ApoA-I mimetic administration, but not increased apoA-I-containing HDL, inhibits tumour growth in a mouse model of inherited breast cancer

Abstract

Low levels of high-density lipoprotein cholesterol (HDLc) have been associated with breast cancer risk, but several epidemiologic studies have reported contradictory results with regard to the relationship between apolipoprotein (apo) A-I and breast cancer. We aimed to determine the effects of human apoA-I overexpression and administration of specific apoA-I mimetic peptide (D-4F) on tumour progression by using mammary tumour virus-polyoma middle T-antigen transgenic (PyMT) mice as a model of inherited breast cancer. Expression of human apoA-I in the mice did not affect tumour onset and growth in PyMT transgenic mice, despite an increase in the HDLc level. In contrast, D-4F treatment significantly increased tumour latency and inhibited the development of tumours. The effects of D-4F on tumour development were independent of 27-hydroxycholesterol. However, D-4F treatment reduced the plasma oxidized low-density lipoprotein (oxLDL) levels in mice and prevented oxLDL-mediated proliferative response in human breast adenocarcinoma MCF-7 cells. In conclusion, our study shows that D-4F, but not apoA-I-containing HDL, hinders tumour growth in mice with inherited breast cancer in association with a higher protection against LDL oxidative modification.

Figure 1. Effects of hApoA-I overexpression on tumour development in PyMT mice.

(a) Tumour latency in PyMT and PyMT-hApoA-I mice. (b) Mammary gland weight at the end of the study. (c) Total tumour burden in PyMT and PyMT-hApoA-I mice. (d) Each section from right inguinal mammary gland was graded according to the maximum lesion grade and classified as follows: normal, hyperplasia, adenoma and carcinoma. The percentage of mice in each histopathologic stage is shown. In (b,c), the values shown represent the mean ± SEM for 10 individual animals per group, and *indicates p ≤ 0.05 vs. the PyMT group.

Figure 2. Effects of D-4F treatment on tumour development in PyMT mice.

(a) Tumour latency in PyMT mice treated with 10 mg/kg of D-4F or vehicle; significant (p ≤ 0.05) differences between curves are indicated by a connecting solid line and an asterisk. (b) Mammary gland weight at the end of the study. Two-way ANOVA showed that D-4F treatment reduced mammary gland weight without interaction between mammary gland localization and treatment. (c) Total tumour burden in PyMT mice treated with D-4F or vehicle. (d) Each section from right inguinal mammary gland was graded according to the maximum lesion grade and classified as follows: normal, hyperplasia, adenoma or carcinoma. The percentage of mice in each histopathologic stage is shown. In (b,c), the values shown represent the mean ± SEM for 8 individual animals per group, and *indicates p ≤ 0.05 vs. the vehicle group.

Figure 3. Effects of hApoA-I overexpression or D-4F treatment on 27-HC levels in PyMT mice.

The 27-HC levels in serum and left thoracic mammary tissue in (a) PyMT and PyMT-hApoA-I mice and in (b) PyMT mice treated with 10 mg/kg of D-4F or the vehicle. The values shown are the mean ± SEM for 5-8 individual animals per group. *indicates p ≤ 0.05 and ^#p ≤ 0.1 vs. PyMT or the vehicle group, respectively.

Figure 4. Effects of hApoA-I overexpression or D-4F treatment on mammary gland gene expression in PyMT mice.

Right cervical mammary gland gene expression in (a) PyMT and PyMT-hApoA-I mice and in (b) PyMT mice treated with 10 mg/kg of D-4F or vehicle, at the end of the study. The signal in the PyMT and vehicle group was set at a normalized value of 1 arbitrary unit (AU). Gapdh was used as the internal control. The values represent the mean ± SEM for 5-7 individual animals per group, and *indicates p ≤ 0.05 vs. PyMT or the vehicle group, respectively.

Figure 5. Effects of hApoA-I or D-4F on the serum oxLDL levels in PyMT mice and oxLDL-mediated proliferative response in MCF-7 cells.

(a) Serum oxLDL levels in PyMT and PyMT-hApoA-I mice. The values represent the mean ± SEM for 8-10 individual animals. *Indicates p ≤ 0.05 vs. PyMT. (b) Serum oxLDL levels in PyMT mice treated with D-4F or the vehicle. The values represent the mean ± SEM for 4–5 individual animals per group, and *indicates p ≤ 0.05 vs. the vehicle. (c) Activity of HDL isolated from wild-type (WT) or hApoA-I mice against LDL oxidative modification. Results are expressed as the lag phase of conjugated diene formation kinetics, which is presented as the relative lag phase of LDL oxidized without HDL. The values represent the mean ± SEM for 5 replicates per group. *Indicates p ≤ 0.05 vs. LDL oxidized without HDL, and ^†indicates p ≤ 0.05 vs. LDL oxidized with HDL from WT. (d) Activity of D-4F against LDL oxidative modification. The results are expressed as the lag phase of conjugated diene formation kinetics, which is presented as the relative lag phase of LDL oxidized without D-4F. The values represent the mean ± SEM for 5 replicates per group. *indicates p ≤ 0.05 vs. LDL oxidized without D-4F. (e) Cell viability of MCF-7 cells treated with 100 mg/L of human oxLDL and human oxLDL combined with 100 mg/L of HDL isolated from WT or hApoA-I mice. The signal in the control group was set at a normalized value of 1 AU. The values represent the mean ± SEM for replicates per group and, *indicates p ≤ 0.05 vs. untreated cells. (f) Cell viability of MCF-7 cells treated with 100 mg/L of human oxLDL with or without 20 mg/L of D-4F. The signal of the control group was set at a normalized value of 1 AU. The values represent the mean ± SEM for 12 replicates per group. *indicates p ≤ 0.05 vs. untreated cells, and ^†indicates p ≤ 0.05 vs. cells treated with oxLDL alone.