Receptor Positive Cell Line MCF-7 Research Articles

Synthesis and Evaluation of Piperazine-Tethered Derivatives of Alepterolic Acid as Anticancer Agents.

Alepterolic acid is a natural diterpenoid isolated from Aleuritopteris argentea with potential anti-cancer activity. In this study, alepterolic acid was modified to construct a series of arylformyl piperazinyl derivatives (3a-3p). The synthesized derivatives were fully characterized with HRMS, NMR, and IR. Four compounds with inhibition rate higher than 30 % at 10 μM (3f, 3n, 3g and 3k) were further measured to obtain the IC50 values against four cancer cell lines, including hepatoma cell lines HepG2, lung cancer cell lines A549, estrogen receptor-positive cell lines MCF7, and triple-negative breast cancer (TNBC) cell lines MDA-MB-231 by MTT assay. It was found that these compounds were more effective to HepG2 and MDA-MB-231 cells, while less toxic to A549 and MCF7 cells, and compound 3n as the most toxic derivatve against MDA-MB-231 cell lines, with IC50 value of 5.55±0.56 μM. Trypan blue staining and colony formation assay showed that compound 3n inhibited the growth of MDA-MB-231 cells and prevented colony formation. Hoechst staining, flow cytometry and western blot analysis revealed that compound 3n induced caspase-dependent apoptosis in MDA-MB-231 cells. Conclusively, compound 3n was demonstrated to be a potential anti-cancer lead compound for further investigation.

TRPC3 Regulates the Proliferation and Apoptosis Resistance of Triple Negative Breast Cancer Cells through the TRPC3/RASA4/MAPK Pathway.

Currently, there is no effective molecular-based therapy for triple-negative breast cancer (TNBC). Canonical transient receptor potential isoform 3 (TRPC3) was previously shown to be upregulated in breast cancer biopsy tissues when compared to normal breast tissues. However, the biological role of TRPC3 in breast cancer still remains to be elucidated. In this study, subcellular fractionation followed by Western blot and immunocytochemistry showed that TRPC3 was over-expressed on the plasma membrane of TNBC line MDA-MB-231 when compared to an estrogen receptor-positive cell line MCF-7. TRPC3 blocker Pyr3 and dominant negative of TRPC3 attenuated proliferation, induced apoptosis and sensitized cell death to chemotherapeutic agents in MDA-MB-231 as measured by proliferation assays. Interestingly, Ras GTPase-activating protein 4 (RASA4), a Ca2+-promoted Ras-MAPK pathway suppressor, was found to be located on the plasma membrane of MDA-MB-231. Blocking TRPC3 decreased the amount of RASA4 located on the plasma membrane, with concomitant activation of MAPK pathways. Our results suggest that, in TNBC MDA-MB-231 cells, Ca2+ influx through TRPC3 channel sustains the presence of RASA4 on the plasma membrane where it inhibits the Ras-MAPK pathway, leading to proliferation and apoptosis resistance. Our study reveals the novel TRPC3-RASA4-MAPK signaling cascade in TNBC cells and suggests that TRPC3 may be exploited as a potential therapeutic target for TNBC.

Abstract 374: Direct measurements of cellular ATP levels in tumor cell lines using real-time, quantitative live-cell analysis

Abstract Metabolic reprogramming is a hallmark of tumor cells, owing to the increased energetic and biosynthetic demands associated with maintaining high rates of proliferation. Therapeutic strategies targeting unique, critical metabolic pathways of tumor cells are under investigation and in clinical trials. Standard approaches to monitoring drug induced metabolic perturbations are limited to endpoint assays that provide population-based measurements and limited kinetic information. We developed a genetically encoded ATP sensor to enable direct, automated analysis of cellular ATP levels using IncuCyte® S3. In this study, our live cell imaging approach was utilized to evaluate the effect of cancer therapeutics on ATP levels in tumor cell lines, focusing primarily on compounds that target metabolic vulnerabilities. Cell lines stably expressing a genetically encoded, fluorescent ATP sensor or a control (non-ATP binding) sensor were generated. ATP levels were monitored and analyzed using an IncuCyte® S3 equipped with a specialized filter set and data acquisition module. Cellular ATP levels were measured over the course of hours to days following compound treatment. Reductions in ATP levels could be observed within an hour of administration, highlighting the sensitivity and temporal resolution of the IncuCyte® S3 ATP sensor assay. Transient reductions in ATP followed by recovery, which would have been missed by typical endpoint assays, were noted using our live cell analysis approach. Sustained decreases in ATP were associated with enhanced antiproliferative efficacy compared to conditions under which recovery of ATP levels were observed. For example, triple-negative breast cancer (TNBC) cell lines have been shown to be more dependent on activity of glutaminase 1 (GLS1), which catalyzes the first step in utilization of glutamine to fuel mitochondrial metabolism, than their receptor-positive counterparts. Using our live cell analysis approach, we observed a rapid drop in ATP upon glutamine deprivation or inhibition of GLS1 by CB-839 in the TNBC cell line MDA-MB-231. ATP levels remained below that of vehicle-treated cells for the duration of the three-day time course. In contrast, the estrogen receptor-positive cell line MCF-7 displayed a more modest decrease in ATP and full recovery within 48 hours. Quantification of phase confluence confirmed that CB-839 had a stronger antiproliferative effect in MDA-MB-231 cells compared to MCF-7 cells. Visualization of morphology in tandem with automated ATP sensor analysis provide additional insight into the heterogeneity of cellular responses to compound addition. Overall, these data highlight the ability of IncuCyte® S3 to provide direct, kinetic measurement of ATP by live-cell analysis. Citation Format: Cicely L. Schramm, Grigory S. Filonov, Michael L. Bowe, Yong X. Chen, Laura A. Skerlos, Dyke P. McEwen, Daniel M. Appledorn. Direct measurements of cellular ATP levels in tumor cell lines using real-time, quantitative live-cell analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 374.

