Inhibition of proliferation and induction of apoptosis in human breast cancer cells by lauryl gallate

Lauryl gallate is an antioxidant food additive showing low toxicity to normal cells. Here, its antiproliferative effect has been studied on three human breast cancer cell lines: estrogen-dependent, wild-type p53, MCF7; estrogen-independent, non-functional p53, MDA-MB-231 and MCF7 ADR, which overexpresses P-glycoprotein (P-gp) and displays a multidrug-resistant phenotype. Lauryl gallate inhibited proliferation and induced cell cycle alterations in all three cell lines without altering P-gp functionality in the drug-resistant cells. A stable arrest in G(1) phase was observed in MCF7, while a slow-down of cell cycle progression was induced in the other two cell lines. Lauryl gallate increased p53 expression only in MCF7, and upregulated p21(Cip1) and reduced cyclin D1 levels in all three cell lines. The induction of apoptosis, demonstrated by annexin V-FITC labeling, PARP cleavage and mitochondrial membrane depolarization and morphological alterations, were clearly detected in MCF7 ADR and MDA-MB-231 and to a minor extent in MCF7. Overexpression of Bcl-2 in MCF7 ADR cells demonstrated its protective role against morphological alterations and apoptosis. Lauryl gallate induction of p21(Cip1) and apoptosis observed in all three cell lines was regulated by Erk1/2 activation. These findings suggest a potential use of lauryl gallate against tumors harboring p53 mutations and drug-resistant phenotypes.     

Inhibition of K cell function by human breast cancer sera.

Sera from breast cancer patients and from female controls were tested for inhibition of lysis of antibody-coated target cells by human leukocytes (K cells). Sera from 39% of breast cancer patients, but from only 8% of controls, inhibited lysis by more than 30%. This inhibition was unrelated to the stage of the disease, the patient''s age or whether the patient was pre- or post-operative. Inhibition was apparently not due to anti-HLA antibodies and did not correlate with the IgG level or anti-complementary activity of the serum. On fractionation by gel-filtration, inhibitory activity was found in fractions of higher molecular weight than IgG. As no IgG could be detected in these fractions, inhibition is probably not due to immune complexes containing IgG antibody. The inhibitory factor may well contribute to the immunosuppressed status of a proportion of breast cancer patients.     

Prolonged retention of estradiol by human breast cancer cells in tissue culture.

Conditions are described under which prolonged estradiol retention and estrogenic activity are observed in human breast cancer cells in tissue culture. The cells were incubated for three hr with a physiological concentration of [3H]estradiol (3 to 5 nM) and then were washed with 3 successive exchanges of medium 3, 17, and 24 or 48 hr following incubation with [3H]estradiol. The total wash period was 78 hr. The following parameters were monitored to assess the duration of estrogen action in MCF-7 human breast cancer cells in tissue culture; (a) the concentration of [3H]estradiol and [3H]estradiol metabolites in the media washes; (b) the intracellular concentration of [3H]estradiol and [3H]estradiol metabolites; and (c) the time course of estradiol-enhanced rates of radiolabeled thymidine incorporation. The [3H]estradiol concentration in the final medium wash was approximately 0.05 nM. The total intracellular concentration of tritium was about 50 nM prior to wash and 9 nM following 78 hr of wash. The intracellular concentration of specifically bound [3H]estradiol was initially 18 nM, and after 78 hr of wash, it was 2.8 nM. After 48 hr of wash, nearly all specifically bound [3H]estradiol was present in the nucleus. Following incubation of the cells with 5 nM estradiol and an identical wash procedure, estrogenic activity as measured by a stimulation of thymidine incorporation was observed throughout the 78 hr monitored. When 10(-6) M tamoxifen or 10(-7) M unlabeled estradiol was included in the medium washes, the washout of nonspecific binding was unaffected; however, specifically bound [3H]estradiol was essentially eliminated within 24 hr. When bovine serum albumin was included in the medium washes, total, nonspecific, and specific [3H]estradiol binding was reduced in a parallel and dose-dependent fashion. After 48 hr, cells washed with medium containing 3.5 or 7% bovine serum albumin contained one-tenth of the [3H]estradiol present in cells washed with medium alone. We conclude that medium exchanges alone do not effectively remove estradiol from MCF-7 cells, and suggest that estrogen retention by estrogen-responsive cells may mask in vitro assessments of such responsiveness in this and other systems. Inclusion of bovine serum albumin in the washes may alleviate this problem.     

