MCF-7 Human Tumor Cell Research Articles

Structural studies and cytotoxic activity of N(4)-phenyl-2-benzoylpyridine thiosemicarbazone Sn(IV) complexes

Structural studies and an investigation of the cytotoxic activity of Sn(IV) complexes with N(4)-phenyl-2-benzoylpyridine thiosemicarbazone (H2Bz4Ph) were carried out. The crystal and molecular structures of [Sn(2Bz4Ph)Cl 3]·CH 3CH 2OH ( 1) and [Sn(2Bz4Ph)BuCl 2]·H 2O (Bu = butyl group) ( 2) were determined. Both compounds present octahedral coordination geometry with the 2Bz4Ph anionic ligand behaving as tridentate on the metal ion. A comparative study of the structures of these compounds along with that of [Sn(2Bz4Ph)Bu 2Cl] ( 3) determined before is presented. The cytotoxicity of H2Bz4Ph and its Sn(IV) complexes was investigated against the MCF-7, TK-10 and UACC-62 human tumor cell lines. Among the three complexes, 3 proved to be better as cytotoxic agent than the clinically used drug etoposide. H2Bz4Ph and all complexes were able to induce apoptosis in UACC-62 cells.

Palladium(II) complexes of 2-benzoylpyridine-derived thiosemicarbazones: spectral characterization, structural studies and cytotoxic activity

Palladium(II) complexes of 2-benzoylpyridine thiosemicarbazone (H2Bz4DH) and its N(4)-methyl (H2Bz4M) and N(4)-phenyl (H2Bz4Ph) derivatives were obtained and fully characterized. [Pd(2Bz4DH)Cl] ( 1) crystallizes in the monoclinic space group P2 1/ c with a = 11.671(1), b = 10.405(1), c = 13.124(1), β = 115.60(1)° and Z = 4; [Pd(2Bz4M)Cl] ( 2) in the monoclinic space group P2 1/ c with a = 9.695(1), b = 15.044(1), c = 10.718(1) Å, β = 105.38(1)° and Z = 4 and [Pd(2Bz4Ph)Cl] ( 3) in the triclinic space group P 1 ¯ with a = 9.389(1), b = 13.629(1), c = 15.218(1) Å, α = 70.25(1), β = 73.46(1), γ = 83.57(1)° and two independent molecules per asymmetric unit ( Z = 4). All complexes show a quite similar planar fourfold environment around palladium(II). A negatively charged organic molecule acts as a tridentate ligand and binds to the metal through the pyridine nitrogen, the imine nitrogen and the sulfur atom. A chloride ion occupies the fourth coordination site. The planar complexes stack nearly parallel to one another in the lattice conforming a layered crystal structure. The cytotoxic activity of the thiosemicarbazones and their metal complexes was tested against the MCF-7, TK-10 and UACC-62 human tumor cell lines. The ligands exhibit lower values of GI 50 and LC 50 than the complexes, H2Bz4Ph being the most active with GI 50 < 0.003 μM; LC 50 = 13.4 μM; GI 50 = 9.3 μM, LC 50 = 12.9 μM; GI 50 < 0.003, LC 50 = 13.8 μM in the MCF-7, TK-10 and UACC-62 cell lines, respectively. Among the complexes, [Pd(2Bz4Ph)Cl] ( 3) exhibited the lowest values of GI 50 in the three studied cell lines.

Bioactive diterpenoids, a new jatrophane and two ent-abietanes, and other constituents from Euphorbia pubescens.

A new jatrophane diterpene, pubescenol (1), known ent-abietane lactones, helioscopinolide A (2) and B (3), and taraxerone, 24-methylenecycloartanol, and vanillin have been isolated from Euphorbia pubescens. Diterpenes 1-3 and previously described pubescene D (4) were shown to be moderate inhibitors of the growth of MCF-7, NCI-H460, and SF-268 human tumor cell lines, whereas compounds 2 and 3 also exhibited significant antibacterial activity against Staphylococcus aureus.

