重组人IL-2抑制乳腺癌细胞增殖及其机制的研究

目的：观察重组人IL-2对体外培养乳腺癌细胞生长的影响,及对洛铂体外抑瘤效果的影响。方法：选择乳腺癌细胞株MCF-7和MDA-MB-231作为实验对象。利用MTT实验检测重组人IL-2和对照药物洛铂对肿瘤细胞生长的抑制率,利用RT-PCR方法分析MCF-7细胞中Syk基因在不同处理因素作用下表达情况。结果：洛铂能明显抑制两种乳腺癌细胞体外生长,重组人IL-2对两种癌细胞有一定的抑制作用。重组人IL-2和洛铂同时处理时,对肿瘤细胞的抑制率比单独应用洛铂时明显升高,并且同时处理时MCF-7细胞中Syk mRNA表达水平比洛泊单独处理时高。结论：重组人IL-2能增强洛铂对乳腺癌细胞生长抑制效果。重组人IL-2单独作用乳腺癌细胞时也有一定的抑制效果,针对MCF-7细胞,重组人IL-2刺激信号传递可能有Syk参与。提示在体内时IL-2不仅对机体免疫细胞起重要的刺激激活作用,还可能对瘤细胞本身也有抑制作用。     

三羟异黄酮对人乳腺癌MCF-7/ADM细胞体外抑瘤效应、细胞周期及凋亡的影响

 目的探讨三羟异黄酮（Genistein GEN）对体外培养人乳腺癌耐药细胞MCF-7/ADM抑瘤作用、细胞周期及细胞凋亡的影响。方法采用MTT法检测GEN单独及联合阿霉素对体外培养人乳腺癌MCF-7/ADM细胞的抑制作用;荧光分光光度法检测GEN对阿霉素在MCF-7/ADM细胞的蓄积作用;用流式细胞仪（FCM）检测细胞周期及凋亡率的变化。结果GEN单独及联合阿霉素对体外培养MCF-7/ADM细胞均有明显生长抑制作用,GEN单独作用48 h后表现为随时间的延长抑制作用明显增强,当GEN浓度达到60 μg/ml时,其抑制作用急剧上升（P<0.01）。联合阿霉素后与对照组相比抑制率明显上升（P<0.01）,并随GEN浓度的加大其抑制作用增强,细胞内阿霉素的浓度也随之升高。与对照组相比,细胞周期均有G2/M期阻滞作用,细胞凋亡百分比以联合组最高（P<0.01）,G₁期前出现典型的亚二倍体凋亡峰。结论GEN单独及联合阿霉素对体外培养人乳腺癌MCF-7/ADM细胞具有抑瘤增效作用,可以提高阿霉素在MCF-7/ADM细胞内的蓄积、对细胞周期具有G₂/M期阻滞作用,显著诱导MCF-7/ADM细胞凋亡,可能是其发挥逆转多药耐药分子生物学机制之一。     

重组人NDRG2蛋白对肿瘤细胞抑制作用研究

目的:考察原核表达的重组人NDRG2蛋白对肿瘤细胞的作用.方法:通过设置不同浓度的蛋白剂量并设置对照组,采用细胞MTT比色法和细胞计数检测NDRG2蛋白对7901、HHCC细胞的抑制效果,流式细胞仪检测细胞周期的变化,体内实验采用裸鼠抑瘤试验.结果:重组人NDRG2对肿瘤细胞生长有较强的抑制作用,约50μg/ml浓度时对肿瘤细胞生长的抑制作用明显,流式细胞术结果显示肿瘤细胞G1期变化,体外能抑制裸鼠肿瘤的生成.结论:重组人NDRG2蛋白在体内体外具有一定的抑制肿瘤细胞生长作用,在肿瘤治疗方面将具有一定的意义.     

腺病毒介导CDES融合基因对乳腺癌细胞的抑制作用

目的:构建一种重组腺病毒rAdCDES,在体内外进行抑瘤和放疗增敏作用实验研究.方法:用细菌内同源重组法与腺病毒骨架质粒重组构建出重组腺病毒质粒,经脂质体介导转染293包装细胞扩增获取重组腺病毒rAdCDES.用测定生长曲线和MTT法检测其对人乳腺癌细胞株MCF-7的生长抑制情况;建立津白号鼠乳腺癌模型,观察rAdCDES和放疗对瘤体大小及鼠存活期的影响.结果:成功构建了rAdCDES;rAdCDES对MCF-7细胞生长抑制率达(83.1±8.1)%,与对照rAdLacZ(19.2±7.8)%相比较有显著性差异(P＜0.01);体内rAdCDES对小鼠乳腺癌MA737细胞有明显生长抑制作用,延长了小鼠的存活期,且对放疗有肯定的增敏作用.结论:本文所构建的rAdCDES在体内外对乳腺癌细胞均有明显的抑制作用和对放疗的增敏作用.     

