingosine-1-phosphate

Macrophage polarization contributes to a number of human pathologies. This is exemplified for tumor-associated macrophages (TAMs), which display a polarized M2 phenotype, closely associated with promotion of angiogenesis and suppression of innate immune responses. We present evidence that induction of apoptosis in tumor cells and subsequent recognition of apoptotic debris by macrophages participates in the macrophage phenotype shift. During coculture of human primary macrophages with human breast cancer carcinoma cells (MCF-7) the latter ones were killed, while macrophages acquired an alternatively activated phenotype. This was characterized by decreased tumor necrosis factor (TNF)-alpha and interleukin (IL) 12-p70 production, but increased formation of IL-8 and -10. Alternative macrophage activation required tumor cell death because a coculture with apoptosis-resistant colon carcinoma cells (RKO) or Bcl-2-overexpressing MCF-7 cells failed to induce phenotype alterations. Interestingly, phenotype alterations were achieved with conditioned media from apoptotic tumor cells, arguing for a soluble factor. Knockdown of sphingosine kinase (Sphk) 2, but not Sphk1, to attenuate S1P formation in MCF-7 cells, restored classical macrophage responses during coculture. Furthermore, macrophage polarization achieved by tumor cell apoptosis or substitution of authentic S1P suppressed nuclear factor (NF)-kappaB signaling. These findings suggest that tumor cell apoptosis-derived S1P contributes to macrophage polarization.     

Synthesis and cytotoxicity evaluation of 22,23-oxygenated stigmastane derivatives

Starting from (22E)-3alpha,5alpha-cyclo-6beta-methoxystigmast-22-ene eighteen derivatives of (22S,23S)-22,23-oxidostigmastane, (22R,23R)-22,23-oxidostigmastane, and (22R,23R)-22,23-dihydroxystigmastane were synthesized and screened for cytotoxicity in human hepatoma Hep G2 cells and human breast carcinoma MCF-7 cells using MTT assay. Four compounds of this series exhibited high cytotoxicity in both cells; three compounds were selectively toxic in MCF-7 cells, one compound was toxic in Hep G2 cells, rather than in MCF-7 cells; four compounds at low concentrations increased MTT test values over the control.     

Scaffold-Free Coculture Spheroids of Human Colonic Adenocarcinoma Cells and Normal Colonic Fibroblasts Promote Tumorigenicity in Nude Mice

The aim of this study was to form a scaffold-free coculture spheroid model of colonic adenocarcinoma cells (CACs) and normal colonic fibroblasts (NCFs) and to use the spheroids to investigate the role of NCFs in the tumorigenicity of CACs in nude mice. We analysed three-dimensional (3D) scaffold-free coculture spheroids of CACs and NCFs. CAC Matrigel invasion assays and tumorigenicity assays in nude mice were performed to examine the effect of NCFs on CAC invasive behaviour and tumorigenicity in 3D spheroids. We investigated the expression pattern of fibroblast activation protein-α (FAP-α) by immunohistochemical staining. CAC monocultures did not form densely-packed 3D spheroids, whereas cocultured CACs and NCFs formed 3D spheroids. The 3D coculture spheroids seeded on a Matrigel extracellular matrix showed higher CAC invasiveness compared to CACs alone or CACs and NCFs in suspension. 3D spheroids injected into nude mice generated more and faster-growing tumors compared to CACs alone or mixed suspensions consisting of CACs and NCFs. FAP-α was expressed in NCFs-CACs cocultures and xenograft tumors, whereas monocultures of NCFs or CACs were negative for FAP-α expression. Our findings provide evidence that the interaction between CACs and NCFs is essential for the tumorigenicity of cancer cells as well as for tumor propagation.     

Influence of interferons on the repair of UV-damaged DNA

The capacity for nucleotide excision repair of a normal (WISH) and three tumour (MCF-7, HeLa, Namalva) cell lines treated with human recombinant interferons (hrIFN-alpha and hrIFN-gamma) was compared by the host cell reactivation assay. The cells were transfected with in vitro UV-damaged plasmid DNA (pEGFP-N1). The repair capacity was determined by measuring the fluorescence intensity of the expressed marker protein in total cell lysates. The correlation between the interferon-induced NO content and the suppressive effect of interferons on DNA repair was shown. The decrease of repair activity and NO induction by hrIFN-alpha were greatest in WISH, followed by MCF-7, Namalva and HeLa cells, whereas hrIFN-gamma was the best NO inducer and inhibitor for the repair of Namalva, followed by WISH, MCF-7 and HeLa cells. Our data clearly show that the two types of interferon have a strong inhibitory effect on the repair of UV-damaged DNA and this effect is cell type-dependent.     

