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Results of colony survival assay. Colonies developed from MCF7, MB231 or MCF15 cells 10-15 days after being treated with 4µM NPCD, or untreated, in a representative experiment were shown, with each cell line in three wells of the 6-well culture plate. Quantitative data from three different experiments were presented in the histogram.
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Figure 2: Results of colony survival assay. Colonies developed from MCF7, MB231 or MCF15 cells 10-15 days after being treated with 4µM NPCD, or untreated, in a representative experiment were shown, with each cell line in three wells of the 6-well culture plate. Quantitative data from three different experiments were presented in the histogram.

Mentions: A decrease in the number of viable cells detected by MTT may be attributable to decreased cell proliferation, increased cell death, or both. To determine which of these two elements is the main mechanism, MCF7, MB231 and MCF15, the three more sensitive cell lines, were treated with 4 μM of NPCD and then subjected to a clonogenic survival assay. The number of colonies developed 10-15 days after NPCD treatment was quantified and calculated as a percentage of the corresponding non-treated control. This was used as the survival rate, which was 7.48±1% for MCF7, 23.59±0.2% for MB231, and 38.00±6% for MCF15 cells, respectively (fig 2). The photos of colonies of treated or non-treated cells from a representative experiment are also shown in figure 2. Interestingly, not only the number, but also the size, of the colonies in NPCD-treated groups was decreased, especially in MCF7 cells. Because some colonies in treated groups were too small to appear in the macroscopic photos shown in figure 2 but could be quantified under the microscope, the difference in quantitative data between the treated and non-treated groups seemed to be smaller than what was reflected by the photos, especially in MCF7 cells. Because the clonogenic assay was terminated 10-15 days after cessation of the treatment and because a smaller colony size indicates fewer cells propagated from a single survival cell in a given time period, this result indicates a persistent or long-lasting growth arrest caused by NPCD.

Effects of an Indolocarbazole-Derived CDK4 Inhibitor on Breast Cancer Cells

Sun Y, Li YX, Wu HJ, Wu SH, Wang YA, Luo DZ, Liao DJ - J Cancer (2011)

Bottom Line: NPCD could induce or reduce the D1 and CDK4 protein levels, depending on the cell line, but this effect was not correlated with its efficacy.Phosphorylation of D1 at Thr286 was decreased but it unexpectedly did not correlate with the change in D1 level in the cell lines studied.Phosphorylation of the Rb protein was decreased as expected whereas the p27kip1 protein level was decreased unexpectedly.

Affiliation: 1. Hormel Institute, University of Minnesota, Austin, MN 55912, USA.

ABSTRACT
Introduction: Cyclin D1 (D1) binds to cyclin-dependent kinases (CDK) 4 or 6 to form a holoenzyme that phosphorylates the Rb protein to promote cell cycle progression from G1 to S phase. Therefore, targeting CDK4/6 may be a good strategy for chemotherapy of cancer. We performed a proof-of-principle study to determine the effect of Naphtho [2, 1-α] pyrrolo [3, 4-c] carbazole-5, 7 (6H, 12H)-dione (NPCD), a novel CDK4 inhibitor, on breast cancer cell lines.Methods: NPCD was synthesized and purified to over 99% purity verified by HPLC. MCF7, MB231, MCF15, T47D and GI101Ap human breast cancer cells were analyzed for the efficacy of NPCD with MTT and clonogenic assays, with FACS and staining for ethidium bromide and acridine orange for cell death and cell cycle profile. Western blot, reverse transcription and PCR were used for studies of gene expression, and co-immunoprecipitation for protein-complex formation.Results: MTT assay showed that NPCD caused growth arrest and apoptosis of MCF7, MDA-MB231, T47D, MCF15 and GI101Ap cells with an IC50 ranging between 3 to 8 µM given as a single dose. The growth arrest persisted for many days after cessation of the treatment, as shown in a clonogenic assay. NPCD could induce or reduce the D1 and CDK4 protein levels, depending on the cell line, but this effect was not correlated with its efficacy. Phosphorylation of D1 at Thr286 was decreased but it unexpectedly did not correlate with the change in D1 level in the cell lines studied. Phosphorylation of the Rb protein was decreased as expected whereas the p27kip1 protein level was decreased unexpectedly. Protein levels of p21cip1, CDK2 and cyclin E were also decreased in some, but not all, of the cell lines, whereas the mRNA levels of D1, CDK4, cyclin E, CDK2, p27kip1 and p21cip1 were increased in different cell lines.Conclusions: NPCD can cause long-lasting growth arrest and cell death of breast cancer cell lines at an IC50 of 3-8 µM. Decreased phosphorylation of Rb by D1-CDK4/6 and decreased p27kip1 protein level may be part of the underlying mechanism.

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