咖啡因对乳腺癌化疗增效作用的体外实验研究

目的:体外研究咖啡因(caffe-ine,CF)联合多柔比星(adriamycin,ADM)对乳腺癌细胞株MCF-7的抑制效应。方法:用MTT法评估瘤细胞的生长抑制情况,通过电镜检测瘤细胞的凋亡,并用流失式细胞仪确定瘤细胞的凋亡率。结果:在0~8·0mg/mL浓度范围内,CF对MCF-7的抑制作用和其浓度呈正相关,且CF联合ADM(0·5~2·5μg/mL)对MCF-7的生长抑制显著大于ADM的单独作用,P<0·05;先加入CF或CF与ADM同时作用,对MCF-7的生长抑制高于后加入CF组或ADM单独使用组,P<0·05;CF和ADM联合作用对MCF-7的凋亡诱导,和CF或ADM单独作用比,凋亡率未有明显改变。结论:CF对MCF-7的生长有抑制作用,与ADM联合应用的抑制作用明显大于单独使用ADM,先加入CF或CF与ADM同时作用的给药顺序优于后加入CF;ADM和CF抑制MCF-7生长的机制之一是诱导凋亡,两者共同作用并未显著增加MCF-7的凋亡率,但有协同杀伤MCF-7的作用,其中化学性杀伤仍起主要作用。     

黄荆子提取物EVn-50对人乳腺癌MCF-7细胞增殖和凋亡的影响

目的:研究黄荆子提取物Evn-50对人乳腺癌MCF-7细胞生长抑制和凋亡的诱导作用.方法:在体外培养人乳腺癌MCF-7细胞,采用表柔比星、他莫西芬和不同浓度的Evn-50处理MCF-7细胞,软琼脂和平皿克隆法测定Evn-50抑制MCF-7细胞增殖的量-效关系,TUNEL法、流式细胞术分析其细胞凋亡率和周期分布.结果:Evn-50对MCF-7细胞增殖的抑制作用随药物浓度的提高而更加明显,而在低浓度时却表现出轻微促细胞增殖现象(<10.0μg/mL),100.0 μg/mL时抑制率为48.68%,实验组与对照组比较,差异有统计学意义,P<0.01.TUNEL法和流式细胞术分析结果表明,高浓度的Evn-50能诱导MCF-7细胞凋亡,阻滞细胞于G2/M期,降低细胞增殖指数,且也存在量-效关系.结论:Evn-50在高浓度时可抑制人乳腺癌MCF-7细胞增殖并诱导其凋亡,具有明显的抗肿瘤作用.     

人端粒酶逆转录亚单位(hTERT)基因特异性siRNA对MCF-7细胞生长抑制作用的研究

目的 研究人端粒酶逆转录亚单位（hTERT）特异性siRNA对MCF-7细胞体外生长及体内肿瘤形成能力的抑制作用。方法 设计并化学合成针对hTERT的siRNA，用脂质体转染法将其导入MCF-7细胞内，通过软琼脂克隆形成试验及接种裸鼠的肿瘤形成实验，观察hTERT-siRNA对人乳腺癌MCF-7细胞体外及体内的生长抑制作用。结果 软琼脂克隆形成试验显示，hTERT-siRNA转染的MCF-7细胞克隆形成数量明显少于对照组，抑制率达到84．1％。裸鼠体内实验结果显示，hTERT-siRNA转染组肿瘤生长速度也明显慢于对照组。结论 hTERT-siRNA在体内外均显示可以有效、特异地抑制肿瘤细胞的生长。     

氧化苦参碱对人乳腺癌 MCF -7 细胞的生长抑制作用及其机制研究

目的：了解中药苦参的有效成分氧化苦参碱对人乳腺癌细胞系MCF-7肿瘤细胞的增殖及其相关的调控机制的干预作用.方法：以乳腺癌细胞株（MCF-7）为研究对象,利用MTT法检测氧化苦参碱的体外杀伤活性;RT-PCR法、Western blot法、流式细胞技术、细胞免疫荧光计数检测基因、蛋白表达水平,对其相关调控基因的表达水平进行检测.结果：氧化苦参碱对体外培养的MCF-7细胞的生长有明显的抑制作用;可以诱导乳腺癌 MCF-7细胞凋亡,而且这种生长抑制及促进凋亡的作用可能是通过抑制Wnt/β-catenin信号转导通路的活性来实现.结论：氧化苦参碱可以通过抑制Wnt/β-catenin信号转导通路的活性来抑制人乳腺癌MCF-7细胞系的增殖,促进其凋亡.     

