三苯氧胺对乳腺癌和宫颈癌细胞增殖的影响

目的：研究三苯氧胺(tamoxifen,TAM)对人乳腺癌Bcap-37和宫颈癌HeLa细胞增殖的影响并探讨其可能的机制。方法：采用细胞培养、细胞计数、MTT、流式细胞术和激光共聚焦显微镜技术。结果：TAM(10^-6mol／L)使Bcap-37细胞的生长曲线下移,使HeLa细胞的生长曲线上移。TAM(10^-8～10^-6mol／L)剂量依赖性的抑制Beap-37细胞的增殖,促进HeLa细胞的增殖作用。TAM(10^-6mol／L)使Bcap-37细胞发生凋亡,凋亡率达到97．5％,而使HeLa细胞周期由G1期加速向S期转化,G1期的DNA含量由对照组的55．5％下降到加药组的32．8％,S期的DNA含量由对照组的29．0％上升到加药组的49．4％。激光共聚焦检测到TAM(10^-6mol／L)可使Bcap-37细胞和HeLa细胞内的Ca^2 浓度显著升高。结论：TAM可以通过调节细胞周期各阶段DNA含量和胞内Ca^2 浓度水平,从而调节Bcap-37细胞和HeLa细胞的增殖活动,提示使用TAM治疗乳腺癌时可能会对子宫颈产生副作用。     

Effect of tamoxifen and 2-methoxyestradiol alone and in combination on human breast cancer cell proliferation

Endocrine therapy is widely accepted for the treatment of hormone receptor-positive breast cancer. However, in many cases eventually resistance will develop and tumor regrows. Combination therapy may be one way to resolve this problem. In the present study we investigated the effect of a combination of the widely used antiestrogen tamoxifen with the endogenous estradiol metabolite 2-methoxyestradiol (2-ME) on the proliferation of human estrogen receptor-positive and receptor-negative breast cancer cells.The receptor-positive cell line MCF-7 and the receptor-negative cell line BM were treated with 4-hydroxytamoxifen (4-OHTam) and 2-methoxyestradiol in the concentration range of 0.8-25 microM alone and equimolar combinations for 4 days. The proliferation of the cells was determined using the ATP-chemosensitivity test.4-Hydroxytamoxifen inhibited proliferation of MCF-7 and BM cells with IC(50) values of 31 and 10 microM, the corresponding figures for 2-methoxyestradiol were 52 and 8 microM. The combination showed IC(50) values of 6 microM and 4 microM.These results indicate that a combination of tamoxifen with 2-methoxyestradiol showed an additive inhibitory effect concerning the proliferation of estrogen receptor-positive and receptor-negative breast cancer cell lines. Thus a combination of these substances may allow ameliorating of adverse events of tamoxifen by reducing its concentrations and probably also drug resistance and should be tested in clinical trials.     

Reversible inhibition by human serum lipoproteins of cell proliferation

Normal human serum or plasma was studied for the presence of inhibitors of cell proliferation by assaying inhibition of incorporation of labeled thymidine into acid-insoluble fraction using human FL cells. Lipoprotein fraction obtained by gel filtration through Sepharose 4B and by KBr density gradient centrifugation was found to play a major part of the inhibitory activity of the serum. It was also shown that the inhibitory activity resides in low-density lipoprotein (LDL). The addition of the lipoprotein fraction to growing FL cells caused an early decrease in the transport of uridine and thymidine across the membrane. This change in the permeability of membrane was followed by the preferential inhibition of DNA synthesis and a reduction in the percentage of mitotic cells in the cell population. The inhibition of the growth was reversible and was observed in various types of cells irrespective of species.     