Growth inhibition of human tumor cell lines by withanolides from Withania somnifera leaves

Ayurvedic medicines prepared in India consist of Withania somnifera roots as one of the main ingredients. It is consumed as a dietary supplement around the world. The leaves of W. somnifera were used in the treatment of tumors and inflammation in several Asian countries. We have isolated twelve withanolides such as withaferin A ( 1), sitoindoside IX ( 2), 4-(1-hydroxy-2, 2-dimethylcyclpropanone)-2, 3-dihydrowithaferin A ( 3), 2, 3-dihydrowithaferin A ( 4), 24, 25-dihydro-27-desoxywithaferin A ( 5), physagulin D (1→6)-β-D-glucopyranosyl- (1→4)-β-D-glucopyranoside ( 6), 27- O-β-D-glucopyranosylphysagulin D ( 7), physagulin D ( 8), withanoside IV ( 9), and 27- O-β-D-glucopyranosylviscosalactone B ( 10), 4, 16-dihydroxy-5β, 6β-epoxyphysagulin D ( 11), viscosalactone B ( 12) from the leaves of this species. Compounds 1– 12 and diacetylwithaferin A ( 13) were tested for their antiproliferative activity on NCI-H460 (Lung), HCT-116 (Colon), SF-268 (Central Nervous System; CNS and MCF-7 (Breast) human tumor cell lines. The inhibitory concentration to afford 50% cell viability (IC 50) for these compounds was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Withaferin A and its derivatives exhibited inhibitory concentrations (50%) ranging from 0.24±0.01 to 11.6±1.9 μg/mL. Viscosalactone B ( 12) showed the 50% inhibition at concentrations ranging from 0.32±0.05 to 0.47±0.15 μg/mL whereas its 27- O-glucoside derivative ( 10) exhibited IC 50 between 7.9±2.9 and 17.3±3.9 μg/ml. However, Physagulin D type withanolides showed either weak or no activity at 30 μg/mL. Therefore, incorporation of withanolides in the diet may prevent or decrease the growth of tumors in human.

Imaging of luciferase and GFP-transfected human tumours in nude mice.

Studies were performed to compare green fluorescent protein (GFP)-transfected and fi re fl y luciferase (Luc)-transfected MCF-7 human breast tumour cells both in vitro and in vivo. For in vitro studies, cells were serially diluted in 96-well microplates and analysed using a NightOwl LB 981 Molecular Light Imager and a Victor multilabel reader. For in vivo studies, nude mice were injected either intraperitoneally, intravenously or subcutaneously with transfected cells and then imaged using the NightOwl Imager after intraperitoneal injection of d-luciferin for Luc tumours, or excitation at 470 nm for GFP tumours. In vitro imaging studies revealed that both GFP and Luc transfectants were quantifiable. However, the Luc-transfected cells were detectable at a significantly lower concentration compared to GFP transfectants. In vivo studies demonstrated that GFP-transfected tumours were detectable as subcutaneous and intraperitoneal tumours but not as deep tissue lesions, whereas Luc-transfected tumours were detectable as subcutaneous and intraperitoneal tumours and as deep tissue lesions resulting from intraperitoneal or intravenous inoculation. These findings demonstrate that GFP-transfected cells may be useful for imaging studies of superficial tumours where both excitation and emission wavelengths are able to penetrate tissues, whereas luciferase-transfected cells appear superior for imaging studies of primary and metastatic tumours in distant sites and deep tissues.

A fluorescence-based high performance liquid chromatographic method for the characterization of palmitoyl acyl transferase activity

Although protein palmitoylation is essential for targeting many important signaling proteins to the plasma membrane, the mechanism by which palmitoylation occurs is uncharacterized, since the enzyme(s) responsible for this modification remain unidentified. To study palmitoyl acyl transferase (PAT) activity, we developed an in vitro palmitoylation (IVP) assay using a fluorescently labeled substrate peptide, mimicking the N-terminal palmitoylation motif of proteins such as non-receptor Src-related tyrosine kinases. The palmitoylated and non-palmitoylated forms of the peptide were resolved by reverse-phase HPLC and detected by fluorescence. The method was optimized for PAT activity using lysates from the MCF-7 and Hep-G2 human tumor cell lines. The PAT activity was inhibited by boiling, reducing the incubation temperature, or adding 10 μM 2-bromopalmitate, a known palmitoylation inhibitor. This IVP assay provides the first method that is suitable to study all facets of the palmitoylation reaction, including peptide palmitoylation by PAT(s), depalmitoylation by thioesterases, and evaluation of potential palmitoylation inhibitors.

Presentation of native TROP-2 tumor antigens to human cytotoxic T lymphocytes by engineered antigen-presenting cells.