黏蛋白1基因转染DC对乳腺癌细胞MCF-7裸鼠移植瘤的免疫抑制作用

目的:探讨黏蛋白1 (mucin 1,MUC1基因转染DC对人乳腺癌MCF-7细胞裸鼠移植瘤的抑制作用.方法:体外诱导培养健康成人DC,应用脂质体转染法将pcDNA3.1-MUC1转染DC,ELISA法检测转染后DC分泌细胞因子IL-12和YNF-α的能力,LDH释放法检测基因转染后DC诱导特异性CTL对乳腺癌MCF-7细胞的杀伤活性.应用MU C1基因转染DC、空质粒转染DC、及生理盐水皮下注射治疗人乳腺癌MCF-7细胞裸鼠移植瘤,观测其对肿瘤生长的抑制作用.结果:转染pcDNA3.1-MUC1的DC分泌IL-12、TNF-α的能力较转染空质粒DC明显增强[IL-12:(202.52±29.61)vs(10.83±1.02)pg/ml;TNF-α:(349.07±79.42)vs(9.26±1.52)pg/ml,均P＜0.01];转染pcDNA3.1-MUC1的DC诱导产生特异性CTL,对人乳腺癌MCF-7细胞具有更明显的杀伤活性,效靶比为10∶1、5∶1和2.5∶1时的杀伤率分别达到56.2％、38.9％和25.8％,显著高于对照组CTL(均P＜0.01).MUC1基因转染DC对乳腺癌MCF-7裸鼠移植瘤生长抑制作用明显强于空质粒转染DC组(P＜0.05).结论:MUC1基因转染DC可以诱导特异性CTL,对乳腺癌MCF-7细胞具有更强的抗肿瘤免疫效应.     

Interleukin-2 differentially affects the proliferation of a hormone-dependent and a hormone-independent human breast cancer cell line in vitro and in vivo

Direct in vitro and in vivo effects of the lymphokine, interleukin-2 (IL-2), on hormone-dependent (MCF-7) and- independent (MDA-231) human breast cancer cell proliferation were investigated. In vitro, picomolar concentrations of IL-2 directly inhibited MCF-7 cell proliferation after 12 days of culture, while nanomolar doses of IL-2 significantly stimulated MCF-7 cell growth over the same time period. In addition, micromolar concentrations of IL-2 had virtually no effect on the in vitro proliferation of MCF-7 cells. In parallel in vitro growth experiments, the hormone-independent cells, MDA-231, were not affected by IL-2 regardless of concentration. IL-2 treatment of overiectomized, estrogen-treated nude mice, burdened with MCF-7 or MDA-231 tumors, inhibited MCF-7 tumor growth, but had no effect on MDA-231 tumors. Examination of T, B and natural killer (NK) cell function in these animals indicated that the interleukin-2-mediated effect on MCF-7 cell growth in vivo is independent of the proliferative abilities of these lymphoid cells, suggesting that IL-2 may directly affect the growth of these hormone-dependent human breast cancer cells.     

IGF-IR反义基因抑制乳腺癌细胞内源性IGF-IR表达的研究

目的探讨重组的人胰岛素样生长因子I型受体(IGFIR)的反义基因,对乳腺癌细胞内源性IGFIR表达的抑制作用。方法体外构建人IGFIR的反义基因真核表达载体pIGFIR/As,并转染人MCF7乳腺癌细胞,经G418筛选而获得稳定表达外源性反义基因的新细胞株MCF7/As,在基因水平和蛋白质水平检测内源性IGFIR的表达情况。结果人IGFIR的反义基因真核表达载体pIGFIR/As构建成功。RTPCR显示MCF7转染前后IGFIRmRNA的表达水平分别为(0.71±0.04)和(0.43±0.06),表达明显下降(P<0.05);Westernblot显示其蛋白质的表达水平呈相同的变化趋势(P<0.01)。结论本实验构建的载体pIGFIR/As能够稳定转染MCF7细胞,所表达的IGFIR的反义基因能够在基因和蛋白质水平显著抑制内源性IGFIR的表达。     