Genistein and erbstatin inhibition of normal mammary epithelial cell proliferation is associated with EGF-receptor down-regulation

Epidermal growth factor (EGF) is a potent mitogen for normal mouse mammary epithelial cells grown in primary culture. EGF activation of the EGF-receptor (EGF-R) induces intrinsic tyrosine kinase activity which results in EGF-R autophosphorylation and tyrosine phosphorylation of other intracellular substrates involved in EGF-R signal transduction. Genistein and erbstatin are anticancer agents which have been shown to be potent tyrosine kinase inhibitors. However, the effects of these compounds in modulating EGF-dependent normal mammary epithelial cell proliferation is presently unknown. Therefore, studies were conducted to determine the effects of genistein and erbstatin on EGF-dependent proliferation, and EGF-R levels and autophosphorylation in normal mouse mammary epithelial cells grown in primary culture and maintained in serum-free media. Chronic treatment with 6.25–100 μM genistein or 1–16 μM erbstatin significantly decreased EGF-dependent mammary epithelial cell proliferation in a dose-responsive manner. However, the highest doses of genistein (100 μM ) and erbstatin (16 μM ) were found to be cytotoxic. Additional studies showed that acute treatment with 6.25–400 μM genistein did not affect EGF-R levels or EGF-induced EGF-R autophosphorylation, while acute treatment with 1–64 μM erbstatin caused a slight reduction in EGF-R levels, but had no effect on EGF-dependent EGF-R autophosphorylation in these cells. In contrast, chronic treatment with similar doses of genistein or erbstatin resulted in a large dose-responsive decrease in EGF-R levels, and a corresponding decrease in total cellular EGF-R autophosphorylation intensity. These results demonstrate that the inhibitory effects of chronic genistein and erbstatin treatment on EGF-dependent mammary epithelial cell proliferation is not due to a direct inhibition of EGF-R tyrosine kinase activity, but results primarily from a down-regulation in EGF-R levels and subsequent decrease in mammary epithelial cell mitogenic-responsiveness to EGF stimulation.     

Inhibition of cellular proliferation and modulation of insulin-like growth factor binding proteins by retinoids in a bovine mammary epithelial cell line

Retinoids are potent inhibitors of growth and tumor progression in many mammary carcinoma cell lines, though regulation of growth in nontumorigenic mammary epithelial cells by retinoids is less clear. Here, we have characterized the inhibition of MAC-T (a nontransformed bovine mammary epithelial cell line) cellular proliferation by retinoids and their role in regulating insulin-like growth factor binding proteins (IGFBPs). Retinoic acid (RA) (100 nM) was a potent inhibitor of MAC-T cell proliferation. Retinol was 10–100 times less effective. Neither retinoid could completely arrest growth at noncytotoxic concentrations. Retinoic acid inhibited cellular proliferation by 1 h (P < .05), but inhibition was fivefold greater by 24 h (P < .01). This second stage of growth inhibition (after 12 h) was dependent upon protein synthesis. However, RA-induced inhibition of cellular proliferation did not persist, with thymidine incorporation increasing toward control levels by 4 days in culture. Retinoic acid was less effective in inhibiting thymidine incorporation when cells were stimulated with insulin, des(1–3) IGF-I, or Long(R³) IGF-I when compared to cells stimulated with native IGF-I or serum. Inhibition of proliferation by RA was associated with increased levels of IGFBP-2 in conditioned media and in plasma membrane preparations. Treatment with insulin or des(1–3) IGF-I resulted in the appearance of IGFBP-3 in conditioned media and on the cell surface. However, RA significantly reduced IGFBP-3 levels in conditioned media and eliminated IGFBP-3 associated with the plasma membrane. Thus, RA is a potent but transient inhibitor of bovine mammary epithelial cell proliferation, and this growth inhibition is correlated with increased IGFBP-2 accumulation and inhibition of IGF-I stimulated IGFBP-3 protein secretion. © 1996 Wiley-Liss, Inc.     

High intensity ERK signal mediates hepatocyte growth factor-induced proliferation inhibition of the human hepatocellular carcinoma cell line HepG2

