Evaluation of Cell Cycle Arrest in Estrogen Responsive MCF-7 Breast Cancer Cells: Pitfalls of the MTS Assay

Abstract

Endocrine resistance is a major problem with anti-estrogen treatments and how to overcome resistance is a major concern in the clinic. Reliable measurement of cell viability, proliferation, growth inhibition and death is important in screening for drug treatment efficacy in vitro. This report describes and compares commonly used proliferation assays for induced estrogen-responsive MCF-7 breast cancer cell cycle arrest including: determination of cell number by direct counting of viable cells; or fluorescence SYBR®Green (SYBR) DNA labeling; determination of mitochondrial metabolic activity by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay; assessment of newly synthesized DNA using 5-ethynyl-2′-deoxyuridine (EdU) nucleoside analog binding and Alexa Fluor® azide visualization by fluorescence microscopy; cell-cycle phase measurement by flow cytometry. Treatment of MCF-7 cells with ICI 182780 (Faslodex), FTY720, serum deprivation or induction of the tumor suppressor p14ARF showed inhibition of cell proliferation determined by the Trypan Blue exclusion assay and SYBR DNA labeling assay. In contrast, the effects of treatment with ICI 182780 or p14ARF-induction were not confirmed using the MTS assay. Cell cycle inhibition by ICI 182780 and p14ARF-induction was further confirmed by flow cytometric analysis and EdU-DNA incorporation. To explore this discrepancy further, we showed that ICI 182780 and p14ARF-induction increased MCF-7 cell mitochondrial activity by MTS assay in individual cells compared to control cells thereby providing a misleading proliferation readout. Interrogation of p14ARF-induction on MCF-7 metabolic activity using TMRE assays and high content image analysis showed that increased mitochondrial activity was concomitant with increased mitochondrial biomass with no loss of mitochondrial membrane potential, or cell death. We conclude that, whilst p14ARF and ICI 182780 stop cell cycle progression, the cells are still viable and potential treatments utilizing these pathways may contribute to drug resistant cells. These experiments demonstrate how the combined measurement of metabolic activity and DNA labeling provides a more reliable interpretation of cancer cell response to treatment regimens.

Anticancer potential of aqueous extract of alocasia macrorrhiza against hepatic cancer in vitro and in vivo

“…reduced significantly (p < 0.01) in a dose-dependent manner (Fig. 3), which correlates well with cell proliferation (McGowan et al, 2011;Qu et al, 2011). Moreover, the cell numbers in G 0 /G 1 and G 2 /M phases increased (p < 0.05), and decreased in S phase significantly (p < 0.05) in water-soluble AME treated cancer cells (Fig.…”

Sphingosine Kinase 1 Isoform-Specific Interactions in Breast Cancer

“…Downstream consequences of inhibiting DPP with Ala-BoroPro was evaluated by observing the effect on p21, a cell cycle regulator in MCF-7 cells (38), and the trypan blue exclusion cell proliferation assay (51). The Ala-BoroPro inhibitor had little or no effect on p21 protein expression in cell lines stably expressing the SK43 and SK51, whereas Ala-BoroPro increased SK51 expression under the same experimental conditions as shown by Western blot ( Figure 8A).…”

Modulation of NOXA and MCL-1 as a Strategy for Sensitizing Melanoma Cells to the BH3-Mimetic ABT-737

“…Proliferation inhibition assays were conducted as described (35,36), on 4 or more occasions. Sensitization to ABT-737 by melphalan or flavopiridol, and vice versa, was assessed by proliferation inhibition assays testing all combinations of a range of ABT-737 with flavopiridol or melphalan concentrations, as shown, applied simultaneously.…”