Interleukin-3: A putative protective factor against breast cancer which is secreted by male but not female breast fibroblasts

The enzyme 17β-hydroxysteroid dehydrogenase (17-HSD) is a key regulator of intracellular 17β-estradiol (E2), which is associated with breast cancer and is influenced by paracrine factors released by breast-cancer fibroblasts. Since the incidence of breast cancer is much higher in females than in males, we have used an in vitro ceil culture system to investigate whether male fibroblasts may inhibit breast-cancer genesis by restricting the intracellular accumulation of E2. Fibroblasts were obtained from normal males and females undergoing reduction mammoplasty, and from females with benign or malignant breast lesions. Fibroblast-conditioned medium (CM) was incubated with the established breast-cancer cell line, MCF-7, and its effects on 17-HSD activity were assessed. CM (25% v/v) from male breast fibroblasts had a significant inhibitory effect on reductive i 7-HSD, decreasing E2 production. This was in direct contrast to the effects of CM from female breast fibroblasts, which had a powerful stimulatory effect on reductive 17-HSD. RT-PCR allowing simultaneous detection of a range of cytokines was performed on each type of fibroblast. IL-3 mRNA was consistently detected in fibroblasts from male but not female breast tissue. Addition of rhIL-3 to cultures of MCF-7 caused a reduction in 17-HSD activity and addition of a polyclonal antibody directed against IL-3 to male CM completely reversed the inhibitory effects of CM. Thus, male breast fibroblasts may be responsible for secreting IL-3-like factors which, given the considerably lower incidence rates of breast cancer in men, may have a protective effect against breast cancer. © 1995 Wiley-Less, Inc.     

Regulation by protein kinase C of TGF-beta 1 expression in cultured cells of breast adenocarcinoma]

In human breast carcinoma MCF-7 cells, phorbol diesters inhibit proliferation and induce cell maturation. We investigated the involvement of TGF-beta 1 in the PCK-mediated inhibition of breast cancer cell proliferation. Using an RNase protection assay, we showed that TPA induced a dose-dependent increase in levels of TGF-beta 1 mRNA that paralleled the inhibitory effect on MCF-7 proliferation. Similar results were obtained with another TPA-sensitive breast cancer cell line (BT-20). TPA did not increase TGF-beta 1 mRNA levels in the MCF-7:RPh-4 and T47D cell lines, which are both insensitive to the growth inhibitory effects of phorbol esters. In addition, the increase in TGF-beta 1 mRNA level was not observed after treatment of the MCF-7 cell with other inducers of cell differentiation such as forskolin, DMF, HMBA and sodium butyrate. The induction of TGF-beta 1 mRNA by TPA along with its inhibitory effect on cell proliferation suggests that TGF-beta 1 mediates, at least in part, the inhibitory effect of PKC activation.     

槲皮素增强乳腺癌细胞化疗敏感度的研究

目的探讨槲皮素(quercetin,Que)对乳腺癌细胞化疗药物敏感度的影响及其可能的机制。方法MTT 法检测不同浓度 Que 对人乳腺癌 MCF-7 细胞生长的影响,单用阿霉素(adriamycin,ADR）或 ADR 联合无毒剂量 Que 作用于人乳腺癌 MCF-7 细胞,MTT 法检测药物对细胞的增殖抑制作用,流式细胞法检测细胞凋亡率;Transwell小室法检测细胞侵袭能力的变化;RT-PCR法及Western blot法分别检测缺氧诱导因子-1α (HIF-1α) mRNA水平和蛋白水平的表达变化。结果不高于 0.7 μM Que对细胞生长无影响。与单用ADR组相比较,0.7 μM Que与ADR联用可提高ADR对人乳腺癌MCF-7细胞的增殖抑制率、诱导其细胞凋亡、降低其体外侵袭和转移能力、下调HIF-1αmRNA和蛋白的表达。结论Que能够增强人乳腺癌MCF-7细胞化疗敏感度,其机制可能与下调该细胞HIF-1α的mRNA和蛋白表达有关。     

Down-regulation of expression of interleukin-6 and its receptor results in growth inhibition of MCF-7 breast cancer cells