Hepatocyte growth factor (HGF) induces growth stimulation of a variety of cell types, but it also induces growth inhibition of several types of tumor cell lines. The molecular mechanism of the HGF-induced growth inhibition of tumor cells remains obscure. We have investigated the intracellular signaling pathway involved in the antiproliferative effect of HGF on the human hepatocellular carcinoma cell line HepG2. HGF induced strong activation of ERK in HepG2 cells. Although the serum-dependent proliferation of HepG2 cells was inhibited by the MEK inhibitor PD98059 in a dose-dependent manner, 10 microM PD98059 reduced the HGF-induced strong activation of ERK to a weak activation; and as a result, the proliferation inhibited by HGF was completely restored. Above or below this specific concentration, the restoration was incomplete. Expression of constitutively activated Ha-Ras, which induces strong activation of ERK, led to the proliferation inhibition of HepG2 cells, as was observed in HGF-treated HepG2 cells. This inhibition was suppressed by the MEK inhibitor. Furthermore, HGF treatment and expression of constitutively activated Ha-Ras changed the hyperphosphorylated form of the retinoblastoma tumor suppressor gene product pRb to the hypophosphorylated form. This change was inhibited by the same concentration of MEK inhibitor needed to suppress the proliferation inhibition. These results suggest that ERK activity is required for both the stimulation and inhibition of proliferation of HepG2 cells; that the level of ERK activity determines the opposing proliferation responses; and that HGF-induced proliferation inhibition is caused by cell cycle arrest, which results from pRb being maintained in its active hypophosphorylated form via a high-intensity ERK signal in HepG2 cells.     

Stimulation and inhibition of human T-lymphocyte colony cell proliferation by hemopoietic cell factors.

Human lymphocytes, isolated from peripheral blood and stimulated with phytohemagglutinin M (PHA) prior to being seeded on a two-layer medium of soft agar which contained the mitogen, developed into colonies 3–4 days after seeding in the culture system. The cloning potential of PHA-treated lymphocytes is significantly enhanced by adding, to the soft agar culture, culture fluid (CF) obtained from mitogen-treated lymphocytes or a feeder layer (FL) prepared either from lymphocytes isolated from peripheral blood or from T-cell enriched populations. PHA seems to stimulate the release of lymphocyte colony enhancing factor (LCEF) from the T-sensitized lymphocytes. The addition of CF or FL to the culture medium appears to increase the amount of LCEF, resulting in enhancement of the number and size of lymphocyte colonies. When CF derived from spleen cells or from the peripheral blood adherent-cell population was added to the lower layer of the soft agar culture, the growth and development of lymphocyte colonies was inhibited. This suggests that monocyte-macrophages release a lymphocyte colony inhibiting factor (LCIF) into the CF. The extent of inhibition or stimulation of colony formation is a function of the number and type of cells used to prepare the CF or FL and the concentration of CF in the culture medium. The presence of FL or CF derived from spleen non-adherent cells, white blood cells, bone marrow cells, or a B-cell enriched population had no effect on colonies growing in the culture. This may possibly be due to the paucity of T lymphocytes and monocyte-macrophages present in these materials. A control system in which LCIF, produced by monocyte-macrophages, and LCEF, produced by T lymphocytes, participate in the regulation of lymphocyte production is postulated.     

重组人乳铁蛋白对食管癌细胞体外增殖的影响

目的:研究重组人乳铁蛋白(rhLF)对体外培养的食管癌细胞的生长特性的影响.方法:MTT法评定rhLF对人食管癌细胞Eca-109、人肝脏细胞L0-2、CHO中国昌鼠细胞系的生长抑制作用.结果:重组人乳铁蛋白对人食管癌细胞系Eca-109的增殖有抑制作用并呈现剂量依赖性,对正常细胞无抑制作用.结论:重组人乳铁蛋白作为新的抗肿瘤化学治疗荆,可以抑制食管癌细胞Eca-109的增殖.     

Interaction of retinoids and bilirubin with the binding of arachidonic acid to human alpha-fetoprotein

Human alpha-fetoprotein (HAFP) has three binding sites for polyunsaturated fatty acids with association constant Ka = 1.8 X 10(7) M-1. One of these binding sites overlaps with a retinoid binding site with Ka = 2.6 X 10(6)M-1. Competition experiments with bilirubin showed that this compound does not compete neither with fatty acids nor with retinoids. Thus, the two bilirubin binding sites previously demonstrated appear as two additional binding sites on HAFP. Nevertheless, the close proximity of two fatty acid binding sites and two bilirubin binding sites resulted in a modification of the binding constants for fatty acids. It is hypothesised that the binding properties of HAFP reflect the three domain structures of the protein recently deduced from the study of the nucleotide sequence of HAFP mRNA and AFPcDNA segments.     