Evaluation of chemotherapeutics in a three-dimensional breast cancer model.

Utilization of miniaturized three-dimensional (3D) cell culture-based assays enables investigation into the anticancer activity of drug candidates and further elucidation of the anticancer profile of standard-of-care chemotherapeutic agents against tumor cells. Drug discovery assays established using 3D cell culture, which better recapitulate the tumor microenvironment, may more accurately reflect the antitumor activity of compounds. Several standard-of-care anticancer drugs, epirubicin, paclitaxel and vinorelbine, were evaluated against a panel of breast cancer cell lines grown in a 3D cell culture microenvironment in the presence of extracellular matrix. A comparison of this antitumor activity in 3D conditions was made with that observed in traditional two-dimensional (2D) monolayer conditions. Examination of the above mentioned drugs against breast tumor cells cultured in 3D conditions demonstrated significantly altered potency and efficacy in comparison with cells propagated in a 2D monolayer system. The differences observed were cell line-dependent and drug-specific; the triple-negative cell line MDA-MB-231 and the endocrine receptor-positive cell line MCF-7 consistently displayed resistance to therapeutics with distinct modes of action (i.e., topoisomerase II and microtubules) in 3D cell culture in comparison with ErbB2 receptor-positive BT-474 cells. The data presented herein demonstrates the cellular viability and physical changes observed within the 3D spheroid following exposure to drug, which is not always reflected in 2D cell culture models.

Cytotoxic small molecule dimers and their inhibitory activity against human breast cancer cells

Small molecules based upon natural product dimers that exhibit cytotoxic activity were synthesized and evaluated for their anti-proliferative activity in human breast cancer cell lines. A central isophthalic core structure linking aromatic amines containing 3,5-disubstitutions produced the most active compounds. This series of compounds was found to be more active against the estrogen receptor positive cell line MCF-7 than the estrogen receptor negative cell line, SKBr3.

Boric acid induces apoptosis in some breast cancer cell lines

Boric acid has recently been reported to inhibit growth in prostate cancer cell lines. The following investigation demonstrates the ability of 1 mM boric acid to inhibit growth in some breast cancer cell lines. Estrogen receptor negative human breast cancer cell lines MDA-MB-231 and MDA-MB-435 cultured in MEM supplemented with 10% FBS and 25 mM Hepes did not show growth inhibition in the presence of boric acid. Estrogen receptor positive cell lines MCF-7 and T47-D cultured in MEM media or RPMI1640 media supplemented with 10% FBS and 25 mM Hepes were also growth inhibited when exposed to 1 mM boric acid. However, growth inhibition was detected in estrogen receptor positive ZR-75-1 (40% inhibition) and estrogen receptor negative SK-BR-3 (15% inhibition) human breast cancer cell lines cultured in similarly supplemented RPMI1640 media. To characterize growth inhibition, flow cytometric analysis was performed on attached and detached ZR-75-1 cells. Attached cells were not affected by boric acid while cells that had detached displayed apoptosis. Fluorescent microscopy also revealed the presence of cells undergoing apoptosis following treatment with boric acid. Boric acid appears capable of inducing apoptosis in some but not all human breast cancer cell lines independent of estrogen receptivity. Further studies will be needed to determine if boric acid treatment will be suitable for clinical application in breast cancer patients.

Effect of tamoxifen and 2-methoxyestradiol alone and in combination on human breast cancer cell proliferation

Endocrine therapy is widely accepted for the treatment of hormone receptor-positive breast cancer. However, in many cases eventually resistance will develop and tumor regrows. Combination therapy may be one way to resolve this problem. In the present study we investigated the effect of a combination of the widely used antiestrogen tamoxifen with the endogenous estradiol metabolite 2-methoxyestradiol (2-ME) on the proliferation of human estrogen receptor-positive and receptor-negative breast cancer cells. The receptor-positive cell line MCF-7 and the receptor-negative cell line BM were treated with 4-hydroxytamoxifen (4-OHTam) and 2-methoxyestradiol in the concentration range of 0.8–25 μM alone and equimolar combinations for 4 days. The proliferation of the cells was determined using the ATP-chemosensitivity test. 4-Hydroxytamoxifen inhibited proliferation of MCF-7 and BM cells with IC 50 values of 31 and 10 μM, the corresponding figures for 2-methoxyestradiol were 52 and 8 μM. The combination showed IC 50 values of 6 μM and 4 μM. These results indicate that a combination of tamoxifen with 2-methoxyestradiol showed an additive inhibitory effect concerning the proliferation of estrogen receptor-positive and receptor-negative breast cancer cell lines. Thus a combination of these substances may allow ameliorating of adverse events of tamoxifen by reducing its concentrations and probably also drug resistance and should be tested in clinical trials.

