Cytotoxicity In MCF-7 Cells Research Articles

Reconstitutable charged polymeric (PLGA) 2- b-PEI micelles for gene therapeutics delivery

This study investigated the potential of creating a charged polymeric micelle-based nucleic acid delivery system that could easily be reconstituted by the addition of water. (PLGA 36kDa) 2- b-bPEI 25kDa (PLGA MW 36 kDa, bPEI M w 25 kDa, PLGA:bPEI block ratio = 2) was synthesized and used to prepare cationic micelles. The copolymer retained proton-buffering capability from the bPEI block within the endosomal pH range. Micelle/pDNA complexes retained their particle size (100–150 nm) and surface charge (30–40 mV) following reconstitution. It was found that adding a small amount of low molecular weight bPEI (1.8 kDa) completely shielded pDNA in the micelle/pDNA complexes and enhanced transfection efficiency 50–100 fold for both fresh and reconstituted complexes without affecting complex size. Transfection efficiency for “reconstituted” micelle/pDNA/bPEI 1.8kDa (WR 1) complexes was 16-fold higher than its “fresh” counterpart. Although transfection levels achieved using “reconstituted” micelle/pDNA/bPEI 1.8kDa complexes were 3.6-fold lower than control “fresh” bPEI 25kDa/pDNA (N/P 5) complexes, transfection levels were 39-fold higher than “reconstituted” bPEI 25kDa/pDNA (N/P 5) complexes. The micelle/pDNA/bPEI 1.8kDa system showed very low cytotoxicity in MCF7 cells even with pDNA doses up to 20 μg, and transfection levels increased linearly with increasing pDNA dose. These results indicate that this PLGA- b-bPEI polymeric micelle-based system is well suited as a reconstitutable gene delivery system, and has high potential for use as a delivery system for gene therapy applications.

Prolylcarboxypeptidase Regulates Proliferation, Autophagy, and Resistance to 4-Hydroxytamoxifen-induced Cytotoxicity in Estrogen Receptor-positive Breast Cancer Cells

Endocrine therapy with tamoxifen (TAM) significantly improves outcomes for patients with estrogen receptor-positive breast cancer. However, intrinsic (de novo) or acquired resistance to TAM occurs in a significant proportion of treated patients. To identify genes involved in resistance to TAM, we introduced full-length cDNA expression library into estrogen receptor-positive MCF7 cells and exposed them to a cytotoxic dose of 4-hydroxytamoxifen (4OHTAM). Four different library inserts were isolated from surviving clones. Re-introduction of the genes individually into naive MCF7 cells made them resistant to 4OHTAM. Cells overexpressing these genes had an increase in acidic autophagic vacuoles induced by 4OHTAM, suggesting their role in autophagy. One of them, prolylcarboxypeptidase (PRCP), was investigated further. Overexpression of PRCP increased cell proliferation, boosted several established markers of autophagy, including expression of LC3-2, sequestration of monodansylcadaverine, and proteolysis of BSA in an ER-α dependent manner, and increased resistance to 4OHTAM. Conversely, knockdown of endogenous PRCP in MCF7 cells increased cell sensitivity to 4OHTAM and at the same time decreased cell proliferation and expression of LC3-2, sequestration of monodansylcadaverine, and proteolysis of BSA. Inhibition of enzymatic activity of PRCP enhanced 4OHTAM-induced cytotoxicity in MCF7 cells. Cells with acquired resistance to 4OHTAM exhibited increased PRCP activity, although inhibition of PRCP prevented development of 4OHTAM resistance in parental MCF7 cells and restored response to 4OHTAM in MCF7 cells with acquired resistance to 4OHTAM. Thus, we have for the first time identified PRCP as a resistance factor for 4OHTAM resistance in estrogen receptor-positive breast cancer cells.