Retinoic acid receptor alpha mediates growth inhibition by retinoids in human colon carcinoma HT29 cells.

Although retinoids have been suggested to inhibit chemically induced colon carcinogenesis, the molecular mechanisms underlying retinoid-mediated growth regulation in colon carcinoma cells are unknown. Therefore, we investigated the biological effects of retinoids on growth in HT29 colon carcinoma cells. All-trans retinoic acid (ATRA) treatment of HT29 cells resulted in a profound inhibition of anchorage-independent growth without biochemical or morphological evidence for induction of differentiation. Treatment with the selective RARalpha agonist Ro 40-6055 completely mimicked the effects of ATRA on growth and transactivation of a betaRAREx2-luciferase reporter construct, while RARbeta- and gamma-specific analogues were ineffective. Furthermore, ATRA-regulated growth and transactivation could be completely blocked by a RARalpha-selective receptor antagonist. Thus, ATRA potently inhibits anchorage-independent growth in HT29 cells and this effect is mainly if not exclusively mediated by the retinoic acid receptor alpha.     

Influence of luteinizing hormone-releasing hormone agonists on human mammary carcinoma cell lines and their xenografts

The specific binding of luteinizing hormone-releasing hormone (LH-RH) agonist in estradiol-dependent MCF-7 and estradiol-independent MDA-MB-231 human breast cancer cells has been studied using [3H]Ovurelin [(D-3H-Phe6),des-Gly10-LH-RH- ethylamide]. The results of Scatchard analyses suggest the presence of a single class of receptor sites, both in cell suspensions and membrane fractions. Evaluation of these peptide receptors appears to reflect additional characteristics of biological behaviour of these human breast cancer cells. The synthetic LH-RH agonist Ovurelin [(D-Phe6),des-Gly10-LH-RH-ethylamide] can directly interfere (25-30%) with the proliferation of MDA-MB-231 human breast cancer cells in culture. The inhibitory effect of Ovurelin in vitro was negligible in the MCF-7 cell line. In the in vivo experiments the treated immunosuppressed mice bearing either MCF-7 or MDA-MB-231 xenografts responded to the high-dose LH-RH analogue Zoladex depot and Decapeptyl depot therapy. Since the MDA-MB-231 tumour was found to be ER-negative it seems possible that the regression of this xenograft results from the direct antitumor action of the LH-RH agonist.     

Folic acid inhibition of EGFR-mediated proliferation in human colon cancer cell lines

Althoughaccumulating evidence suggests a chemopreventive role for folic acid incolon cancer, the regulation of this process in unknown. We hypothesizethat supplemental folic acid exerts its chemopreventive role byinhibiting mucosal hyperproliferation, an event considered to becentral to the initiation of carcinogenesis in the gastrointestinaltract. The present investigation examines the effect of supplementalfolic acid on proliferation of Caco-2 and HCT-116 colon cancer celllines. Furthermore, because certain tyrosine kinases, particularlyepidermal growth factor receptor (EGFR), play a role in regulating cellproliferation, we also examined the folic acid-induced changes intyrosine kinase activity and expression of EGFR. In Caco-2 and HCT-116cells, maintained in RPMI 1640 medium containing 1 µg/ml folic acid,we observed that the supplemental folic acid inhibited proliferation ina dose-dependent manner. Pretreatment of HCT-116 and Caco-2 cell lineswith supplemental folic acid (1.25 µg/ml) completely abrogated transforming growth factor- (TGF-)-induced proliferation in bothcell lines. Tyrosine kinase activity and the relative concentration ofEGFR were markedly diminished in both cell lines following a 24-hexposure to supplemental folic acid. The folic acid-induced inhibitionof EGFR tyrosine kinase activity in colon cancer cell lines was alsoassociated with a concomitant reduction in the relative concentrationof the 14-kDa membrane-bound precursor form of TGF-. In conclusion,our data suggest that supplemental folic acid is effective in reducingproliferation in two unrelated colon cancer cell lines and that EGFRtyrosine kinase appears to be involved in regulating this process.

