Homoharringtonine, an approved anti-leukemia drug, suppresses triple negative breast cancer growth through a rapid reduction of anti-apoptotic protein abundance

Abstract

Triple negative breast cancers (TNBC) without BRCA1/2 gene mutation or BRCAness are nowadays the breast malignancies most difficult to treat. Improvement of their treatment, for all phases of the disease, is an important unmet medical need. We analyzed the effect of homoharringtonine (HHT), a natural protein synthesis inhibitor approved for treatment of chronic myeloid leukemia, on four cell lines representing aggressive, BRCA1/2 non-mutated, TNBC genomic categories. We show that HHT inhibits in vitro growth of all cell lines for more than 80%, after 48-72 h exposure to 20-100 ng/mL, the concentrations achievable in human plasma after subcutaneous administration of the drug. HHT, at 100 ng/mL, strongly reduced levels of a major TNBC survival factor, anti-apoptotic protein Mcl-1, after only 2 h of exposure, in all cell lines except MDA-MB-231. Other anti-apoptotic proteins, Bcl-2, survivin and XIAP, were also strongly downregulated. Moreover, in vivo growth of the least sensitive cell line to HHT in vitro, MDA-MB-231, was inhibited for 36.5% in mice, by 1 mg/kg of the drug, given subcutaneously, bi-daily, over 7 days. These results demonstrate marked antineoplastic activity of homoharringtonine in TNBC, making further development of the drug in this disease highly warranted.

Keywords: Homoharringtonine; Mcl-1; breast cancer; protein synthesis; translation inhibitor.

Figure 1

Cell viability after exposure to homoharringtonine in vitro. HHT, homoharringtonine; VF, viable fraction. As HHT-tartrate is soluble in water, the control samples were omitted the drug and the volume was replaced with the culture media. The concentrations are given as the HHT equivalent in HHT tartrate. The values represent a mean ± SD of three independent experiments.

Figure 2

Distribution of cells in the cell cycle phases after exposure to homoharringtonine for 48 h. HHT, homoharringtonine. As HHT-tartrate is soluble in water, the control samples were omitted the drug and the volume was replaced with the culture media. The concentrations are given as the HHT equivalent in HHT tartrate. The figure represents the most representative of three independent experiments.

Figure 3

Apoptotic fractions in the cell lines exposed to homoharringtonine. HHT, homoharringtonine; AF, apoptotic fraction; T0, beginning of the experiment (before adding the drug). As HHT tartrate is soluble in water, the control samples were omitted the drug and the volume was replaced with the culture media. The concentrations are given as the HHT equivalent in HHT tartrate. The values represent a mean ± SD of three independent experiments.

Figure 4

Levels of anti-apoptotic proteins in cell lines exposed to homoharringtonine. ctl, control, beginning of the experiment (before adding the drug). All cells were incubated with 100 ng/ml of HHT. The figure represents the most representative of three independent experiments.

Figure 5

Mcl-1 protein level after exposure of cell lines to MCL1 siRNA, to homoharringtonine or to both components. ctl, control, beginning of the experiment (before adding the drug); HHT, homoharringtonine. All cells were incubated with 100 ng/ml of HHT. The figure represents the most representative of two independent experiments.

Figure 6

Myc protein level after exposure of cell lines to homoharringtonine. ctl, control, beginning of the experiment (before adding the drug). All cells were incubated with 100 ng/ml of HHT. The figure represents the most representative of two independent experiments.

Figure 7

Determination of Maximal Tolerated Dose of homoharringtonine in mice after subcutaneous bi-daily administration over 7 days. D0, beginning of the experiment; D4, D8, D10, days 4, 8 and 10 after the treatment start.

Figure 8

Effect of bi-daily subcutaneous administration of 1 mg/kg homoharringtonine over 7 days on MDA-MB-231 xenografts in mice (experiment 1). A and B: Tumor volumes, absolute values. C: Tumor volume before and after treatment, mean ± SD. D and E: Mouse weight, absolute values. CTL, control, mice treated with saline water only; HHT, homoharringtonine; D0, beginning of the experiment; D10, day 10 after the treatment start.

Figure 9

Effect of bi-daily subcutaneous administration of 1 mg/kg homoharringtonine over 7 days on MDA-MB-231 xenografts in mice (experiment 2). A and B: Tumor volumes, absolute values. C: Tumor volume before and after treatment, mean ± SD. D and E: Mouse weight, absolute values. CTL, control, mice treated with saline water only; HHT, homoharringtonine; D0, beginning of the experiment; D3, D6, D8, D10, day 3, 6, 8 and 10 after the treatment start.

Figure 10

Effect of bi-daily subcutaneous administration of 0.5 mg/kg homoharringtonine over 7 days on MDA-MB-468 xenografts in mice. A and B: Tumor volumes, absolute values. C: Tumor volume before and after treatment, mean ± SD. D and E: Mouse weight, absolute values. CTL, control, mice treated with saline water only; HHT, homoharringtonine; D0, beginning of the experiment; D3, D6, D8, D10, day 3, 6, 8 and 10 after the treatment start.