白藜芦醇对不同乳腺癌细胞增殖的抑制作用

Objective To investigate the effects of resveratrol (Res) on the proliferation, cell cycle and apoptosis of three breast cell lines DU4475, MDA-MB-231 and MCF-7 with different estrogen receptor (ER) expressions. Methods Res was added to the drug treatment group at 6.25, 12.5, 25, 50, 100 and 200 μmol/L, respectively. Three observation periods at 24, 48 and 72 hours were set up respectively. MTT assay was used to detect the effects of Res on proliferation of breast cells. Cell cycle and apoptosis were analyzed by flow cytometry (FCM). The concentrations of drugs were 0, 12.5, 25, 50 μmol/L. The results were analysed by the statistical software SPSS17.0. Results After Res intervention, the proliferations of three cell lines were inhibited to different extent. After 48 and 72 hours of Res, inhibitions of Res with different concentration were significant different (F=15.26, P﹤0.05). Inhibition rates of DU4475 and MDA-MB-231 with ER-negative were higer than that of MCF-7 with ER-positive. FCM results prompted that these three kinds of cells were blocked in S phase after 48 hours treatment of 12.5-50 μmol/L Res and the block percentages had significant difference (F=34.81, P﹤0.05). The percentages of S phase for DU4475 and MDA-MB-231 arresting were higher than that of MCF-7. For DU4475, the apoptotic and necrosis percentage were higher than that of MCF-7 at 100 μmol/L (t=16.082, P<0.05). Conclusion Res can inhibit proliferation, induce cell cycle changing and apoptosis of DU4475, MDA-MB-231 and MCF-7 cells. The inhibitions of Res on DU4475 and MDA-MB-231 are better than that of MCF-7 with ER-positive.     

Apoptosis-mediated inhibition of human breast cancer cell proliferation by lemon citrus extract

Dietary phytochemicals have a variety of antitumor properties. In the present study, the antitumor activity of methanolic extract of lemon fruit (lemon extract; LE) (LE) on the MCF-7 breast cancer cell line was investigated in vitro. Apoptotic cell death was analyzed using the TUNEL assay. In addition, the apoptosis mediated by LE extract in the MCF-7 cells was associated with the increased expression of the tumor suppressor p53 and caspase-3. Additionally, the expression of a pro-apoptotic gene, bax, was increased, and the expression of an anti-apoptotic gene, bcl-2, was decreased by LE extract treatment, resulting in a shift in the Bax:Bcl-2 ratio to one that favored apoptosis. The expression of a major apoptotic gene, caspase-3, was increased by LE extract treatment. In light of the above results, we concluded that LE extract can induce the apoptosis of MCF-7 breast cancer cells via Bax-related caspase-3 activation. This study provides experimental data that are relevant to the possible future clinical use of LE to treat breast cancer.     

Inhibition of triple-negative and Herceptin-resistant breast cancer cell proliferation and migration by Annexin A2 antibodies

Background:

Annexin A2 (AnxA2), a calcium-dependent phospholipid binding protein, is abundantly present at the surface of triple-negative and Herceptin-resistant breast cancer cells. Interactions between cell-surface AnxA2 and tyrosine kinase receptors have an important role in the tumour microenvironment and act together to enhance tumour growth. The mechanism supporting this role is still unknown.

Methods:

The membrane function of AnxA2 was blocked by incubating cells with anti-AnxA2 antibodies. Western blotting, immunoprecipitation, immunofluorescence, 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), flow cytometry, Clonogenic, and wound-healing assays were performed in this study.