     

Effect of selenite combined with chemotherapeutic agents on the proliferation of human carcinoma cell lines

Selenite is frequently used in combination with cancer chemotherapeutic agents to reduce side effects. However, the cytoprotective activity of selenite may also reduce the efficacy of chemotherapeutic drugs on tumor cells. This study was designed to examine the effects of selenite combined with cytotoxic agents used in clinical protocols [e.g., doxorubicine, docetaxel, 5-fluorouracil (5-FU), methotrexate (MTX), mafosphamide, mitomycin C, gemcitabine, etoposide, cisplatin, irinotecan, and oxaliplatin] on the proliferation of various carcinoma cell types. The data demonstrated that selenite had no marked effects on the antiproliferative activity of docetaxel, doxorubicine, 5-FU, MTX, and mafosphamide in MDA-MB-231 breast cancer cells. Likewise, no consistent changes were observed in A549 lung cancer cell proliferation when selenite was combined with cisplatin, etoposide, gemcitabine, or mitomycin C. On the other hand, selenite potentiated the cytotoxicity of 5-FU, oxaliplatin, and irinotecan in HCT116 colon cancer cells by approx 1.1-fold, 2.7-fold, and 2.6-fold, respectively. In SW620 colon cancer cells, selenite induced a 1.5-fold and 4.3-fold increase of the antiproliferative activity of 5-FU and oxaliplatin, respectively. Whereas irinotecan showed no effects on SW620 cell growth, a combination with selenite resulted in 23% inhibition. Our results indicate that selenite did not reduce the antiproliferative activity of chemotherapeutic agents in vitro. In addition, selenite was able to increase the inhibitory activity of docetaxel in A549 lung cancer cells, and of 5-FU, oxaliplatin, and irinotecan in HCT116 and SW620 colon cancer cells implying selenite is potentially useful as an adjuvant chemotherapeutic agent.     

BCG对乳腺癌细胞增殖和凋亡的影响

目的从细胞增殖和凋亡两方面观察BCG对乳腺癌细胞MDA—MB-231的抑制作用。方法用MTT法检测BCG作用后MDA—MB-231细胞的增殖能力,采用TUNEL法检测凋亡。结果BCG能明显降低MDA-MB-231细胞的增殖代谢活性而抑制其增殖。TUNEL法染色显示BCG可显著增加凋亡细胞。结论BCG不仅能显著抑制MDA—MB-231细胞增殖,还能有效诱导其凋亡。     

Inhibition of human breast cancer cell proliferation with estradiol metabolites is as effective as with tamoxifen.

The anti-estrogenic substance tamoxifen is effective in the adjuvant therapy applied in human breast cancer. Since it partly exhibits estrogenic activity and has serious side-effects, however, pure anti-estrogenic compounds are being sought. In our experimental study, we compared the anti-proliferative effect of estradiol and 13 endogenous estradiol metabolites on human breast cancer cells with the effect of tamoxifen. We used MCF-7 and MDA-MB 231, the well-established estrogen receptor-positive and -negative cell lines. 4-hydroxytamoxifen, the active metabolite of tamoxifen, estradiol and 13 estradiol metabolites were tested in concentrations ranging from 3.1 to 100 microM. Incubation time was 4 days and cell proliferation was measured by means of the ATP chemosensitivity test. 4-hydroxytamoxifen showed an IC50 value of 27 microM and 18 microM in MCF-7 and MDA-MB 231 cells, respectively. Estradiol and its metabolites were anti-proliferative in both cell lines. A few A-ring metabolites were more effective in inhibiting cell proliferation than D-ring metabolites and the parent substance 17beta-estradiol. 4-OHE1, 2-MeOE1 and 2-MeOE2 were as effective in both cell lines as tamoxifen. For the first time it has been demonstrated that endogenous estradiol metabolites are equally anti-proliferative as tamoxifen in the context of human breast cancer cells. Since some of these metabolites exhibit no estrogenic activity, they are likely to be valuable in clinical studies of chemoprevention and adjuvant therapy of breast cancer.