Professional antigen-presenting cells (APC), e.g. dendritic cells, express immuno-proteasome components and process proteins for MHC presentation differently from non-immune cells. Thus, they induce reactivities against sets of peptides that do not overlap with those generated by non-professional APC, i.e., tumor cells, and stimulate cytotoxic T lymphocytes (CTL) that may not recognize them. The goal of this work was to establish a system for antigen presentation and in vitro stimulation of human CTL using "tumor-cell-like" engineered APC. Murine fibroblasts were transfected with human HLA Class I alleles, together with the B7.1, ICAM-1 and germ-line TROP2 genes. The last encodes a cell surface glycoprotein widely expressed by human cancers. Unseparated peripheral blood mononuclear cells from HLA Class I-matched individuals were stimulated in vitro by the engineered APC. These efficiently induced the activation and proliferation of antigen-specific HLA-restricted CTL lines and clones. The Trop-2-specific CTL demonstrated high specific cytotoxicity against the appropriate transfected target cells. They also efficiently lysed MCF-7 human tumor cells expressing endogenous HLA-A2.1, Trop-2 together with ICAM-1. These results demonstrate that Trop-2 is a target molecule recognized by human CTL. Moreover, they demonstrate that non-immune engineered APC efficiently process and present native tumor-specific proteins in the context of human MHC Class I, and stimulate the growth and cytotoxicity of specific anti-tumor CTL.

Effect of ploidy, recruitment, environmental factors, and tamoxifen treatment on the expression of sigma-2 receptors in proliferating and quiescent tumour cells.

Recently, we demonstrated that sigma-2 receptors may have the potential to be a biomarker of tumour cell proliferation (Mach et al (1997) Cancer Res57: 156–161). If sigma-2 receptors were a biomarker of tumour cell proliferation, they would be amenable to detection by non-invasive imaging procedures, thus eliminating many of the problems associated with the flow cytometric measures of tumour cell proliferation presently used in the clinic. To be a good biomarker of tumour cell proliferation, the expression of sigma-2 receptors must be essentially independent of many of the biological, physiological, and/or environmental properties that are found in solid tumours. In the investigation reported here, the mouse mammary adenocarcinoma lines, 66 (diploid) and 67 (aneuploid), 9L rat brain tumour cells, and MCF-7 human breast tumour cells were used to study the extent and kinetics of expression of sigma-2 receptors in proliferative (P) and quiescent (Q) tumour cells as a function of species, cell type, ploidy, pH, nutrient depletion, metabolic state, recruitment from the Q-cell compartment to the P-cell compartment, and treatment with tamoxifen. In these experiments, the expression of sigma-2 receptors solely reflected the proliferative status of the tumour cells. None of the biological, physiological, or environmental properties that were investigated had a measurable effect on the expression of sigma-2 receptors in these model systems. Consequently, these data suggest that the proliferative status of tumours and normal tissues can be non-invasively assessed using radiolabelled ligands that selectively bind sigma-2 receptors. © 1999 Cancer Research Campaign

BN 80927: A novel homocamptothecin with inhibitory activities on both topoisomerase I and topoisomerase II

BN 80927, a novel homocamptothecin derivative, inhibits both topoisomerase I and topoisomerase II mediated DNA relaxation and shows pronounced cytotoxicity against HT29, SKOV-3, DU145 and MCF7 human tumor cell lines.

Induction of p53 dependent apoptosis upon overexpression of a nuclear protein tyrosine phosphatase

Two ubiquitously expressed protein tyrosine phosphatases, PTP-S2 and PTP-S4 (also known as TC 45 and TC 48, respectively), are alternately spliced products of the same gene. Overexpression of PTP-S2 by transient transfection induced chromatin condensation and nuclear fragmentation, typical of apoptosis. Expression of PTP-S4 resulted in a much lower number of cells with apoptotic phenotype. PTP-S2 induced apoptosis in MCF7 and A549 human tumor cell lines which are p53 positive but not in HeLa and SW620 cells which are p53 negative. Apoptosis induced by PTP-S2 in MCF7 cells was inhibited by cotransfection with mutant p53 (Arg-273→His) but not by wild type p53. PTP-S2 induced apoptosis was inhibited by antiapoptotic protein Bcl2 and certain inhibitors of caspases. These results suggest that the nuclear tyrosine phosphatase PTP-S2 induces p53 dependent, serum starvation independent and caspase mediated apoptosis.

