Glutathione S-transferases in wild-type and doxorubicin-resistant MCF-7 human breast cancer cell lines

1. Overexpression of glutathione S-transferases (GST) in breast cancer cells is hypothesized to be a component of the multifactorial doxorubicin-resistant phenotype. 2. We have characterized the expression of GST enzymes at the catalytic activity, protein and mRNA levels in wild-type MCF-7 (MCF-7/WT) human breast cancer cells and a line selected for resistance to doxorubicin (MCF-7/ADR), with the goal of modulating GST activity to overcome resistance. 3. The MCF-7/ADR cells were 30-65-fold more resistant to doxorubicin than the MCF-7/WT cells. 4. Total cytosolic GST catalytic activity was elevated 23-fold in the MCF-7/ADR cells as compared with the MCF-7/WT cells, and the MCF-7/ADR cells also showed 3-fold increases in catalytic activity toward GST mu and alpha class-selective substrates. Neither cell line showed detectable catalytic activity with a GST mu class-selective substrate. 5. MCF-7/ADR cells showed pronounced overexpression of GST mu protein and GST P1 mRNA in comparison with the wild-type cell line. Neither cell line displayed detectable GST alpha or mu at the protein level. 6. A glutathione analogue that functions as a selective GST alpha inhibitor was more potent at inhibiting total cytosolic GST catalytic activity in the MCF-7/ADR cell line than GST alpha and mu class-selective inhibitory glutathione analogues and the non-selective GST inhibitor ethacrynic acid. 7. The multidrug resistance-associated protein, which can function as a glutathione-conjugate transporter, appeared weakly overexpressed in the MCF-7/ADR cells in comparison with the MCF-7/WT cells.     

The cytotoxic protein can induce autophagocytosis in addition to apoptosis in MCF-7 human breast cancer cells

Phagocytic clearance of dying cells is found in many phagocytes. It has been shown that dying cells can be phagocytosed by other phagocytic cells through autophagic or apoptotic cellular death. To date, whether cancer cells have such phagocytic activity has not been studied. In this study, our data shows that RC-RNase can trigger cell death in human breast cancer MCF-7 cells through the apoptotic pathway. Interestingly, when treated with cytotoxic protein, the remaining MCF-7 cells can phagocytose the dying MCF-7 cells via autophagocytic activity, demonstrated directly by real-time image observation and electron microscopy analysis. To sum up, this study demonstrates for the first time that RC-RNase can trigger apoptosis and autophagocytosis in MCF-7 cancer cells.     

Evaluation of chemopreventive and cytotoxic effect of lemon seed extracts on human breast cancer (MCF-7) cells

Extracts from lemon seed were investigated for the radical scavenging activity and apoptotic effects in human breast adenocarcinoma (MCF-7) cells and non-malignant breast (MCF-12F) cells for the first time. Defatted seed powder was successively extracted with ethyl acetate (EtOAc), acetone, methanol (MeOH), and MeOH:water (80:20). The chemical constituents were identified and quantified by LC-MS and HPLC analysis, respectively. The highest radical scavenging activity of 62.2% and 91.3% was exhibited by MeOH:water (80:20) at 833μg/mL in 1,1-diphenyl-2-picryl hydrazyl (DPPH) and 2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS(+)), respectively. In addition, the MeOH:water (80:20) extract showed the highest (29.1%, P<0.01) inhibition of MCF-7 cells in MTT assay. Treatment of the MeOH:water (80:20) extract induced DNA fragmentation and poly(ADP-ribose) polymerase (PARP) cleavage. Increased levels of Bax and cytosolic cytochrome C and decreased levels of Bcl2 were also observed in MeOH:water (80:20) treated MCF-7 cells. In conclusion, the MeOH:water (80:20) extract from lemon seed has potent antioxidant activity and induces apoptosis in MCF-7 cells, leading to the inhibition of proliferation. These results suggest that aglycones and glucosides of the limonoids and flavonoid present in MeOH:water (80:20) extract may potentially serve as a chemopreventive agent for breast cancer.     