The potentiation of estrogen on insulin-like growth factor I action in MCF-7 human breast cancer cells includes cell cycle components

To gain insight into the mechanisms involved in the cross-talk between IGF-1 receptor (IGF-1R) and estrogen receptor signaling pathways, we used MCF-7-derived cells (SX13), which exhibit a 50% reduction in IGF-1R expression. Growth of NEO cells (control MCF-7 cells) was stimulated by both IGF-1 and estradiol (E2), and the addition of both mitogens resulted in a synergistic response. Estrogen enhanced IGF-1R signaling in NEO cells, but this effect was markedly diminished in SX13 cells. Estrogen was also able to potentiate the IGF-1 effect on the expression of cyclin D1 and cyclin E and on the phosphorylation of retinoblastoma protein in control but not in SX13 cells. IGF-1 increased the protein level of p21 and the luciferase activity of the p21 promoter, whereas it only reduced the protein level of p27 without affecting p27 promoter activity. Estrogen did not affect the p21 inhibitor, but it decreased the protein level of p27 and the p27 promoter luciferase activity. These effects of both mitogens were also observed at the level of association of both cyclin-dependent kinase inhibitors with CDK2 suggesting that IGF-1 and E2 affect the activity of both p21 and p27. Taken together, these data suggest that in MCF-7 cells, estrogen potentiates the IGF-1 effect on IGF-1R signaling as well as on the cell cycle components. Moreover, IGF-1 and E2 regulate the expression of p21 and p27 and their association with CDK2 differently.     

Regulation of steroid sulphatase expression and activity in breast cancer

Steroid sulphatase (STS) catalyzes the conversion of oestrone sulphate (E1S) to oestrone (E1) and its action in breast tumours makes a major contribution to in situ oestrogen production in this tissue. Although expression of STS mRNA and STS activity are increased in malignant breast tissues compared with that in non-malignant tissues, little is known about the regulation of its expression or activity. In the present study we have used a RT-PCR technique to investigate the regulation of STS mRNA expression in cultured breast tissue fibroblasts and MCF-7 cells. STS mRNA expression was readily detectable in fibroblasts derived from breast tissue proximal to tumours, breast tumour tissue and reduction mammoplasty tissue. For two pre-menopausal subjects, STS mRNA expression was similar in proximal and tumour fibroblasts whereas for a third, post-menopausal subject, expression in breast tumour fibroblasts was 2.4-fold that in proximal fibroblasts. The cytokine tumour necrosis factor alpha (TNFalpha) or the STS inhibitor, 2-methoxyoestrone-3-O-sulphamate, had no effect on STS mRNA expression in fibroblasts. STS mRNA was detectable in MCF-7 cells but neither TNFalpha nor interleukin 6 (IL-6) affected its expression. Transient transfection of COS-1 and MCF-7 cells with a STS cDNA lacking STS 5' and 3' sequences increased activity 17-fold and 2-fold, respectively. TNFalpha plus IL-6 increased STS activity in mock transfected MCF-7 cells and further increased STS activity in transfected MCF-7 cells. This indicates that activation can occur independently of STS promoter and enhancer elements. In conjunction with the lack of regulation of STS mRNA it suggest that TNFalpha and IL-6 may increase STS activity via a post-translational modification of the enzyme or by increasing substrate availability.     

Exosomes from Drug-Resistant Breast Cancer Cells Transmit Chemoresistance by a Horizontal Transfer of MicroRNAs

Adriamycin and docetaxel are two agents commonly used in treatment of breast cancer, but their efficacy is often limited by the emergence of chemoresistance. Recent studies indicate that exosomes act as vehicles for exchange of genetic cargo between heterogeneous populations of tumor cells, engendering a transmitted drug resistance for cancer development and progression. However, the specific contribution of breast cancer-derived exosomes is poorly understood. Here we reinforced other''s report that human breast cancer cell line MCF-7/S could acquire increased survival potential from its resistant variants MCF-7/Adr and MCF-7/Doc. Additionally, exosomes of the latter, A/exo and D/exo, significantly modulated the cell cycle distribution and drug-induced apoptosis with respect to S/exo. Exosomes pre-treated with RNase were unable to regulate cell cycle and apoptosis resistance, suggesting an RNA-dependent manner. Microarray and polymerase chain reaction for the miRNA expression profiles of A/exo, D/exo, and S/exo demonstrated that they loaded selective miRNA patterns. Following A/exo and D/exo transfer to recipient MCF-7/S, the same miRNAs were significantly increased in acquired cells. Target gene prediction and pathway analysis showed the involvement of miR-100, miR-222, and miR-30a in pathways implicated in cancer pathogenesis, membrane vesiculation and therapy failure. Furthermore, D/exo co-culture assays and miRNA mimics transfection experiments indicated that miR-222-rich D/exo could alter target gene expression in MCF-7/S. Our results suggest that drug-resistant breast cancer cells may spread resistance capacity to sensitive ones by releasing exosomes and that such effects could be partly attributed to the intercellular transfer of specific miRNAs.     

