Anti-tumor effects of low-dose metronomic vinorelbine in combination with alpelisib in breast cancer cells

Abstract

In metastatic breast cancer (MBC), PIK3CA mutations, activating the phosphatidylinositol 3-kinase (PI3K) signaling pathway seem to be associated with chemotherapy resistance and poor outcome. Inhibition of the PI3K signaling pathway may lead to sensitization and prevention of the development of resistance to cytotoxic drugs. The present study aimed to investigate the anti-tumor activity of low-dose vinorelbine (VRL) combined with alpelisib, an α-selective PI3K inhibitor and degrader, in breast cancer (BC) cells. Human BC cell lines MCF-7, T-47D [both hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative, PIK3CA-mutated], MDA-MB-231 and BT-549 (both triple-negative, wild-type PIK3CA) were exposed to a combination of low-dose VRL and alpelisib for 3 and 7 days. Cell viability was detected by the Alamar blue assay, and cell proliferation was determined by the BrdU incorporation. The effect of the substances on the p110α protein expression that is encoded by PIK3CA gene was investigated by Western blot. Low-dose VRL plus alpelisib showed synergistic anti-tumor effects and significantly inhibited cell viability and proliferation of MCF-7 and T-47D cells. Even lower alpelisib concentrations (10 ng/ml and 100 ng/ml) combined with low-dose metronomic VRL led to a significant reduction of cell viability of PIK3CA-mutated cells, and the anti-tumor activity was comparable with the effects at 1000 ng/ml alpelisib. Cell viability and proliferation of MDA-MB-231 and BT-549 cells were inhibited by VRL but not by alpelisib alone. This indicates that alpelisib did not significantly affect the cell growth of triple-negative, PIK3CA wild-type BC cells. The p110α expression was downregulated or not affected in PIK3CA-mutated cell lines, and not significantly upregulated in PIK3CA wild-type cell lines. In conclusion, combination of low-dose metronomic VRL and alpelisib showed synergistic anti-tumor effects and significantly inhibited the growth of HR-positive, HER2-negative, PIK3CA-mutated BC cells, providing a rationale for further efforts to evaluate this combination in vivo.

Keywords: PIK3CA mutation; alpelisib; breast cancer; low-dose metronomic chemotherapy; vinorelbine.

Figure 1. Effects of low-dose vinorelbine plus alpelisib on cell viability of the MCF-7 (a, b), T-47D (c, d), MDA-MB-231 (e, f) and BT-549 (g, h) cell line. Alamar blue assay was used to measure cell viability after 3 and 7 days of treatment with low-dose vinorelbine plus alpelisib. The results are shown as the mean ± standard deviation of three separate experiments. Statistical significance was assumed at *p<0.05, **p<0.01 and ***p<0.001.

Figure 2. Effects of DMSO on cell viability of MCF-7, T-47D, MDA-MB-231 and BT-549 cells after 7 days of treatment at a concentration corresponding to the highest alpelisib concentration (0.5 % DMSO≙1000 ng/ml alpelisib) and in a concentration corresponding to a lower alpelisib concentration (0.1 % DMSO≙100-500 ng/ml alpelisib). The results are shown as the mean ± standard deviation of three separate experiments. Statistical significance was assumed at *p<0.05, **p<0.01 and ***p<0.001.

Figure 3. Effects of low-dose vinorelbine plus alpelisib on cell proliferation of the MCF-7 (a), T-47D (b) and MDA-MB-231 (c) cell line. BrdU incorporation was used to measure cell proliferation after 7 days of treatment with low-dose vinorelbine plus alpelisib. The results are shown as the mean ± standard deviation of three separate experiments. Statistical significance was assumed at *p<0.05, **p<0.01 and ***p<0.001.

Figure 4. Dose-response curves of vinorelbine and alpelisib regarding cell viability: vinorelbine (a, b), alpelisib (c, d) after 3 and 7 days of treatment and regarding cell proliferation: vinorelbine (e), alpelisib (f) after 7 days of treatment. The results are shown as the mean of three separate experiments. The standard deviation results are shown in Figure 1 and Figure 3, respectively, and have been omitted from this figure for clarity.

Figure 5. Combination index-isobologram for cell viability of the MCF-7 (a, b) and T-47D (c, d) cell line after 3 and 7 days of treatment and for cell proliferation of the MCF-7 (e) and T-47D (f) cell line after 7 days of treatment with low-dose vinorelbine plus alpelisib. The inhibitory concentrations (IC)₅₀ and IC₈₀ were calculated by interpolation and used as a reference. The combination index (CI) was calculated according to the formula shown in formula [1] and was interpreted as follows: CI<1, synergism; CI=1, additive effect, and CI>1, antagonism (range ± 5 %). The graphical presentation was performed as classical isobologram with isoboles of the IC₅₀ or IC₈₀ and presentation of the corresponding CI values in relation to the corresponding isoboles within the diagram.

Figure 6. Western blot analyses of the p110α expression in the PIK3CA-mutated cell lines MCF-7 and T-47D (a) and the PIK3CA wild-type cell lines MDA-MB-231 and BT-549 (b) after 7 days of treatment with low-dose vinorelbine plus alpelisib. 12.5 µg protein was separated in a 10 % polyacrylamide gel. The order of the concentrations of substances differs between the bars and bands.