Results:

We demonstrate that AnxA2 interacts with epidermal growth factor receptor (EGFR) at the cell surface and has an important role in cancer cell proliferation and migration by modulating EGFR functions. Blocking AnxA2 function at the cell surface by anti-AnxA2 antibody suppressed the EGF-induced EGFR tyrosine phosphorylation and internalisation by blocking its homodimerisation. Furthermore, addition of AnxA2 antibody significantly inhibited the EGFR-dependent PI3K-AKT and Raf-MEK-ERK downstream pathways under both EGF-induced and basal growth conditions, resulting in lower cell proliferation and migration.

Conclusions:

These findings suggest that cell-surface AnxA2 has an important regulatory role in EGFR-mediated oncogenic processes by keeping EGFR signalling events in an activated state. Therefore, AnxA2 could potentially be used as a therapeutic target in triple-negative and Herceptin-resistant breast cancers.     

Human pancreatic cancer cell proliferation in tissue culture is tonically inhibited by opioid growth factor

Pancreatic adenocarcinoma is a fatal malignancy that ranks as the fourth most common cause of cancer-related mortality in the United States. The median survival after diagnosis is 3-6 months, with a 5-year survival rate of 3% or less. In spite of treatment efforts of surgery, radiation, and chemotherapy, the survival rate remains unchanged. In this study, we discovered that an endogenous opioid peptide, [Met5]-enkephalin, inhibited the growth of human pancreatic cancers in vitro; in view of this pentapeptide's action it has been termed opioid growth factor (OGF). OGF was found to be constitutively expressed, autocrine produced, and tonically capable of suppressing cell replication in an opioid receptor mediated manner. Growth inhibition was dose-related, reversible, not cytotoxic, and independent of serum. All 4 pancreatic cancer cell lines examined, representative of poor to well-differentiated neoplasias, exhibited growth regulation by OGF. Immunocytochemical studies detected both OGF and its related receptor, zeta, in the cytoplasm of log phase cells. Radioimmunoassays revealed that OGF was produced and secreted by the cells. These data suggest that a native opioid peptide, OGF, interacts with a novel opioid receptor, zeta, to arrest the growth of human pancreatic cancer.     

Methylation status of the Ep-CAM promoter region in human breast cancer cell lines and breast cancer tissue

We examined the methylation status of the Ep-CAM promoter region of human breast cancer cell lines and breast cancer tissue using MethyLight technology and bisulfite sequencing. We found the promoter of Ep-CAM-negative breast cancer cell lines Hs 578T to be methylated to a higher degree as compared to positive cell lines MCF-7. Demethylation of cell lines was performed using 5-aza-2'-deoxycytidine. Ep-CAM RNA and protein expression could be partially restored by treating cells with 5-Aza-2'-deoxycytidine. In most primary breast cancer tissue, methylation of the Ep-CAM gene could be detected at a low level and no correlation was found with Ep-CAM protein expression in tumour tissue. Taken together, these data suggest that methylation of the Ep-CAM promoter is not a crucial mechanism for regulation of Ep-CAM expression in breast cancer. Thus, most important regulatory mechanisms have to be supposed in vivo.     

Inhibition of human leukemia cell proliferation by dimethyl sulfoxide

The treatment with 1% and 2% DMSO produced reversible structural and functional changes in the K-562 human myelogenous cell line. These changes include: (a) inhibition of cell proliferation with decreased RNA and DNA syntheses; (b) morphological features that are compatible with less primitive leukemia cells; (c) diminished plating efficiency in agar; and (d) loss of their malignancy as determined by the capacity of K-562 cells to develop myelosarcomas in lasat mice. The cytochemical pattern and antigenic markers of the leukemic cells were not modified by treatment with DMSO. These results suggest that the effect of DMSO on K-562 cells is consistent with cytotoxicity rather than a stimulation of cell differentiation.     

