calcein-am and Breast-Neoplasms

Tumor spheroid models have proven useful in the study of cancer cell responses to chemotherapeutic compounds by more closely mimicking the 3-dimensional nature of tumors in situ. Their advantages are often offset, however, by protocols that are long, complicated, and expensive. Efforts continue for the development of high-throughput assays that combine the advantages of 3D models with the convenience and simplicity of traditional 2D monolayer methods. Herein, we describe the development of a breast cancer spheroid image cytometry assay using T47D cells in Aggrewell™400 spheroid plates. Using the Celigo

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Fluoresceins; Fluorescent Dyes; High-Throughput Screening Assays; Humans; Image Cytometry; Propidium; Spheroids, Cellular

Cancer is a proliferation disease affecting a genetically unstable cell population, in which molecular alterations can be somatically inherited by genetic, epigenetic or extragenetic transmission processes, leading to a cooperation of neoplastic cells within tumoural tissue. The efflux protein P-glycoprotein (P-gp) is overexpressed in many cancer cells and has known capacity to confer multidrug resistance to cytotoxic therapies. Recently, cell-to-cell P-gp transfers have been shown. Herein, we combine experimental evidence and a mathematical model to examine the consequences of an intercellular P-gp trafficking in the extragenetic transfer of multidrug resistance from resistant to sensitive cell subpopulations.. We report cell-to-cell transfers of functional P-gp in co-cultures of a P-gp overexpressing human breast cancer MCF-7 cell variant, selected for its resistance towards doxorubicin, with the parental sensitive cell line. We found that P-gp as well as efflux activity distribution are progressively reorganized over time in co-cultures analyzed by flow cytometry. A mathematical model based on a Boltzmann type integro-partial differential equation structured by a continuum variable corresponding to P-gp activity describes the cell populations in co-culture. The mathematical model elucidates the population elements in the experimental data, specifically, the initial proportions, the proliferative growth rates, and the transfer rates of P-gp in the sensitive and resistant subpopulations.. We confirmed cell-to-cell transfer of functional P-gp. The transfer process depends on the gradient of P-gp expression in the donor-recipient cell interactions, as they evolve over time. Extragenetically acquired drug resistance is an additional aptitude of neoplastic cells which has implications in the diagnostic value of P-gp expression and in the design of chemotherapy regimens.

Topics: Algorithms; ATP Binding Cassette Transporter, Subfamily B, Member 1; Breast Neoplasms; Cell Communication; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Computer Simulation; Doxorubicin; Drug Resistance, Multiple; Female; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Humans; Kinetics; Models, Biological; Protein Transport

Breast cancer is the leading cause of cancer deaths among non-smoking women worldwide. At the moment the treatment regime is such that patients receive different chemotherapeutic and/or hormonal treatments dependent on the hormone receptor status, the menopausal status and age. However, in vitro sensitivity testing of tumor biopsies could rationalize and improve the choice of chemo- and hormone therapy. Lab-on-a-Chip devices, using microfluidic techniques, make detailed cellular analysis possible using fewer cells, enabling working with a patients' own cells and performing chemo- and hormone sensitivity testing in an ex vivo setting. This article describes the development of two microfluidic devices made in poly(dimethylsiloxane) (PDMS) to validate the cell culture properties and analyze the chemosensitivity of MCF-7 cells (estrogen receptor positive human breast cancer cells) in response to the drug staurosporine (SSP). In both cases, cell viability was assessed using the life-stain Calcein-AM (CAAM) and the death dye propidium iodide (PI). MCF-7 cells could be statically cultured for up to 7 days in the microfluidic chip. A 30 min flow with SSP and a subsequent 24 h static incubation in the incubator induced apoptosis in MCF-7 cells, as shown by a disappearance of the aggregate-like morphology, a decrease in CAAM staining and an increase in PI staining. This work provides valuable leads to develop a microfluidic chip to test the chemosensitivity of tumor cells in response to therapeutics and in this way improve cancer treatment towards personalized medicine.

Topics: Antineoplastic Agents; Apoptosis; Biological Assay; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Dimethylpolysiloxanes; Drug Evaluation, Preclinical; Female; Fluoresceins; Fluorescent Dyes; Humans; Microfluidic Analytical Techniques; Polymers; Reproducibility of Results; Sensitivity and Specificity; Staurosporine; Time Factors

A series of 4,6-dimethoxyaurones were synthesized by reacting 4,6-dimethoxybenzofuran-3(2H)-one with various benzaldehydes in a base-catalyzed aldol reaction. A Z configuration was assigned to the aurones based on spectroscopic and crystallographic data. The aurones were tested for their ability to modulate ABCG2 (breast cancer resistance protein)-mediated multidrug resistance in vitro. Several members (0.5 microM) increased the accumulation of mitoxantrone (MX) in human breast cancer cells (MDA-MB-231) transfected with ABCG2 and re-sensitized these cells to the cytotoxic effects of MX. In the re-sensitization assay, aurones at 0.5 microM reduced the resistance of the transfected cells to MX to just twice that of the parental cells, exceeding fumitremorgin C (FTC) tested at the same concentration. The aurones (10 microM) also increased calcein-AM accumulation in MDCKII/MDR1 cells that were transfected with ABCB1 (P-glycoprotein), at levels comparable to verapamil tested at the same concentration. Structure-activity analysis showed that substitution of the benzylidene ring B of the aurone template was less important for ABCG2 inhibition, with little variation in activity noted for compounds with an unsubstituted ring B or one that was substituted. In contrast, substitution of ring B gave rise to better inhibitors of ABCB1. A preference for the 3' position of ring B was noted. There was also some indication from the data that aurones with good ABCG2 inhibitory activity were poor ABCB1 inhibitors and vice versa, but further confirmation would be required. Limited antiproliferative activity (>70% cell survival) was observed for many aurones on four different cell lines. Thus, functionalized 4,6-dimethoxyaurones are promising ABCG2 inhibitors that combine good activity at submicromolar concentrations with limited antiproliferative activity.