Autophagy is involved in cytotoxic effects of crotoxin in human breast cancer cell line MCF-7 cells

AIM: To investigate the role of crotoxin (CrTX)-induced autophagy in the death of MCF-7 cells, a caspase-3-deficient, human breast cancer cell line. METHODS: Cultured MCF-7 cells were treated with various doses of CrTX, a phospholipase A2 (PLA2) isolated from the venom of the South American rattlesnake, Crotalus durissus terrificus. The cytotoxicity of CrTX in the presence and absence of caspase inhibitors was measured with methyl thiazolyl tetrazolium (MTT) and lactate dehydrogenase (LDH) leakage assays. The activation of autophagy was determined with transmission electron microscope and monodansylcadaverin (MDC) labeling. The upregulation of lysosomal enzymes, the release of cytochrome c (cyto-c), and the nuclear translocation of the apoptosis inducing factor (AIF) were examined by immunoblotting and immunofluorescence. RESULTS: CrTX inhibited the viability of MCF-7 cells in a dose- and time-dependent manner. CrTX-activated autophagy was revealed by punctuate MDC labeling, and an increase in the formation of autophagosomes as well as apoptosis, as evidenced by nuclear condensation and fragmentation. The activation of cathepsin B, D, and L, in addition to the release of cytochrome c and the relocation of AIF into nuclei, were observed after CrTX treatment. Autophagy inhibitors 3-methyladenine (3-MA), NH4Cl, and the pan-caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone (Z-Vad-fmk), attenuated CrTX-induced cell death. CONCLUSION: An autophagic mechanism contributes to the apoptosis of MCF-7 cells induced by CrTX.     

Salinomycin对乳腺癌阿霉素耐药细胞株MCF-7/DOX的增殖抑制作用及机制

目的本研究旨在探讨Salinomycin对乳腺癌阿霉素耐药细胞株MCF-7/DOX增殖和凋亡的影响及可能作用机制。方法 MTS实验检测Salinomycin对MCF-7/DOX细胞增殖的影响;Annexin V-FITC/PI染色检测Salinomycin对MCF-7/DOX耐药细胞凋亡的影响;DCFH-DA染色检测Salinomycin对MCF-7/DOX耐药细胞活性氧(reactive oxygen species,ROS)产生的影响;JC-1法测定细胞线粒体膜电位;Western blot法检测细胞凋亡相关蛋白BAX、BCL-2、caspase-3和caspase-9的表达变化。结果 Salinomycin能明显抑制MCF-7/DOX耐药细胞增殖,且具有浓度依赖性;流式分析发现Salinomycin能够诱导MCF-7/DOX细胞凋亡,增加细胞内ROS水平,降低细胞线粒体膜电位;与对照组相比较,Salinomycin处理明显抑制BCL-2的表达,上调BAX、cleaved caspase-3和cleaved caspase-9的蛋白表达;抗氧化剂N-acetylcysteine(NAC)则逆转上述作用。结论 Salinomycin能够诱导MCF-7/DOX细胞凋亡,其机制可能与Salinomycin诱导ROS的产生,激活线粒体凋亡途径有关。     

Autophagy preceded apoptosis in oridonin-treated human breast cancer MCF-7 cells

Recent studies have shown that MCF-7 cells undergo autophagy under some conditions, such as tamoxifen treatment and starvation. In this study, we investigated autophagy in MCF-7 cells under oridonin treatment and further examined the relationship between autophagy and apoptosis. After 3-MA (the specific inhibitor of autophagy) pre-culture, MCF-7 cells were exposed to oridonin, and the growth inhibitory ratio, morphologic changes, DNA fragmentation, proteins expression, autophagic ratio and apoptotic ratio were evaluated. Oridonin inhibited the proliferation of MCF-7 cells and induced autophagy in vitro. MDC (a specific dye for autophagosome) recruitment and typical apoptotic features, including apoptotic bodies, membrane blebbing as well as nuclear condensation, were induced by oridonin. Oridonin downregulated the phosphorylation of ERK, whereas those of JNK and P38 kinase were upregulated. In the condition of oridonin treatment, 3-MA significantly reduced the autophagic level, and the apoptotic cell ratio was also declined. Furthermore, combined treatment with oridonin and 3-MA upregulated ERK phosphorylation and downregulated JNK and P38 kinases phosphorylation compared with oridonin alone treatment groups, indicating that autophagy facilitated apoptosis in oridonin-induced MCF-7 cells. In addition, 3-MA application downregulated DNA ladder and Bax expression but upregulated Bcl-2 expression, compared with oridonin alone treatment. Taken together, oridonin simultaneously induced MCF-7 cells both apoptosis and autophagy, and in this settings, inhibition of autophagy induced lowered apoptotic level, therefore, autophagy participated in upregulation of apoptosis.     