Estrogenic Activity of Naturally Occurring Anthocyanidins

Anthocyanins, which are natural plant pigments from the flavonoid family, represent substantial constituents of the human diet. Because some other bioflavonoids are known to have estrogenic activity, the aim of this study was to determine the estrogenic activity of the anthocyanine aglycones. Binding affinity to the estrogen receptor-α was 10,000- to 20,000-fold lower than that of the endogenous estrogen estradiol. In the estrogen receptor-positive cell line MCF-7, the anthocyanidins induced expression of a reporter gene. The tested anthocyanidins showed estrogen-inducible cell proliferation in two cell lines (MCF-7 and BG-1), but not in the receptor-negative human breast cancer cell line MDA-MB-231. The phytoestrogen-induced cell proliferation could be blocked by addition of the receptor antagonist 4-hydroxytamoxifen. Combination treatments with the endogenous estrogen estradiol resulted in a reduction of estradiol-induced cell proliferation. Overall, the tested anthocyanidins exert estrogenic activity, which might play a role in altering the development of hormone-dependent adverse effects.

Assaying the estrogenicity of phytoestrogens in cells of different estrogen sensitive tissues

There is currently much concern that a wide range of both synthetic and naturally occuring enviromental chemicals may act as endocrine disruptors (ED), and may adversely affect humans and wildlife. We examined the estrogenic effects of the phytoestrogens daidzein (DAI), equol (EQU) and O-desmethylangolensin (O-DMA), two metabolites of DAI, in three different assays. Binding affinity to the estrogen receptor α was 1000–10,000-fold lower compared with the endogenous estrogen estradiol. In the receptor positive cell line MCF-7 the phytoestrogens induced the expression of a reporter gene. The E-SCREEN is based on the estrogen-receptor binding induced proliferation of the human breast cancer cell line MCF-7. We also adapted the E-SCREEN for the estrogen-receptor positive human ovarian cancer cell line BG-1. The tested phytoestrogens induced cell proliferation in both cell lines, but not in the receptor negative human breast cancer cell line MDA-MB-231. The phytoestrogen-induced cell proliferation could be blocked by addition of the receptor antagonist 4-hydroxytamoxifen (OHT). Combination treatments with the endogenous estrogen estradiol showed competitive effects in MCF-7 cells. These studies demonstrated that the tested phytoestrogens exerted estrogenic responses in cells derived from two different tissues, breast and ovary. Furthermore, we demonstrated that BG-1 cells are a suitable additional cell system to investigate estrogenicity of test compounds.

Transforming growth factors type beta 1 and beta 2 are equipotent growth inhibitors of human breast cancer cell lines.

At least one member of the TGF-beta family, TGF-beta 1, has been previously shown to inhibit the anchorage-independent growth of some human breast cancer cell lines (Knabbe et al., 1987; Arteaga et al., 1988). Members of the TGF-beta family might, therefore, provide new strategies for breast cancer therapy. We have studied the inhibitory effects of TGF-beta 1 and TGF-beta 2 on the anchorage-independent growth of the oestrogen receptor-negative cell lines MDA-MB-231, SK-BR-3, Hs578T, MDA-MB-468, and MDA-MB-468-S4 (an MDA-MB-468 clone not growth inhibited by EGF) and the estrogen receptor-positive cell lines MCF7, ZR-75-1, T-47D. TGF-beta 1 and TGF-beta 2 caused a 75-90% growth inhibition of MDA-MB-231, SK-BR-3, Hs578T, and MDA-MB-468 cells and a 50% growth inhibition of ZR-75-1 and early passage (less than 100) MCF7 cells. T-47D cells responded to TGF-beta only in serum-free conditions in the presence of IGF-1 or EGF. The growth of MDA-MB-468-S4 cells and late passage (greater than 500) MCF7 cells was not inhibited by TGF-beta 1 or TGF-beta 2. TGF-beta-sensitive MCF7 and MDA-MB-231 cells did not respond to Muellerian inhibiting substance (MIS), a TGF-beta-related polypeptide. TGF-beta 1 or TGF-beta 2 were mutually competitive for receptor binding with a similar affinity (Kd 25-130 pM, 1,000-13,000 sites per cell). To determine the time course of the TGF-beta effect, an anchorage-dependent growth assay was carried out using MDA-MB-231 cells. Growth inhibition occurred at 6 days, and cell-cycle changes were seen 12 hr after the addition of TGF-beta. Cells accumulated in the G1 phase and were thus inhibited from entering the S-phase. These data indicate that TGF-beta is a potent growth inhibitor in most breast cancer cell lines and provide a basis for studying TGF-beta effects in vivo.