Toxicity of (22R,23R)-22,23-dihydroxystigmastane derivatives to cultured cancer cells

Toxicity of eight 22,23-dihydroxystigmastane derivatives (four pairs of (22R,23R)- and (22S,23S)-isomers differing in steroid backbone structure) to human breast carcinoma MCF-7 cells was compared. For every pair of structurally related compounds, (22R,23R) isomer was found to be significantly more toxic than (22S,23S) isomer. Computational analysis showed that side chain of (22R,23R)-22,23-dihydroxystigmastane derivatives is rigid, whereas that of (22S,23S)-isomers is rather flexible. Structure of steroid backbone significantly affects cytotoxicity of (22R,23R)-22,23-dihydroxystigmastane derivatives to human breast carcinoma MCF-7 cells, human ovary carcinoma CaOv cells, and human prostate carcinoma LnCaP cells. (22R,23R)-3β,22,23-trihydroxystigmast-5-ene and (22R,23R)-3β,22,23-trihydroxystigmast-5-en-7-one, both comprising equatorial 3β-hydroxyl group, exhibited the highest cytotoxicity, while the most polar 28-homobrassinolide and 28-homocastasterone, both comprising 2α,3α-dihydroxy groups, exhibited the lowest toxicity. Binding of (22R,23R)-22,23-dihydroxystigmastane derivatives to plasmatic membrane was suggested to be important for cytotoxicity.     

Fibroblast growth factor 8 increases breast cancer cell growth by promoting cell cycle progression and by protecting against cell death

Fibroblast growth factor 8 (FGF-8) is expressed in a large proportion of breast cancers, whereas its level in normal mammary gland epithelium is low. Previous studies have shown that FGF-8b stimulates breast cancer cell growth in vitro and in vivo. To explore the mechanisms by which FGF-8b promotes growth, we studied its effects on cell cycle regulatory proteins and signalling pathways in mouse S115 and human MCF-7 breast cancer cells. We also studied the effect of FGF-8b on cell survival. FGF-8b induced cell cycle progression and up-regulated particularly cyclin D1 mRNA and protein in S115 cells. Silencing cyclin D1 with siRNA inhibited most but not all FGF-8b-induced proliferation. Inhibition of the FGF-8b-activated ERK/MAPK pathway decreased FGF-8b-stimulated proliferation. Blocking the constitutively active PI3K/Akt and p38 MAPK pathways also lowered FGF-8b-induced cyclin D1 expression and proliferation. Corresponding results were obtained in MCF-7 cells. In S115 and MCF-7 mouse tumours, FGF-8b increased cyclin D1 and Ki67 levels. Moreover, FGF-8b opposed staurosporine-induced S115 cell death which effect was blocked by inhibiting the PI3K/Akt pathway but not the ERK/MAPK pathway. In conclusion, our results suggest that FGF-8b increases breast cancer cell growth both by stimulating cell cycle progression and by protecting against cell death.     

Radiation response of drug-resistant variants of a human breast cancer cell line

The radiation response of drug-resistant variants of the human tumor breast cancer cell line MCF-7 has been investigated. Two sublines, one resistant to adriamycin (ADRR) and the other to melphalan (MLNR), have been selected by exposure to stepwise increasing concentrations of the respective drugs. ADRR cells are 200-fold resistant to adriamycin and cross-resistant to a number of other drugs and are characterized by the presence of elevated levels of selenium-dependent glutathione peroxidase and glutathione-S-transferase. MLNR cells are fourfold resistant to melphalan and cross-resistant to some other drugs. The only mechanism of drug resistance established for MLNR cells to date is an enhancement of DNA excision repair processes. While the spectrum of drug resistance and the underlying mechanisms differ for the two sublines, their response to radiation is qualitatively similar. Radiation survival curves for ADRR and MLNR cells differ from that for wild-type cells in a complex manner with, for the linear-quadratic model, a decrease in the size of alpha and an increase in the size of beta. There is a concomitant decrease in the size of the alpha/beta ratio which is greater for ADRR cells than for MLNR cells. Analysis of results using the multitarget model gave values of D0 of 1.48, 1.43, and 1.67 Gy for MCF-7 cells are not a consequence of cell kinetic differences between these sublines. Results of split-dose experiments indicated that for both drug-resistant sublines the extent of sublethal damage repair reflected the width of the shoulder on the single-dose survival curve. For MCF-7 cells in the stationary phase of growth, the drug-resistant sublines did not show cross-resistance to radiation; however, delayed subculture following irradiation of stationary-phase cultures increased survival to a greater extent for ADRR and MLNR cells than for wild-type cells.