Phthalates inhibit tamoxifen-induced apoptosis in MCF-7 human breast cancer cells

Environmental estrogens represent a class of compounds that can mimic the function or activity of the endogenous estrogen 17 -estradiol (E2). Phthalates including butyl benzyl phthalate (BBP), di(n-butyl) phthalate (DBP), and di(2-ethylhexyl) phthalate (DEHP) are used as plasticizers, and also widely used in food wraps and cosmetic formulations. Phthalates have been shown to mimic estrogen and are capable of binding to the estrogen receptor (ER). It has been demonstrated that estrogen promotes drug resistance to tamoxifen (TAM) in breast cancer. In order to further evaluate the potential role of the phthalates as environmental estrogens, the effect of phthalates was investigated on TAM-induced apoptosis in MCF-7 human breast cancer cells. Our results show that phthalates, BBP (100 M), DBP (10 M), and DEHP (10 M), significantly increased cell proliferation in MCF-7, but not in MDA-MB-231 cells. In addition, BBP, DBP, and DEHP mimicked estrogen in the inhibition of TAM-induced apoptosis in MCF-7 cells. Our data suggest that the inhibitory effect of phthalates on TAM-induced apoptosis involves an increase in intracellular Bcl-2 to Bax ratio. Given that the phthalates are widely used in cosmetics mainly for women, our findings that revealed the promoting effect of BBP, DBP, and DEHP on chemotherapeutic drug resistance to TAM in breast cancer may be of biological relevance.     

Lack of glutathione conjugation to adriamycin in human breast cancer MCF-7/DOX cells. Inhibition of glutathione S-transferase p1-1 by glutathione conjugates from anthracyclines

One of the proposed mechanisms for multidrug resistance relies on the ability of resistant tumor cells to efficiently promote glutathione S-transferase (GST)-catalyzed GSH conjugation of the antitumor drug. This type of conjugation, observed in several families of drugs, has never been documented satisfactorily for anthracyclines. Adriamycin-resistant human breast cancer MCF-7/DOX cells, presenting a comparable GSH concentration, but a 14-fold increase of the GST P1–1 activity relative to the sensitive MCF-7 cells, have been treated with adriamycin in the presence of verapamil, an inhibitor of the 170 P-glycoprotein (P-gp) drug transport protein, and scrutinized for any production of GSH–adriamycin conjugates. HPLC analysis of cell content and culture broths have shown unequivocally that no GSH conjugates are present either inside the cell or in the culture broth. The only anthracycline present inside the cells after 24 hr of incubation was > 98% pure adriamycin. Confocal laser scanning microscopic observation showed that in MCF-7/DOX cells adriamycin was localized mostly in the Golgi apparatus rather than in the nucleus, the preferred site of accumulation for sensitive MCF-7 cells. These findings rule out GSH conjugation or any other significant biochemical transformation as the basis for resistance to adriamycin and as a ground for the anomalous localization of the drug in the cell. Adriamycin, daunomycin, and menogaril did not undergo meaningful conjugation to GSH in the presence of GST P1–1 at pH 7.2. Indeed, their synthetic C(7)-aglycon–GSH conjugates exerted a strong inhibitory effect on GST P1–1, with K_i at 25° in the 1–2 μM range, scarcely dependent on their stereochemistry at C(7).     