Redox-Sensitive Disassembly of Amphiphilic Copolymer Based Micelles

Amphiphilic polymers of different hydrophilic-lipophilic ratios were prepared by free radical polymerization using two monomers consisting of triethylene glycol as the hydrophilic part and an alkyl chain connected by disulfide bond as the hydrophobic part. These polymers form micelle-like nanoassemblies in aqueous media and can encapsulate hydrophobic drug molecules up to 14% of their mass. In a reducing environment, these polymeric micelles disassemble and dissolve in water, since the amphiphilic polymers are converted into hydrophilic polymers upon cleavage of the disulfide bond. This disassembly event results in the release of hydrophobic molecules that had been encapsulated inside the micelle, the rate of which was found to be dependent on the concentration of the reducing agent, glutathione (GSH). In vitro experiments also show that the GSH-dependent release of the doxorubicin can be used to effect cytotoxicity in MCF-7 cells.

Dietary mushrooms reduce mitogenesis and induce apoptosis and cytotoxicity in MCF‐7 human breast cancer cells

Cancer is a leading cause of death in the U.S. Studies show that people with diets rich in fruits and vegetables have a reduced risk of cancer. Moreover, an accumulating body of evidence also suggests that consumption of dietary mushrooms, or fungi, can protect against many forms of human cancer. In this study, we tested the ability of five commonly consumed mushrooms to modulate the cancer process in MCF‐7 human breast cancer cells. Hot water extracts (5% v/v) of freeze‐dried maitake (MT), crimini (CRIM), portabella (PORT), king oyster (OYS), and white button (WB) mushrooms or water alone were incubated overnight with MCF7 cells. Mitogenesis, measured by BrdU incorporation, was significantly reduced by all mushrooms up to 33% with MT and OYS being the most potent. Cell number, however, did not significantly differ between groups as measured by hemacytometry. Apoptosis, measured by TUNEL, was increased significantly (p<0.05) by 40% after incubation with MT and ~20% (p>0.05) by other test mushrooms. MTT reduction, a mitochondrion‐dependent marker of proliferation, was unchanged. Cytoplasmic LDH release significantly increased after MT and WB, but not after other test mushrooms, indicating cytotoxicity although trypan blue exclusion appeared marginally elevated (95%) compared to control cells (92%) after 24 hours. Overall, dietary mushrooms reduced mitogenesis and induced apoptosis and cytotoxicity in MCF7 cells.

Role of glutathione S-transferase P1-1 in the cellular detoxification of cisplatin

Cells expressing elevated levels of allelic variants of human glutathione S-transferase P1 (GSTP1) and/or efflux transporters, MRP1 or MRP2, were used to evaluate the role of GSTP1-1 in cisplatin resistance. These studies revealed that GSTP1-1 confers low-level resistance (1.4- to 1.7-fold) to cisplatin-induced cytotoxicity in MCF7 cells. However, expression of MRP1 (MCF7 cells) or MRP2 (HepG2 cells) failed to augment or potentiate GSTP1-1-mediated resistance in either cell line. To understand the mechanism by which variants of GSTP1-1 confer resistance to cisplatin, their relative abilities to catalyze conjugation of cisplatin with glutathione were examined. Enzymes encoded by all three alleles tested, GSTP1a (I(104)A(113)), GSTP1b (V(104)A(113)), and GSTP1c (V(104)V(113)), increased the formation rate of the mono-platinum/glutathione derivative of cisplatin with relative catalytic activities of 1.0 (GSTP1a-1a variant) and 1.8 to 1.9 (GSTP1b-1b and GSTP1c-1c variants). Although these data are consistent with the idea that very low level resistance to cisplatin may be conferred by GSTP1-1-mediated cisplatin/glutathione conjugation, two observations indicate that such catalysis plays a minor role in the protection from cisplatin toxicity. First, the rates of GSTP1-1-mediated conjugation are extremely slow (1.7-2.6 h(-1) at 25 degrees C). Second, despite an 80% to 90% increase in catalysis of cisplatin conjugation by GSTP1b-1b or GSTP1c-1c over GSTP1a-1a, we observed no discernable differences in relative resistances conferred by these alternative variants when expressed in MCF7 cells. We conclude that high-level cisplatin resistance attributed to GSTP1-1 in other studies is not likely due to catalysis of cisplatin conjugation but rather must be explained by other mechanisms, which may include GSTP1-mediated modulation of signaling pathways.

