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. 2020 Dec 10;10:21762. doi: 10.1038/s41598-020-78646-y

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Ipatasertib enhances the antiproliferative activity of fulvestrant and lapatinib combination in a PIK3CA-mutant HER2+/ER+ breast cancer cell line. (a) Antiproliferative activity of BT474 (PIK3CA-wild-type, left) and MDA-MB-361 (PIK3CA-mutant, right) cells treated with DMSO (black), combination of fulvestrant (100 nM) and lapatinib (100 nM) (blue), and the triple-combination of fulvestrant, lapatinib, and ipatasertib (1000 nM) (red) for 0, 2, 4, and 8 days (mean ± SD [n = 3]). Relative ATP amounts were compared with the chemiluminescence value of DMSO treatment on day 0. (b) Comparison of the inhibitory effect in each drug treatment between BT474 and MDA-MB-361 cell lines. Differences on day8 were analyzed using Student’s t-test. The data represent the mean ± SD (n = 3). (c) Representative images of crystal violet staining in BT474 or MDA-MB-361 cells treated with DMSO, the combination of fulvestrant (100 nM) and lapatinib (100 nM), and the triple-combination of fulvestrant, lapatinib, and ipatasertib (1000 nM) for 8 days. (d) Quantified data from Fig. 4c. Crystal violet absorbance indicating the amount of normalized protein was measured with a microplate reader and compared with a DMSO control. The data represent mean ± SD (n = 3). Crystal violet absorbance was statistically analyzed using Student’s t-test to compare the double-combination and the triple-combination treatments. Differences were considered significant at p ≤ 0.05 (*). (e) Effect of combined fulvestrant, lapatinib, and ipatasertib treatment on the apoptosis of BT474 (left) and MDA-MB-361 (right) cells. The cells were treated with DMSO (black), the combination of fulvestrant (100 nM) and lapatinib (100 nM) (blue), and the triple-combination of fulvestrant, lapatinib, and ipatasertib (1000 nM) (red) for 24 h (mean ± SD [n = 3]). Relative caspase-3/7 activities were calculated based on luminescence compared with the DMSO control. Caspase-3/7 activities were analyzed using Student’s t-test to compare the DMSO treatment and the double-combination treatment, and the double-combination and the triple-combination treatments. Differences were considered significant at p ≤ 0.05 (*). F, Fulvestrant; L, Lapatinib; I, Ipatasertib; n.s., not significant. (f) Representative images of 3D culture in BT474 (left) or MDA-MB-361 (right) cells. (g) Antiproliferative activity of 3D-cultured BT474 (PIK3CA-wild-type, left) and MDA-MB-361 (PIK3CA-mutant, right) cells treated with DMSO (black), combination of fulvestrant (100 nM) and lapatinib (100 nM) (blue), and the triple-combination of fulvestrant, lapatinib, and ipatasertib (1000 nM) (red) for 0, 2, 4, and 8 days (mean ± SD [n = 3]). Relative ATP amounts were compared with the chemiluminescence value of DMSO treatment on day 0.