恩替诺特或西达本胺作用人乳腺癌耐多柔比星的MCF-7细胞的特征

组蛋白去乙酰化酶抑制剂西达本胺和恩替诺特均属苯酰胺类,已经批准在临床应用。鉴于耐药性是癌症化疗中的常见问题,本研究以耐多柔比星的人乳腺癌MCF-7细胞为模型,探讨两药的作用特征。CCK-8增殖实验显示:与敏感细胞相比,耐药细胞对该两药均具有一定的耐药性,尤其是对西达本胺更为明显。恩替诺特可增加顺铂的抑制作用。以罗丹明123滞留作为ATP结合盒转运蛋白1 (ATP-binding cassette B1, ABCB1)耐药性的间接指示剂,检测到两药对耐药细胞的罗丹明123的滞留均不明显,说明这些药物不是ABCB1的外排底物。固定浓度的恩替诺特处理耐药细胞,可明显地引起ABCB1蛋白的表达增加。流式细胞术分析细胞周期发现:两药均引起细胞明显的G1期阻滞,轻微地增加G2/M期细胞的数量。细胞凋亡分析发现两药均可引起MCF-7敏感细胞变圆,出现凋亡样形态,凋亡指示蛋白PARP-1出现切割;但对MCF-7耐药细胞均没有诱导凋亡的作用。本研究结果表明:MCF-7耐药细胞对西达本胺或恩替诺特均具有一定的耐药性,抗细胞凋亡可能是其耐药特征之一。     

Docetaxel-loaded PLGA nanoparticles to increase pharmacological sensitivity in MDA-MB-231 and MCF-7 breast cancer cells

This study aimed to develop docetaxel (DTX) loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (DTX-NPs) and to evaluate the different pharmacological sensitivity of NPs to MCF-7 and MDA-MB-231 breast cancer cells. NPs containing DTX or coumarin-6 were prepared by the nanoprecipitation method using PLGA as a polymer and d-α-tocopherol polyethylene glycol 1000 succinate (TPGS) as a surfactant. The physicochemical properties of NPs were characterized. In vitro anticancer effect and cellular uptake were evaluated in breast cancer cells. The particle size and zeta potential of the DTX-NPs were 160.5 ± 3.0 nm and –26.7 ± 0.46 mV, respectively. The encapsulation efficiency and drug loading were 81.3 ± 1.85% and 10.6 ± 0.24%, respectively. The in vitro release of DTX from the DTX-NPs was sustained at pH 7.4 containing 0.5% Tween 80. The viability of MDA-MB-231 and MCF-7 cells with DTX-NPs was 37.5 ± 0.5% and 30.3 ± 1.13%, respectively. The IC₅₀ values of DTX-NPs were 3.92- and 6.75-fold lower than that of DTX for MDA-MB-231 cells and MCF-7 cells, respectively. The cellular uptake of coumarin-6-loaded PLGA-NPs in MCF-7 cells was significantly higher than that in MDA-MB-231 cells. The pharmacological sensitivity in breast cancer cells was higher on MCF-7 cells than on MDA-MB-231 cells. In conclusion, we successfully developed DTX-NPs that showed a great potential for the controlled release of DTX. DTX-NPs are an effective formulation for improving anticancer effect in breast cancer cells.     

保护性自噬对顺铂诱导人乳腺癌 MCF-7细胞凋亡的抑制作用探讨

目的：探讨顺铂对乳腺癌 MCF-7细胞自噬的影响及自噬在顺铂诱导凋亡中的作用。方法顺铂处理乳腺癌 MCF-7细胞,MTT 检测细胞增殖的能力,Hoechst 33342染分析细胞的凋亡,吖啶橙染色分析细胞的自噬,Western blot 分析自噬蛋白 LC3Ⅰ／Ⅱ和 p62的表达和凋亡蛋白多聚 ADP-核糖聚合酶 PARP 表达。结果顺铂呈时间和剂量依赖性抑制乳腺癌 MCF-7细胞的增殖,并且凋亡细胞数量随顺铂浓度的递增而增加;同时顺铂能诱导微管相关蛋白轻链3-Ⅱ（LC3Ⅱ）蛋白的增加,p62蛋白的减少以及酸性自噬溶酶体的增加,顺铂联合氯喹明显增加了 LC3Ⅱ和 p62的蛋白的表达;与单药顺铂相比,自噬抑制剂氯喹明显降低细胞存活率（89．17％±2．56％）vs （74．63％±1．51％）,（P ＜0．05）,而且 PARP 蛋白发生了明显的裂解（P ＜0．05）。结论顺铂诱导乳腺癌 MCF-7细胞保护性自噬,抑制自噬可以增加顺铂诱导乳腺癌 MCF-7细胞凋亡,自噬抑制剂联合顺铂为乳腺癌提供了新的治疗策略。     