Topics: Animals; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Benzofurans; Blotting, Western; Breast Neoplasms; Cell Proliferation; Cell Survival; Crystallography, X-Ray; DNA, Complementary; Dogs; Drug Resistance, Neoplasm; Female; Flow Cytometry; Fluoresceins; Humans; Indicators and Reagents; Mitoxantrone; Models, Molecular; Neoplasm Proteins; Structure-Activity Relationship; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured

Anticoagulant treatment with heparins is frequently used to prevent venous thromboembolism in cancer patients. In the present study, we investigated the ability of unfractionated heparin (UFH) to inhibit P-glycoprotein (Pgp)-mediated multidrug resistance (MDR) on human breast cancer cell line (MDA-MB231) and its doxo-resistant subline. Results were a compared to the classic reversing agent, Verapamil (Ver), used, as reference at 50 microM concentration. We analysed the Pgp function by calcein acetoxymethylester (calcein-AM) uptake, a fluorescent marker substrate, before and after in vitro exposure to UFH at clinically achievable dose of 20 U/ml. The mean percentage of calcein-AM retained into cancer cells after 3 and 12 h were 32 +/- 10.9 and 45 +/- 12.3, respectively, for UFH pretreated cells and 25.3 +/- 8.7 and 29.4 +/- 10.4, respectively, for Ver pretreated cells when compared to control cells, receiving only medium. Pgp activity was studied by measuring intracellular drug accumulation in doxo-resistant subline, treated (2 h) with either UFH or Ver, prior exposure (2 h) at different doxo concentrations (2, 4 and 8 microM). The mean percentage of remaining intracellular doxo were 55.4 +/- 4.5 , 51.4 +/- 3.9 and 50 +/- 1.8 percent, respectively for UFH treated cells, and 44.1 +/- 5.8, 39.3 +/- 4.4 and 19.4 +/- 8.6%, respectively, for Ver treated cells as compared with control cells, receiving only doxo. These results were consistent with the increase of sensitivity to doxo of the same doxo-resistant subline resulting in a 2.2, 2.6 and 2.2-fold increase, respectively, for UFH-doxo combination and 2.2, 2.5 and 2.0-fold respectively, for Ver-doxo combination respect to cells receiving doxo alone, as assessed by MTT test. In conclusion, these findings demonstrate the potentiating effect in vitro of UFH on doxo accumulation and cytotoxicity in the MDA-231 cell line and its doxo-resistant subline and suggest that UFH could to be used, as an potential chemosensitizer, in clinical chemotherapy for increasing in vivo, the efficacy of the anticancer treatment.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Transport; Breast Neoplasms; Cell Line, Tumor; Doxorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Female; Fluoresceins; Heparin; Humans; Verapamil

Large-scale screening strategies aimed at finding anticancer drugs traditionally focus on identifying cytotoxic compounds that attack actively dividing cells. Because progression to malignancy involves acquisition of an aggressively invasive phenotype in addition to hyperproliferation, simple and effective screening strategies for finding compounds that target the invasive aspects of cancer progression may prove valuable for identifying alternative and preventative cancer therapies. Here, we describe a fluorescence-based automated assay for identifying antimigratory compounds, with the ability to discern cytotoxic from noncytotoxic modes of action. With this assay, we analyzed the effects of two drugs on tumorigenic (MDA-MB-435) and nontumorigenic (MCF-10A) human breast cell lines. We chose to compare carboxyamidotriazole (CAI), an experimental compound shown to inhibit migration of various cell types, with tamoxifen, a common preventative and therapeutic anticancer compound. Our assay demonstrated that both these compounds inhibit migration at sublethal concentrations. Furthermore, CAI was more effective than tamoxifen at inhibiting chemotactic and haptotactic migration of both cell lines at all concentrations tested.

Topics: Antineoplastic Agents; Breast Neoplasms; Drug Screening Assays, Antitumor; Fluoresceins; Fluorescent Dyes; Humans; Neoplasm Metastasis; Propidium; Spectrometry, Fluorescence; Tamoxifen; Triazoles; Tumor Cells, Cultured

We have been studying some properties of the new generation photosensitizers--phthalocyanines. The influence of the certain phthalocyanine concentrations at combination with laser irradiation doses (semiconductor laser, lambda = 670 nm, P = 50 mW) have been studied by in vitro fluorescent methods. Cytotoxicity assay LIVE/DEAD for fluorescence microscopy and measurements of cells viability with multi-well plate scanner was used. The optimal phototoxic effect on MCF7--mamma carcinoma cells was observed 8 micrograms/ml ZnPcS2, 10 micrograms/ml ClAlPcS2 in combination with laser irradiation 50 J/cm2 and 100 J/cm2 respectively. This concentration and dose killed MCF7 carcinoma cells.

Topics: Breast Neoplasms; Cell Survival; Female; Fluoresceins; Fluorescent Dyes; Humans; Indoles; Isoindoles; Lasers; Microscopy, Fluorescence; Radiation Dosage; Radiation-Sensitizing Agents; Tumor Cells, Cultured