二甲双胍抑制人MDA-MB-231细胞增殖及迁移的体外实验研究

目的： 研究二甲双胍对体外培养的人三阴性乳腺癌MDA-MB-231细胞增殖及迁移的影响。方法： MTT实验分为4个处理组,分别为RPMI 1640培养基对照组,浓度为1、2、4 mmol/L的二甲双胍处理组,在作用48、72 h后测定各组的细胞活力并进行统计学分析;倒置相差显微镜下观察药物处理72 h后人乳腺癌MDA-MB-231细胞与对照组相比的形态变化;Giemsa染色观察4 mmol/L二甲双胍作用48 h后的细胞形态;采用细胞划痕实验和Transwell实验,检测二甲双胍（2、4 mmol/L）作用人乳腺癌MDA-MB-231细胞24 h后,对细胞迁移能力的影响。结果： MTT实验结果显示4 mmol/L二甲双胍作用72 h可显著抑制MDA-MB-231细胞的活力,抑制率为（29.83±2.25）％,与对照组相比,细胞活力降低（P<0.05）。倒置相差显微镜下形态学观察发现,与对照组相比,4 mmol/L二甲双胍处理组细胞密度降低,变圆的细胞数量增加,细胞与细胞之间的联系减少。Giemsa染色结果显示4 mmol/L二甲双胍作用48 h后,部分细胞出现凋亡细胞核碎裂形态。划痕实验结果表明,2、4 mmol/L二甲双胍作用24 h后细胞汇合度明显低于对照组,其中4 mmol/L处理组的划痕愈合率为（52.67±4.48）%,与对照组间的差异显著（P<0.01）;Transwell实验结果表明,2、4 mmol/L二甲双胍作用细胞24 h后穿膜细胞数量减少,穿膜细胞数量分别为（61.6±1.6）、（51.3±2.6）个,均低于对照组的（99.3±18.9）个（P<0.05）。结论： 二甲双胍在体外可抑制人乳腺癌MDA-MB-231细胞的增殖及迁移。     

Prolactin receptors in human breast cancer cells in long-term tissue culture.

Prolactin receptors have been identified for the first time in a number of human breast cancer cell lines and a normal human breast cell line maintained in long-term tissue culture. Optimal conditions for determining the binding of 125I-labeled human prolactin to these cells were established. Five different tumor cell lines have different content of prolactin receptors ranging from 2,300 to 26,000 sites/cell. All tumor cell lines contained more prolactin receptors than does one normal breast cell line (1700 sites/cell). The prolactin receptors in these human mammary tumor cells not only bind human prolactin but also recognize other lactogenic hormones such as human growth hormone, human placental lactogen, and sheep prolactin, but not animal growth hormone, which are not lactogenic. The affinity (Ka) of binding of human prolactin to these cells is 4 x 10(9) M-1 (Kd = 2.5 x 10(-10)M). The hormone specificity and affinity for hormone of these human mammary tumor cells are very similar to that found for the rabbit mammary gland. These human mammary tumor cell lines in long-term culture should prove very useful to study the biology of prolactin receptors in living human cells and the role of prolactin in the tumorigenesis of the human breast.     

Effect of stromal and epithelial cells derived from normal and tumorous breast tissue on the proliferation of human breast cancer cell lines in co-culture

Stromal and epithelial components surrounding neoplastic cells are believed to be important in tumor regulation. We have studied the effects of stromal and epithelial cells on the proliferation of a variety of breast-cancer epithelial cell lines. Co-culture experiments were performed in which the 2 cell types were separated by a microporous membrane. Under these conditions, fibroblasts from normal breast tissues inhibited the proliferation of MCF-7 cells, but not that of immortalized normal S2T2 cells. In contrast, fibroblasts from cancerous breast tissues did not influence the proliferation of the 2 cell lines tested. Conditioned media (CM) of breast fibroblasts derived from normal tissues were not able to affect MCF-7 cell growth, suggesting complex paracrine interactions between both cell types. Normal breast epithelial cells (NBEC) have also been tested for their ability to regulate the proliferation of breast-cancer epithelial cell lines. Co-culture experiments demonstrated that NBEC inhibited a variety of breast-cancer cell lines. CM from NBEC induced similar results and the inhibitory effect appeared to be specific for epithelial cells from tumorous breast. Moreover, CM from NBEC and normal fibroblasts were shown to contain more TGF_β1 and amphiregulin than those of MCF-7 cells. We conclude that both the tissue origin and the target tumor cell's phenotype will determine the extent of proliferative response. More important, the tumor-cell growth inhibition induced by fibroblasts and epithelial cells of normal breast tissue may constitute a tumor-growth-regulatory mechanism. Int. J. Cancer, 71:42–48, 1997. © 1997 Wiley-Liss, Inc.     