Fate of 3,3'-diindolylmethane in cultured MCF-7 human breast cancer cells

3,3'-Diindolylmethane (DIM) is a major in vivo product of the cancer preventative agent indole-3-carbinol that is found in vegetables of the genus Brassica. Here, we report on the metabolic fate of radiolabeled DIM in MCF-7 cells. DIM was slowly metabolized to several sulfate conjugates of oxidized DIM products that were primarily detected in the medium. The radioactivity detected in cells was predominantly unmodified DIM (81-93%) at all time intervals up to 72 h treatment. Co-treatment of MCF-7 cells with quercetin slowed the rate that oxidized DIM products accumulated in the medium, while indole[3,2-b]carbazole (ICZ) co-treatment accelerated their production. ICZ is an inducer of P450 1A2, while quercetin is a specific inhibitor of this isoform, suggesting that P450 1A2 is primarily responsible for the oxidation of DIM, probably through 2,3-epoxidation similar to 3-methylindole. Sulfate conjugates of oxidized DIM metabolites were cleaved by sulfatase digestion and identified by LC/MS as 3-(1H-indole-3-ylmethyl)-2-oxindole (2-ox-DIM), bis(1H-indol-3-yl)methanol (3-methylenehydroxy-DIM), 3-[hydroxy-(1H-indol-3-yl)-methyl]-1,3-dihydro-2-oxindole (3-methylenehydroxy-2-ox-DIM), and 3-hydroxy-3-(1H-indole-3-ylmethyl)-2-oxindole (3-hydroxy-2-ox-DIM). Derivatives of 2-ox-DIM represented greater than 30% of the radioactivity in the sulfatase-digested medium. Although oxindole formation was the primary metabolic pathway in MCF-7 cells, synthetic 2-ox-DIM was inactive in a 4-ERE-luciferase reporter assay and, therefore, probably not responsible for the estrogenic activity previously observed for DIM. Unmodified DIM rapidly accumulated in the nuclear membranes representing approximately 35-40% of the radioactivity after 0.5-2 h treatment. Uptake of radiolabeled DIM appeared to be a passive partitioning into the nuclear membranes and was not dependent upon the cell cytosol. The nuclear uptake of DIM was not saturable and could not be blocked by pretreatment with unlabeled DIM (100 microM). Further, treatments in serum-free medium increased the uptake of radiolabeled DIM by the MCF-7 cells. These findings show that the uptake of DIM by membranes significantly increases its localized concentration, which may contribute to its biological activities.     

丁酸钠对乳腺癌细胞MCF-7生长抑制的影响

目的探讨丁酸钠对乳腺癌细胞MCF-7生长的抑制作用。方法将乳腺癌细胞MCF-7常规培养至对数生长期,采用浓度为0、2．5、5．0、10．0mmol／L的丁酸钠处理后,观察对MCF生长的抑制作用。结果浓度为2．5、5．0、10．0mmol／L的丁酸钠处理的MCF-7细胞生长速度明显慢于亲本细胞,随着浓度的增加呈减慢趋势,差异有统计学意义（P〈0．05）。浓度为5mmol／L的丁酸钠处理后的MCF-7细胞克隆形成率明显低于MCF亲本细胞,差异有统计学意义（P〈0．05）。丁酸钠处理的细胞G1明显高于亲本细胞,S期及G2／M期明显低于亲本细胞,凋亡细胞百分比明显高于亲本细胞,差异有统计学意义（P〈0．05）。结论丁酸钠可能是通过诱导MCF-7细胞凋亡而起到逆转其恶性生物学行为的作用。     

Resveratrol modulates roscovitine-mediated cell cycle arrest of human MCF-7 breast cancer cells.

Human MCF-7 breast cancer cells are relatively resistant to anti-cancer drugs. Recently, we reported that roscovitine (ROSC), a selective cyclin-dependent kinase (CDK) inhibitor, arrested human MCF-7 breast cancer cells in G2 phase of the cell cycle and concomitantly induced apoptosis. Moreover, we observed that the effect of the CDK inhibitor was dependent on the content of the culture medium. The cell cycle inhibiting action of ROSC was markedly diminished in human MCF-7 cells cultivated in medium supplemented with phenol red. These observations indicated that the therapeutic effects of ROSC can be affected by the components of the tissue medium. Recently, a number of epidemiological and experimental studies indicated that polyphenols (e.g. resveratrol, epicatechins etc.), abundant micronutrients in food, are anti-oxidant agents and could have strong anti-mitotic as well as pro-apoptotic activities. In the present contribution we raised the question whether the ROSC-mediated cell cycle arrest could be additionally modulated by compounds of natural origin, especially by polyphenols. Considering the potential benefits of the dietary components during the post-chemotherapy period, we focused our attention on the effects of resveratrol administration after treatment with ROSC. We analyzed whether the combined treatment with resveratrol would exert any additional effect on the cell cycle status of ROSC-treated human cancer cells. Resveratrol exhibited low direct cytotoxicity. The combined treatment with ROSC enhanced the ROSC-mediated inhibition of cell proliferation and cell cycle arrest. These results indicate that targeted combination of anti-cancer drugs with distinct naturally occurring compounds could increase the efficacy of the therapy and concomitantly reduce the undesired side effects exerted by cytostatic drugs.     