Synthesis and protective effects of coumarin derivatives against oxidative stress induced by doxorubicin

The use of doxorubicin (DOX) in the treatment of solid tumors is limited by cardiotoxicity essentially due to oxidative stress generation. The aim of this study was to identify coumarin derivatives displaying a protective antioxidant activity without affecting DOX antitumoral efficiency. A set of eighteen coumarinic derivatives was synthesized. Their antioxidant power was evaluated in vitro with the FRAP (ferric reducing ability of plasma) method and in human breast adenocarcinoma MCF7 cells using H(2)DCFDA (2',7'-dichlorodihydrofluorescein diacetate) in a cytometric analysis. 4-Methyl-7,8-dihydroxycoumarin was found to exhibit an important antioxidant strength, a low cytotoxicity, and could decrease ROS (reactive oxygen species) production generated by DOX treatment without affecting DOX cytotoxicity in MCF7 cells.

Compensatory effects of the human nucleoside transporters on the response to nucleoside-derived drugs in breast cancer MCF7 cells

Nucleoside transporters (NTs) are involved in the cytotoxicity and transcriptomic response induced by nucleoside analogues. A relationship between the expression of nucleoside transporters and response to therapy has been demonstrated in solid tumours, although the pattern of such expression is highly variable. Thus, a question is whether the transporter expression pattern rather than specific NT proteins might better explain the ability of tumour cells to respond to nucleoside-derived drug therapy. In this study we used the breast cancer cell lines MCF7 and MCF7–hCNT1 (stably transfected with hCNT1) to determine whether hCNT1 expression can complement hENT1 functional loss in the cytotoxicity and transcriptomic response triggered by nucleoside analogues. Expression of hCNT1 slightly increased cell sensitivity to 5′-deoxy-5-fluorouridine (5′-DFUR). Inhibition of the endogenous equilibrative activity blocked 5′-DFUR cytotoxicity in MCF7 cells, but not in MCF7–hCNT1 cells. Moreover, under equilibrative transport inhibition conditions, induction of some transcriptional targets of 5′-DFUR was blocked in MCF7 cells, whereas ENT-inhibition had no effect on the transcriptional response to 5′-DFUR in MCF7–hCNT1 cells. To confirm the role of hCNT1 in 5′-DFUR treatment, a panel of nucleoside derivatives suitable for hCNT1-inhibition was obtained. The molecule T-Ala inhibited hCNT1-mediated transport. Furthermore, the cytotoxic action of 5′-DFUR and the transcriptional changes produced by this nucleoside analogue were partially inhibited by T-Ala in MCF7–hCNT1 cells. These results show a link between NT function and the pharmacogenomic response to nucleoside analogues and further support the hypothesis that the expression pattern rather than specific transporters determines the cytotoxic effect of nucleoside derivatives.

Short-Term Exposure of Cancer Cells to Micromolar Doses of Paclitaxel, with or without Hyperthermia, Induces Long-Term Inhibition of Cell Proliferation and Cell Death In Vitro