Sustained enhancement of liposome-mediated gene delivery and gene expression in human breast tumour cells by ionizing radiation

Purpose: To investigate whether irradiation improves the delivery and expression of liposome-DNA complexes in human breast tumour cells. Materials and methods: MDA-MB231 and MCF-7 human breast tumour cells were transfected with a liposomal SV40-luciferase complex and irradiated immediately after, at 24h after or 24h prior to transfection and in the presence or absence of serum. The amount of luciferase plasmid in the cell was evaluated after extraction by the Hirt procedure, while luciferase expression was measured using a luminescence assay. Results: Ionizing radiation enhanced the liposome-mediated delivery and expression of the SV40-luciferase transgene in MDAMB231 breast tumour cells both in the absence and presence of serum as well as in MCF-7 breast tumour cells. Improved transgene delivery and expression was observed at a clinically relevant dose of 2Gy, and was dose-dependent over a dose range of 2-10Gy. The effects of irradiation on transgene expression were observed with irradiation immediately prior to exposure of the cells to the liposome-transgene complex, with irradiation up to 24h before or up to 24h after initiation of exposure. Conclusions: Irradiation at 24h prior to exposure of breast tumour cells to the liposome-transgene complex appears to be the optimal approach for enhancing transgene delivery and expression. These findings suggest that ionizing radiation could promote the utility of gene therapy in the treatment of breast cancer.

(2E,9E)-pyronaamidine 9-(N-methylimine), a new imidazole alkaloid from the northern Mariana islands sponge Leucetta sp. cf. chagosensis.

A new imidazole alkaloid, (2E,9E)-pyronaamidine 9-(N-methylimine) (4), was isolated along with the known compounds pyronaamidine (3) and kealiiquinone (5) from the yellow sponge Leucetta sp. cf. chagosensis collected near the Island of Rota, Northern Mariana Islands. Single-crystal X-ray diffraction analysis allowed the unambiguous assignment of the structure of compound 4, including the position of the exchangeable proton and the geometry of the two imino double bonds. Compound 4 exhibited mild cytotoxicity toward the A-549, MCF-7, and HT-29 human tumor cell lines.

WR-1065, the active metabolite of amifostine (ethyol®), does not inhibit the cytotoxic effects of a broad range of standard anticancer drugs against human ovarian and breast cancer cells

Amifostine (WR-2721, Ethyol), a phosphorylated thiol, demonstrates the unique ability to protect normal but not tumour tissue from cytotoxic damage induced by radiation therapy and chemotherapy. This study tested the effect of amifostine's active metabolite, the free thiol, WR-1065, on the cytotoxicity of standard anticancer drugs against human A2780 ovarian and MCF7 breast cancer cell lines in vitro, using the well-characterised sulphorhodamine B assay. 50% inhibitory concentration (IC50) values were determined for each of 16 different anticancer drugs in the presence and absence of the highest nontoxic dose of WR-1065 from concentration-response curves constructed in triplicate and based on 18 replicate cell culture plates for each tested drug concentration. Pretreatment with WR-1065 had no statistically significant effect on the IC50 value of any of the 16 drugs tested against either the A2780 or MCF7 human tumour cells. These data expand upon previous reports showing that amifostine does not protect tumours from the cytotoxic effects of anticancer agents. The ability of amifostine to protect against dose-limiting toxicity to a variety of normal tissues without protection of tumour should enhance the efficacy ratio of a wide range of standard anticancer drugs.

New Bioactive Monotetrahydrofuran Annonaceous Acetogenins, Annomuricin C and Muricatocin C, from the Leaves of Annona muricata

The leaves of Annona muricata have yielded two additional monotetrahydrofuran Annonaceous acetogenins, annomuricin C [1] and muricatocin C [2]. Compounds 1 and 2 each possess five hydroxyl groups; two hydroxyl groups are at the C-10/C-11 and C-10/C-12 positions in 1 and 2, respectively. The absolute configurations of 1 and 2, except for positions C-10 and C-11 or C-12, were determined by Mosher ester methodology. The C-10/C-11 and C-10/C-12 acetonides (1c, 2c) suggested relative stereochemistry and significantly enhanced the cytotoxicities against the A-549 human lung and the MCF-7 human beast solid tumor cell lines. One known monotetrahydrofuran acetogenin, gigantetronenin, not described previously from this plant, was also found.

Structural studies on bioactive compounds. 23. Synthesis of polyhydroxylated 2-phenylbenzothiazoles and a comparison of their cytotoxicities and pharmacological properties with genistein and quercetin.