苦参碱对人乳腺癌细胞MCF-7增殖、凋亡的影响

目的探讨苦参碱对人乳腺癌细胞MCF-7的增殖、凋亡的影响。方法实验组MCF-7细胞加入苦参碱溶液,对照组加入等量培养液。MTT比色法检测苦参碱对MCF-7细胞的生长抑制率;镜下观察细胞形态学变化;流式细胞术检测MCF-7细胞Bax、Bcl-2蛋白表达。结果与对照组比较,实验组苦参碱呈时间、剂量依赖性明显抑制MCF-7细胞生长,诱导MCF-7细胞凋亡,上调Bax蛋白表达,下调Bcl-2蛋白表达。结论苦参碱对MCF-7细胞具有明显的生长抑制和促凋亡作用,与上调Bax和下调Bcl-2表达有关。     

Elevation of pi class glutathione S-transferase activity in human breast cancer cells by transfection of the GST pi gene and its effect on sensitivity to toxins

Increased expression of the glutathione S-transferase (GST; E.C.2.5.1.18) pi class isozyme is associated with both malignant transformation and drug resistance, as well as with decreased estrogen receptor content in breast cancer. In order to further characterize the role of this enzyme in drug resistance, we cloned the cDNA encoding the human isozyme GST pi and developed two eukaryotic expression vectors using this cDNA and either the human metallothionein IIa or cytomegalovirus immediate-early promoters. These GST pi expression vectors were cotransfected with pSV2neo into drug-sensitive MCF-7 human breast cancer cells, which have low amounts of GST activity and which do not express GST pi. The transfected cells were selected for G418 resistance and individual clones were screened for GST activity. Three clones that demonstrated increased GST activity were selected for further study. Immunoprecipitation studies demonstrated that the increase in GST activity in these clones was due to expression of GST pi. Although the total GST activity of the positive clones was increased as much as 15-fold over that in wild-type MCF-7 cells, there was no change in glutathione peroxidase activity, as measured using cumene hydroperoxide as a substrate. Immunoblot studies revealed that the increased GST enzyme produced in the transfected cells was identical in size to endogenous GST pi. Southern blot analysis demonstrated the incorporation of the GST pi expression vector into the genome of the positive clones and Northern blot analysis showed that the transfected genes made a hybrid GST pi RNA that was slightly larger than the endogenous GST pi RNA. Primer extension studies demonstrated that this increase in length corresponded to the added length of the 5' leader sequence of the expression vector. The effect of increased GST pi activity on the sensitivity of the transfected clones to several cytotoxic agents was assessed by colony-forming assay. The transfected clones were slightly more resistant (1.3-4.1-fold) to benzo(a)pyrene and its toxic metabolite benzo(a)pyrene-(anti)-7,8-dihydrodiol-9,10-epoxide, as well as to ethacrynic acid (3.1-to 4.4-fold). Although increased GST pi expression is found in MCF-7 cells selected for doxorubicin resistance, the transfected clones were not consistently more resistant to doxorubicin than control cells. In addition, the transfected cells were not resistant to either melphalan or (cis)-platinum, even though conjugation with glutathione is known to play a role in the detoxification of both of these drugs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Metabolism of equilenin in MCF-7 and MDA-MB-231 human breast cancer cells