During intraoperative hyperthermic intraperitoneal chemotherapy for primary or secondary peritoneal malignancies, tumor cells are exposed to high drug concentrations for a relatively short period of time. We investigated in vitro the effect of paclitaxel and hyperthermia on cell proliferation, cell cycle kinetics and cell death under conditions resembling those during intraoperative hyperthermic intraperitoneal chemotherapy. Human breast MCF-7, ovarian SKOV-3 and hepatocarcinoma HEpG2 cells were exposed to 10 and 20 microM paclitaxel at 37, 41.5 or 43 degrees C for 2 h. Cell proliferation, cell cycle kinetics, necrosis and apoptosis were evaluated. Hyperthermia exerted a cytostatic effect to all cell lines and at 43 degrees C a cytotoxic effect on MCF-7 cells. MCF-7 and SKOV-3 cells treated under normothermic conditions with paclitaxel were arrested at G2/M or M phase for at least 3 days. Most of MCF-7 cells and approximately half of SKOV-3 cells were in interphase and became multinucleated without properly completing cytokinesis. Hyperthermia at 41.5 degrees C altered cell cycle distribution and affected the paclitaxel-related effect on cell cycle kinetics of MCF-7 and SKOV-3 cells. Analysis of the mode of cell death showed that cell necrosis prevailed over apoptosis. Hyperthermia at 43 degrees C increased paclitaxel-mediated cytotoxicity in MCF-7 cells and to a lesser extent in SKOV-3 and HEpG2 cells. Short-time treatment of carcinoma cells with high (micromolar) concentrations of paclitaxel in normothermic and hyperthermic conditions is highly efficient for cell growth arrest and could be of clinical relevance in locoregional chemotherapy.

Prodigiosin induces the proapoptotic gene NAG-1 via glycogen synthase kinase-3β activity in human breast cancer cells

Prodigiosin (2-methyl-3-pentyl-6-methoxyprodigiosene) is a bacterial metabolite that has anticancer and antimetastatic properties. However, the molecular mechanisms responsible for these abilities are not fully understood. Gene expression profiling of the human breast cancer cell line MCF-7 treated with prodigiosin was analyzed by cDNA array technology. The majority of the significantly modified genes were related to apoptosis, cell cycle, cellular adhesion, or transcription regulation. The dramatic increase of the nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1) made this gene an interesting candidate regarding the possible mechanism by which prodigiosin induces cytotoxicity in MCF-7 cells. Our results show that prodigiosin triggers accumulation of the DNA-damage response tumor-suppressor protein p53 but that NAG-1 induction was independent of p53 accumulation. Moreover, prodigiosin caused AKT dephosphorylation and glycogen synthase kinase-3beta (GSK-3beta) activation, which correlated with NAG-1 expression. Prodigiosin-induced apoptosis was recovered by inhibiting GSK-3beta, which might be due, at least in part, to the blockade of the GSK-3beta-dependent up-regulation of death receptors 4 and 5 expression. These findings suggest that prodigiosin-mediated GSK-3beta activation is a key event in regulating the molecular pathways that trigger the apoptosis induced by this anticancer agent.

Reactive Oxygen Species Elicit Apoptosis by Concurrently Disrupting Topoisomerase II and DNA-Dependent Protein Kinase

Reactive oxygen species (ROS) are produced by all aerobic cells and have been implicated in the regulation of diverse cellular functions, including intracellular signaling, transcription activation, proliferation, and apoptosis. Salvicine, a novel diterpenoid quinone compound, demonstrates a broad spectrum of antitumor activities. Although salvicine is known to trap the DNA-topoisomerase II (Topo II) complex and induce DNA double-strand breaks (DSBs), its precise antitumor mechanisms remain to be clarified. In this study, we investigated whether salvicine altered the levels of ROS in breast cancer MCF-7 cells and whether these ROS contributed to the observed antitumoral activity. Our data revealed that salvicine stimulated intracellular ROS production and subsequently elicited notable DSBs. The addition of N-acetyl cysteine (NAC), an antioxidant, effectively attenuated the salvicine-induced ROS enhancement and subsequent DNA DSBs. Heat treatment reversed the accumulation of DNA DSBs, and the addition of NAC attenuated the Topo II-DNA cleavable complexes formation and the growth inhibition of salvicine-treated JN394top2-4 yeast cells, collectively indicating that Topo II is a target of the salvicine-induced ROS. On the other hand, when examining the impact of salvicine on DNA repair pathways, we unexpectedly observed that salvicine selectively down-regulated the catalytic subunit of DNA-dependent protein kinase (DNA-PK(CS)) protein levels and repressed DNA-PK kinase activity; both of these effects were attenuated by NAC pretreatment of MCF-7 cells. Finally and most importantly, NAC attenuated salvicine-induced apoptosis and cytotoxicity in MCF-7 cells. These results indicate that apart from its direct actions, salvicine generates ROS that modulate DNA damage and repair, contributing to the comprehensive biological consequences of salvicine treatment, such as DNA DSBs, apoptosis, and cytotoxicity in tumor cells. The finding of salvicine-induced ROS provides new evidence for the molecular mechanisms of this compound. Moreover, the effects of salvicine-induced ROS on Topo II and DNA-PK give new insights into the diverse biological activities of ROS.

