Amyloid β oligomers inhibit growth of human cancer cells

Abstract

Effects of amyloid beta (Aβ) oligomers on viability and function of cell lines such as NB4 (human acute promyelocytic leukemia), A549 (human lung cancer (adenocarcinomic alveolar basal epithelial tumor)) and MCF-7 (human breast cancer (invasive breast ductal carcinoma)) were investigated. Two types of Aβ oligomers were used in the study. The first type was produced in the presence of oligomerization inhibitor, hexafluoroisopropanol (HFIP). The second type of amyloids was assembled in the absence of the inhibitor. The first type preparation was predominantly populated with dimers and trimers, while the second type contained mostly pentadecamers. These amyloid species exhibited different secondary protein structure with considerable amount of antiparallel β sheet structural elements in HFIP oligomerized Aβ mixtures. The effect of the cell growth inhibition, which was stronger in the case of HFIP Aβ oligomers, was observed for all cell lines. Tests aiming at elucidating the effects of the amyloid species on cell cycles showed little differences between amyloid preparations. This prompts us to conclude that the effect on the cancer cell proliferation rate is less specific to the biological processes developing inside the cells during the proliferation. Therefore, cell growth inhibition may involve interactions with the peripheral parts of the cancer cells, such as a phospholipid membrane, and only in case of the NB4 cells, where accumulation of amyloid species inside the cells was detected, one may imply the opposite. In general, cancer cells were much less susceptible to the damaging effects of amyloid oligomers compared to earlier observations in mixed neuronal cell cultures.

Fig 1. SEC–HPLC chromatograms of amyloids preparation.

(A)–Aβ(1–42)–HFIP protocol preparation; (B)–Aβ(1–42)–HFIP-free protocol preparation. The sample concentration was 10 μM. Mobile phase– 0.1 M NaCl, 0.01 M NaH₂PO₄ buffer at pH 7.4 and flow rate of 0.25 ml/min.

Fig 2

Representative AFM images of adsorbed 10 μM Aβ(1–42) oligomers prepared by HFIP protocol (A) and HFIP-free protocol (B). Mica surfaces (1 μm²) were visualized after 10 min incubation with preparations.

Fig 3. The actual height and Gauss approximation distributions of Aβ(1–42) adsorbed on mica.

HFIP-protocol (black box and line) and HFIP-free protocol (box with diagonal lines and dashed line).

Fig 4. FTIR spectra of Aβ(1–42) oligomers of different size.

FTIR spectra of HFIP protocol (a) and HFIP-free protocol (b) Aβ(1–42) oligomers deposited at CaF₂ substrate in the spectral region of 1200−1800 cm⁻¹.

Fig 5. Comparison of Aβ(1–42) oligomers in Amide-I spectral region.

FTIR absorption spectra with fitted Gaussian-Lorentzian form components in Amide-I spectral region: (A) spectra of Aβ(1–42)–HFIP protocol, and (B) Aβ(1–42)–HFIP-free protocol; both deposited at CaF₂ substrate.

Fig 6. Second derivative spectra of Amide-I band.

(a) Amide-I band of HFIP protocol, and (b) HFIP-free protocol of Aβ(1–42) oligomers deposited at CaF₂ substrate in the spectral region of 1590−1760 cm⁻¹.

Fig 7. Evaluation of growth inhibition of cancer cell lines treated with amyloids.

The anti-proliferative activities of amyloids were tested using the MTT assay as previously described. (A)–growth inhibition of cells treated with HFIP protocol amyloids, concentration range 0.5–2 μM. (B)–growth inhibition of cells treated with HFIP-free protocol amyloids, concentration range 1–5 μM. P ≤0.05 (*), P ≤0.01 (**), P ≤0.001 (***) indicate significant differences, if not indicated otherwise the difference was not significant.

Fig 8. Distribution of cell cycle phases of cancer cells treated with amyloids.

Amyloid activity on cell cycle distribution was analyzed by flow cytometry, as detailed in the Materials and methods section. (A)–cell cycle of cells treated with 1 μM HFIP protocol amyloids and solvent for negative control. (B)–cell cycle of cells treated with 2–5 μM HFIP-free protocol amyloids and solvent for negative control.

Fig 9. Induction of cancer cell death by amyloids.

The pathway of cell death induced by amyloids was analyzed by flow cytometry. Representative scatter plots showing Annexin V and PI staining of NB4 cells, I–necrosis (Annexin V-/PI+), II–late apoptosis (Annexin V+/PI+), III–early apoptosis (Annexin V+/PI-). (A)–NB4 cells treated with 2 μM of HFIP protocol amyloids and solvent for negative control. (B)–NB4 cancer cells treated with 5 μM of HFIP-free protocol amyloids and solvent for negative control.

Fig 10. Fluorescence analysis of cancer cells with FAM-labeled amyloids.

Amyloid accumulation in cancer cells after certain periods of time (4, 24, 48 h) and control cells without amyloid treatment. FAM-labeled amyloids are shown in green, DAPI stained nuclei are shown in blue. Magnification x63.