Sulfate conjugates of the B-ring unsaturated estrogens, equilin, equilenin, and 8-dehydroestrone, and their 17alpha- and 17beta-dihydro analogues, constitute about 54% of Premarin (Wyeth-Ayerst), the most commonly prescribed estrogen formulation in estrogen replacement therapy. Despite the wide clinical use of Premarin, there have been very few studies on the metabolism of the B-ring unsaturated estrogens in humans and there is no information regarding the fate of these compounds in breast tissue or tumors. In this study, we investigated the metabolism of equilenin in two lines of human breast-cancer cells, MCF-7 and MDA-MB-231. MCF-7 cells respond to treatment with Ah-receptor agonists with induction of cytochromes P450 1A1 and 1B1, whereas in MDA-MB-231 cells P450 1B1 is predominantly induced. Metabolites of equilenin were identified and quantified by GC/MS utilizing a series of synthetic metabolite standards and deuterium-labeled analogues as internal standards. In the two cell lines, the same pathways of equilenin metabolism were observed. Equilenin was reduced at C-17 to the 17beta-dihydro form, with minimal production of the 17alpha-dihydro isomer. Both equilenin and 17beta-dihydroequilenin were hydroxylated at the C-4 position, and the resultant catechol metabolites were methylated to form 4-methoxyequilenin and 4-methoxy-17beta-dihydroequilenin. Rates of equilenin metabolism were markedly elevated in cultures exposed to the Ah-receptor agonists, 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3,4,4',5-tetrachlorobiphenyl, implicating the activities of P450s 1A1 and 1B1 in the metabolism. The 2-hydroxylation pathways of equilenin and 17beta-dihydroequilenin metabolism were not observed. In microsomal reactions with cDNA-expressed human enzymes, both P450s 1A1 and 1B1 catalyzed the 4-hydroxylation of 17beta-dihydroequilenin, whereas with 17beta-estradiol as substrate P450 1A1 catalyzes predominantly 2-hydroxylation and P450 1B1 predominantly 4-hydroxylation. Since P450 1B1 is constitutively expressed and both P450s 1A1 and 1B1 are inducible in many extrahepatic tissues including the mammary epithelium, these results indicate the potential for 4-hydroxylation of equilenin and 17beta-dihydroequilenin in extrahepatic, estrogen-responsive tissues.     

Prolidase-activated prodrug for cancer chemotherapy cytotoxic activity of proline analogue of chlorambucil in breast cancer MCF-7 cells

Although prolidase [EC 3.4.13.9] is found in normal cells, substantially increased levels are found in some neoplastic tissues. Because prolidase possesses the ability to hydrolyse imido bonds of various low molecular weight compounds coupled to L-proline, we hypothesized that coupling of L-proline through an imido bond to anticancer drugs might create prodrugs which would be locally activated by tumour-associated prolidase and consequently would be less toxic to normal cells that evoke lower prolidase activity. To test this concept we have synthesized a conjugate of chlorambucil-proline (CH-pro) as a possible prodrug. Treatment of this prodrug with prolidase generated the L-proline and the free drug, demonstrating its substrate susceptibility to prolidase. We have compared several aspects of biological actions of chlorambucil (CH) and its prodrug in breast cancer MCF-7 cells. IC50 values for chlorambucil and for CH-pro in DNA synthesis were found to be 54 and 16 microM, respectively. CH-pro also exhibited a lesser ability to inhibit collagen biosynthesis in breast cancer MCF-7 cells compared to the free drug. The IC50 values for chlorambucil and for CH-pro in collagen biosynthesis were found to be about 32 and 80 microM, respectively. This suggests that the targeting of prolidase may serve as a potential strategy for converting antineoplastic prodrugs.     

Response of MCF-7 human breast cancer cells to some binary mixtures of oestrogenic compounds in-vitro.

The effects of simultaneous administration of some binary mixtures of seven natural and synthetic oestrogenic substances (17beta-estradiol, estrone, bisphenol A, butylbenzyl phthalate, endosulfan, methoxychlor and pentachlorophenol) on the cellular proliferation of human breast cancer MCF-7 cells in-vitro (a modified E-screen assay) have been measured. To assess the presence or absence of interactions of the two agents, the data were analysed on the basis of a graphical method in which the types and extents of interactions were described by response-surface diagrams. Of the nine combinations of the agents examined, synergistic interaction was evident for the combination of 17beta-estradiol and bisphenol A, whereas the remaining eight combinations were weakly synergistic, additive and/or weakly antagonistic in the dose-range tested.