The role of p38 MAP kinase in the synergistic cytotoxic action of calcitriol and TNF-α in human breast cancer cells

Calcitriol, the hormonal form of Vitamin D, potentiates the activity of some agents of the anti-cancer immune system including tumor necrosis factor-α (TNF-α). Different signaling pathways activated by TNF-α may be targets for calcitriol action. Activation of p38 MAP kinase was shown to have both pro- and anti-apoptotic actions in TNF-α-induced programmed cell death depending on cell context. Treatment of MCF-7 breast cancer cells with TNF-α resulted in activation of p38 MAP kinase that persisted for at least 24 h. Whereas calcitriol had no effect on the earlier phase of p38 MAP kinase activation (up to 1 h), it inhibited the activation of this pathway between one and 24 h after exposure to TNF-α. Both calcitriol and the p38 MAP kinase inhibitor SB203580 enhanced TNF-α-induced cytotoxicity and drop in mitochondrial membrane potential, but their combined effect was sub-additive. Taken together, these findings suggest that p38 MAP kinase plays an anti-apoptotic role in TNF-α-induced cytotoxicity in MCF-7 cells and that the synergistic interaction between TNF-α and calcitriol, leading to mitochondrial damage and subsequent cell death, is partially due to modulation of this signaling pathway.

Glycoprotein isolated from Solanum nigrum L. inhibits the DNA-binding activities of NF-κB and AP-1, and increases the production of nitric oxide in TPA-stimulated MCF-7 cells

Solanum nigrum L. (SNL) has been used in traditional folk medicine to treat numerous cancers. We isolated a glycoprotein (150 kDa) from SNL and tested its effect on the modulation of transcriptional factors (NF-kappa B and AP-1) and iNO production in TPA induced-MCF-7 cells, which are part of the human breast cancer cell line, without estrogen receptors. However, the mechanism of SNL glycoprotein in pharmacological and biochemical actions in cancer cells has not been studied. To test the effect of SNL glycoprotein on the DNA-binding activities of nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1), and nitric oxide (NO) production, these experiments were carried out using electrophoretic mobility shift assays (EMSA), western blot analysis, and the Griess method. Results in this experiment showed that SNL glycoprotein inhibits 12- O-Tetra decanoylphorbol-13-acetate (TPA; 100 nM)-induced DNA-binding activities of NF-κB and AP-1, and enhances NO production in MCF-7 cells. That is, our results indicated that SNL glycoprotein has the capacity to modulate the TPA-induced DNA-binding activities of transcription factors and NO production, which play a critical role with respect to cytotoxicity in MCF-7 cells. Therefore, SNL glycoprotein might be one of the agents that blocks TPA-mediated signal responses in tumor cells.

High energy shock waves (HESW) enhance paclitaxel cytotoxicity in MCF-7 cells.

High energy shock waves (HESW) produced by a piezoelectric generator were studied for their effect on human breast cancer cell (MCF-7) viability and sensitivity to paclitaxel. A dose-dependent impairment of cell viability was observed after HESW treatment (250-2000 shock waves, rate = 4/s, energy flux density = 0.25 mJ/mm2). Single treatment with shock waves produced no significant growth inhibition. Combined exposure to paclitaxel (ranging 0.1 nM to 20 microM) and shock waves (100, 500 and 1000 shots, respectively) resulted in a significant reduction of MCF-7 cell proliferation at day 3 after treatment in respect with cells treated with paclitaxel alone. Notably, a cell viability reduction of about 50% was obtained after combined treatment with HESW and 10 nM paclitaxel, in front of a reduction of only 40% using 10 microM paclitaxel alone. Moreover, an earlier induction as well as an enhancement of apoptotis was observed in cells subjected to combined treatment with shock waves and paclitaxel (200 nM; 20 microM). In conclusion, HESW can enhance paclitaxel cytotoxicity in MCF-7 cells, thus allowing the treatment with lower doses of drug.