Interleukin-6 (IL-6) plays an important role in the neoplastic process through its action on cancer cell adhesion, motility, proliferation, tumor-specific antigen expression, and thrombopoiesis. IL-6 exerts its activity by binding to a high affinity receptor complex consisting of two membrane glycoproteins: the 80 kDa IL-6 a-receptor subunit (IL-6R) and the 130 kDa signal-transducing protein (GP130). In the present study, MCF-7 breast cancer cells were cultured with human IL-6 and IL-6 soluble receptor (sIL-6R). MCF-7 cells were also treated with either antibodies specific to human IL-6 and IL-6R, or synthetic antisense oligodeoxynucleotides (ODNs) targeted to IL-6 and IL-6R genes. Cell growth was measured, and it was found that human IL-6 and sIL-6R did not significantly increase the proliferation of MCF-7 cells. When IL-6 produced by the MCF-7 cells was bound by rabbit anti-human IL-6 antibody, there was a significant dose-dependent inhibition of cell proliferation. IL-6 and IL-6R antisense ODNs caused a marked and specific decrease in IL-6 and IL-6R mRNA and proteins, respectively. Both IL-6 and IL-6R antisense ODNs significantly inhibited the proliferation of MCF-7 cells, but the inhibitory effect of IL-6R antisense ODN was greater than that of IL-6 antisense ODN (IC??: IL-6R: 1 μM; IL-6: 5 μM, 72-hour incubation). Addition of exogenous IL-6 partially reversed the growth inhibition caused by IL-6 antisense ODN but not the growth inhibition caused by IL-6R antisense ODN. In conclusion, IL-6 plays an important role in maintaining the growth of MCF-7 breast cancer cells. These results suggest careful modulation of IL-6 and IL-6R expression of cells as a potential approach for breast cancer therapy.     

Overexpression of macrophage migration inhibitory factor induces angiogenesis in human breast cancer

Macrophage migration inhibitory factor (MIF) is known to be an important contributor to tumor progression. Overexpression of MIF has been reported in different types of tumors. However, the correlation between MIF expression and tumor pathologic features in patients with breast cancer has not been elucidated. In this study, we examined the expression of MIF, vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) in human tissues with or without tumor. In addition, we investigated the expression of MIF in MDA-MB-231, MCF-7 (breast cancer cell lines) and MCF-10A (epithelial cell line) cells, and its effect on VEGF and IL-8. We found that MIF was overexpressed in breast cancer tissues compared with normal ones. The level of MIF showed the positive correlation between the expression of IL-8 and tumor microvessel density (MVD). The patients with positive MIF expression in tumor tissues showed a significantly worse disease-free survival compared with negative ones. Increased MIF serum levels were also found to correlate with higher levels of IL-8 in the sera of the patients with breast cancer. In vitro experiments successfully detected MIF in breast cell lines. However, the expression level of it by normal epithelial cells was much less than that of cancer cells. Exogenous MIF did not cause endothelial tube formation and migration but induced a dose dependent increase in VEGF and IL-8 secretion in breast cancer cell lines. In summary, our studies show that human breast cancer tissue expresses MIF. Its in vitro effect on VEGF and IL-8 indicates that MIF may contribute to tumor in angiogenesis and thus play an important role in the pathogenesis of breast cancer.     

Synergistic Effects of Exemestane and Aspirin on MCF-7 Human Breast Cancer Cells

Objective: The purpose of this study is to investigate the combined effects of exemestane and aspirin on MCF-7 human breast cancer cells. Methods: Antiproliferative effects of exemestane and aspirin, alone and in combination, on growth of MCF-7 human breast cancer cells were assessed using the MTT assay. Synergistic interaction between the two drugs was evaluated in vitro using the combination index (CI) method. The cell cycle distribution was analyzed by flow cytometry and Western blotting was used to investigate the expression of cyclooxygenase-1, cyclooxygenase-2 and Bcl-2. Results: MTT assays indicated that combination treatment obviously decreased the viability of MCF-7 human breast cancer cells compared to individual drug treatment (CI<1). In addition, the combination of exemestane and aspirin exhibited a synergistic inhibition of cell proliferation, significantly arrested the cell cycle in the G0/G1 phase and produced a stronger inhibitory effect on COX-1 and Bcl-2 expression than control or individual drug treatment. Conclusion: These results indicate that the combination of exemestane and aspirin might become a useful method to the treatment of hormonedependent breast cancer. The combination of the two inhibitors significantly increased the response as compared to single agent treatment, suggesting that combination treatment could become a highly effective approach for breast cancer.