A series of polyhydroxylated 2-phenylbenzothiazoles 3 has been prepared by demethylation of the precursor methoxylated 2-phenylbenzothiazoles 9. The key step in the construction of the benzothiazole nucleus involves a Jacobson cyclization of methoxylated thiobenzanilides 8. The target compounds inhibit WiDr human colon tumor cells and MCF-7 human mammary tumor cells in vitro with IC50 values in the low micromolar range, but the activity against MCF-7 cells is not related to estrogen receptor-binding affinity. None of the compounds showed selective cytotoxicity against Abelson virus-transformed ANN-1 cells encoded with the pp120gag-abl tyrosine kinase compared with the parental 3T3 line. Compounds were only marginally inhibitory to the EGF receptor-associated protein tyrosine kinase from a membrane preparation of A431 cells. The most active compound was 4,6-dihydroxy-2-(4-hydroxyphenyl)benzothiazole (3b) which has the same overall hydroxyl substitution pattern as genistein (1a). The compounds were weakly cytotoxic for an EGF receptor, overexpressing cell line HN5, but when tested for differential toxicity against the EGF receptor tyrosine kinase or the PDGF receptor tyrosine kinase in a standard mitogenesis assay utilizing human fibroblasts, no discrimination was observed. In this assay, the compounds inhibited DNA synthesis when added to cells during S phase. This suggests that inhibition could not be interpreted in terms of tyrosine kinase inactivation but more likely as a relatively broad specificity for the ATP-binding domain of other kinases such as thymidine kinase.

Activity of D-glucarate analogues: Synergistic antiproliferative effects with retinoid in cultured human mammary tumor cells appear to specifically require the D-glucarate structure

D-Glucarate has shown modest chemopreventive and synergistic chemopreventive effects with retinoids in a number of tumor models as well as a similar antiproliferative effect in MCF-7 human tumor cells in culture. It has been postulated that D-glucarate exerts some of its effects by equilibrium conversion to D-glucarolactone, a potent β-glucuronidase inhibitor. In the present study, D-glucarate and a number of its analogues, including D-glucarolactone, were evaluated as antiproliferatives in the MCF-7 model with and without added retinoid. Results suggest that the effects of glucarate are reasonably specific for its structure and may not require conversion to glucarolactone.

Antiproliferative activity of thermosensitive liposome-encapsulated doxorubicin combined with 43°C hyperthermia in sensitive and multidrug-resistant MCF-7 cells

Thermosensitive liposome-encapsulated doxorubicin (TLED) was compared to free doxorubicin, at 37°C or combined with 43°C hyperthermia, in sensitive and multidrug-resistant MCF-7 human tumour cells using clonogenic assays. In the resistant subline, TLED was found to partly circumvent multidrug resistance (MDR). The reversal was comparable to that obtained when verapamil was added to free doxorubicin. When hyperthermic treatment was applied, no difference in thermosensitivity was found between sensitive and resistant cells. The combination of hyperthermia with free doxorubicin did not reverse MDR. Hyperthermia and TLED yielded additive effects in the resistant cells while potentiation was observed in the sensitive cells. These results confirmed the usefulness of the liposome encapsulation of doxorubicin in reversing MDR. The possibility of obtaining additive cytotoxicity using TLED combined with hyperthermia may represent an alternative way of intensification of doxorubicin cytotoxicity concomitant with the circumvention of MDR without using MDR reversing agents, which often generate limiting toxic side-effects.

A lack of detectable modification of topoisomerase II activity in a series of human tumor cell lines expressing only low levels of etoposide resistance

Etoposide (VP-16) resistance is expressed following in vitro exposure of HN-1 and MCF-7 human tumor cells to the drug itself or to fractionated X irradiation. VP-16-selected sublines prove cross-resistant to Adriamycin, amsacrine and actinomycin D, whilst X-ray-pretreated sublines show cross-resistance to only actinomycin D. These differential responses, in the HN-1 series, are not associated with significant differences in amounts of immunoreactive topoisomerase (topo) II, altered topo-II catalytic activity of nuclear extracts or changes in susceptibility of the topo II to VP-16- or amsacrine-induced DNA-protein cross-link formation. Therefore significant modifications in topo II appear not to be implicated in VP-16 resistance in these HN-1 sublines.

Effects of interaction between estradiol-17 beta and progesterone on the proliferation of cloned breast tumor cells (MCF-7 and T47D).

We have previously reported that the proliferation of cloned MCF-7 and T47D human mammary tumor cells can be inhibited by increasing concentrations of charcoal-dextran stripped female human serum (CDFHS). The maximal proliferation rate was restored by the addition of 3 X 10(-11) M estradiol-17 beta to the culture media. These observations suggest that the proliferation of T47D and MCF-7 cells is regulated by a blood-borne inhibitor whose effects are neutralized by estrogens. In the present report we explore the possibility that progesterone alters the estrogenic response. MCF-7 cells were grown in DME containing 2-40% CDFHS. Progesterone, at 3 X 10(-7) M to 3 X 10(-12) M, had no effect on the yield of MCF-7 or T47D cells that were cultured in the presence or absence of estradiol-17 beta.