Prevention of Tumor Necrosis Factor (TNF)-Mediated Induction of p21WAF1/CIP1Sensitizes MCF-7 Carcinoma Cells to TNF-Induced Apoptosis

The MCF-7 breast carcinoma and MRC-5 lung fibroblast cell lines are sensitive and resistant to tumor necrosis factor (TNF)-induced apoptosis, respectively. As the cyclin-dependent kinase inhibitor p21WAF1/CIP1(p21) is involved in cell cycle regulation and has been implicated in apoptosis, we studied the influence of p21 on growth of MRC-5 cells and on growth and apoptosis in MCF-7 cells. TNF induced p21 mRNA and protein in both cell types. p21 induction by >0.5 ng/ml TNF in MRC-5 and MCF-7 cells correlated with the inhibition of cell growth. In contrast, <0.1 ng/ml TNF stimulated MRC-5 (but not MCF-7) cell growth without reduction in p21 levels. TNF-induced apoptosis in MCF-7 cells was first detected after the TNF-mediated increase in p21 and growth arrest had occurred. MCF-7 cells stably transfected with antisense p21 cDNA became more sensitive to TNF-induced apoptosis. Thus, TNF-induced p21 accompanied by growth arrest may counteract or delay TNF cytotoxicity in MCF-7 cells.

Role of cellular glutathione and glutathione S-transferase in the expression of alkylating agent cytotoxicity in human breast cancer cells

Glutathione (GSH) and glutathione S-transferases (GSTs) play an important role in the protection of cells against toxic effects of many electrophilic drugs and chemicals. Modulation of cellular GSH and/or GST activity levels provides a potentially useful approach to sensitizing tumor cells to electrophilic anti-cancer drugs. In this study, we describe the interactions of four representative alkylating agents (AAs), melphalan, 4-hydroperoxy-cyclophosphamide (4HC), an activated form of cyclophosphamide, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), and cisplatin, with GSH and GST in the human breast cancer cell line MCF-7. Depletion of cellular GSH pools by ≈80% by treatment of the cells with the GSH synthesis inhibitor buthionine sulfoximine (BSO) sensitized the tumor cells to each AA to a different extent, with dose-modifying factors of 2.39, 2.21, 1.64, and 1.27 observed for melphalan, 4HC, cisplatin, and BCNU, respectively. Treatment of the cells with the GST inhibitor ethacrynic acid (EA) failed to show any significant effects on the cytotoxicity of these AAs. However, EA did potentiate the cytotoxicity of melphalan when given in combination with BSO, an effect that may be due to a more complete depletion of cellular GSH levels by the combined modulator treatment. Following a 1-hr exposure to cytotoxic-equivalent concentrations of these AAs, GSH levels decreased substantially in the case of 4HC and BCNU, but increased by 30–50% in the case of cisplatin and melphalan. BSO pretreatment largely blocked this effect of cisplatin and melphalan on cellular GSH, while it further enhanced the GSH-depleting activity of both 4HC and BCNU. On the basis of these results, it is concluded that (a) GSH affects the cytotoxicity of different AAs to different extents, (b) basal GST expression in MCF-7 cells does not play a major role in AA metabolism, (c) EA can potentiate the enhancing effect of BSO on melphalan cytotoxicity in MCF-7 cells, and (d) depletion of cellular GSH by pretreatment with BCNU or cyclophosphamide may correspond to a useful strategy for enhancing the anti-tumor activity of other AAs given in a sequential combination.