Inhibition of murine transformed Leydig cell proliferation by leukotrienes in serum-free culture.

We have previously shown that estrogen-dependent growth enhancement of murine transformed Leydig cells (B-1 F) is mediated through inhibition of arachidonic acid metabolite formation. In the present study, the growth-inhibitory ability of leukotrienes (LTs) on B-1 F cells in serum-free culture was directly addressed. All peptidyl LTs (LTC4, LTD4, and LTE4) inhibited B-1 F cell growth in a dose-dependent manner and exhibited maximum inhibition of DNA synthesis (60-80%) compared with that of untreated cells in a range of 10(-9) to 10(-8) M. To examine the mechanism of this LT-dependent inhibition, binding studies of LTD4 toward plasma membrane were conducted. Specific binding sites for LTD4 were identified. Scatchard analyses indicated the presence of a single class of high-affinity sites (Kd = 0.9 +/- 0.2 nM; maximum binding sites, 61 +/- 18 fmol/mg protein). This binding of LTD4 to the high-affinity site was markedly inhibited by ICI 198615, a specific inhibitor for LTD4. These results would suggest that inhibitory effects of LTs, at least LTD4, are elicited as a receptor-mediated event. In addition, this LT-dependent growth inhibition could not be blocked by simultaneous exposure of cells to estrogen, whereas estrogen partially protected arachidonic acid-dependent growth inhibition. Furthermore, treatment of cells with estrogen resulted in marked suppression of 5-lipoxygenase activity. Collectively, the present data clearly show that LTs play an important role as intermediates in an autocrine loop for B-1 F cells to exhibit estrogen-dependent growth.     

α-干扰素与肉桂酸对人肺癌细胞增殖的抑制作用

背景与目的:有研究表明肉桂酸对肿瘤细胞有抑制作用。本实验拟进一步研究α-干扰素(alpha-interferon,α-IFN)与肉桂酸(cinnamicacid,CINN)单独及联合作用对人肺腺癌细胞增殖的抑制作用。方法:用α-IFN和CINN处理人肺腺癌A549细胞,通过绘制生长曲线、软琼脂集落形成实验、甲基绿-派洛宁染色、流式细胞术(FCM)测定等方法来研究其对细胞的增殖抑制作用。结果:与对照组相比,α-IFN和CINN能明显抑制A549细胞增殖(P<0.05),促进细胞分化(P<0.05),α-IFN和CINN联合应用时抑制作用较二者单独作用时强;细胞在软琼脂中集落形成率明显下降(P<0.05);FCM显示细胞周期移行被阻滞于G1/G0期。结论:CINN联合α-IFN能抑制肺腺癌细胞增殖,α-IFN和CINN两药联合作用效应强于α-IFN或CINN单独作用。     

Inhibition of non-small-cell lung cancer cell proliferation by Pbx1

Lung cancer is one of the most deadly human cancers and continues to be a major unsolved health problem worldwide. Here, we evaluate the function of Pbx1 in the proliferation of non-smallcell lung cancer（NSCLC）. In contrast with its known proliferative function, we found that Pbx1 inhibits the proliferation of lung cancer cells. In particular, Pbx1-specific RNA interference resulted in increased proliferation in lung cancer cells. In addition, histone H3 phosphorylation was also increased following inhibition of Pbx1 expression. In contrast, Pbx1 overexpression repressed the proliferation of lung cancer cells and inhibited DNA synthesis. Collectively, our data indicate that Pbx1 inhibits proliferation in lung cancer cells, suggesting a complex role for Pbx1 in modulating the proliferation of cancer cells and making this protein a potential new target for lung cancer therapy.