Multivariate analysis by the minimum spanning tree method of the structural determinants of diphenylethylenes and triphenylacrylonitriles implicated in estrogen receptor binding, protein kinase C activity, and MCF7 cell proliferation

The response profiles of 36 para-substituted diphenylethylenes (DPEs) and triphenylacrylonitriles (TPEs) have been compared by multivariate analysis. The responses measured were (a) relative binding affinity (RBA) for the cytosol estrogen receptor (ER), (b) ability to promote the growth of the human MCF7 breast cancer cell-line, (c) cytotoxicity in MCF7 cells, and (d) ability to stimulate or inhibit protein kinase C (PKC) III activity under three different conditions of enzyme activation. The prime object of the analysis was to observe the simultaneous influence of diverse combinations of substituents on all these in vitro responses. To do this, the minimum spanning tree (MST) method was used to organize the molecules into a network in which proximate molecules are closely related with regard to their responses whereas remote molecules are distinct. The MST of this population of molecules had four main branches. E2 and its TPE mime were located in a central position within the trunk whereas the tips of the branches tended toward molecules of different specificity, i.e., cytotoxic molecules that bind to ER and interfere with PKC, noncytotoxic molecules that also bind to ER and interfere with PKC but promote cell growth, molecules only active on PKC, and molecules active on all parameters except PKC stimulation. A parallel MST analysis of the relationships among the response parameters themselves confirmed previous conclusions: For this population of molecules, RBAs for ER are fairly closely related to ability to promote MCF7 cell growth and only little to cytotoxicity (Bignon et al. J. Med. Chem. 1989, 32, 2092). Cytotoxicity is much more clearly correlated with inhibition of diacylglycerol-stimulated PKC activity than with RBAs for ER. PKC inhibition differs substantially depending upon whether the substrate is H1 histone or protamine sulfate.

Nitrosourea and nitrosocarbamate derivatives of the antiestrogen tamoxifen as potential estrogen receptor-mediated cytotoxic agents in human breast cancer cells.

We have prepared two analogs of the antiestrogen tamoxifen that incorporate known DNA-crosslinking functions, a chloroethyl nitrosourea and a nitrosocarbamate moiety, and we have tested their bioactivities in cultures of human breast cancer cells. Both compounds bind to the estrogen receptor from MCF-7 cells, with relative binding affinities of 0.18% for the nitrosocarbamate derivative and 0.35% for the nitrosourea derivative, while the affinity of tamoxifen is 1.8%, and that of estradiol is set at 100%. The tamoxifen-nitrosocarbamate compound demonstrated a dose-related cytotoxicity by the colony formation and cell proliferation assays that was not blocked by estradiol in either estrogen receptor-positive MCF-7 cells or estrogen receptor-negative MDA-MB-231 cells, and thus, was not studied further. Tamoxifen-nitrosourea (TAM-NU) showed dose-related cytotoxicity in MCF-7 cells that was blocked by estradiol, whereas its activity in MDA-MB-231 cells was unaffected by estradiol. N-2-(4-t-butylphenoxy)ethyl-N'-chloroethyl-N'-nitrosourea (BPE-NU), a control compound which contains the nitrosourea moiety but does not bind to the estrogen receptor, had no effect on cell proliferation or colony formation in MCF-7 cells, but was very inhibitory in the receptor-negative MDA-MB-231 cells. In contrast, TAM-NU was more active in the receptor-positive MCF-7 cells than in the MDA-MB-231 line. Thus, because TAM-NU appears to be active selectively against the receptor-positive cell line, and because this activity is suppressible by estradiol, its cytotoxic effect seems to be mediated via the estrogen receptor. However, since TAM-NU is active only in prolonged treatment protocols, it appears likely that its cytotoxic activity results from the hormone antagonistic effect of the hydrolysis product of TAM-NU (bis-desmethyltamoxifen), rather than from a direct receptor-mediated, DNA-directed cytotoxic action of TAM-NU itself. This study stresses the need for the use of appropriate control compounds and cell systems in order to assess whether the toxic activity displayed by hormone-cytotoxic conjugates is mediated by receptor interactions and whether it operates through the intended toxic mechanism.