Enhancement of cytotoxicity and induction of apoptosis by cationic nano-liposome formulation of n-butylidenephthalide in breast cancer cells

Abstract

Breast cancer is the second most common malignancy in women. Current clinical therapy for breast cancer has many disadvantages, including metastasis, recurrence, and poor quality of life. Furthermore, it is necessary to find a new therapeutic drug for breast cancer patients to meet clinical demand. n-Butylidenephthalide (BP) is a natural and hydrophobic compound that can inhibit several tumors. However, BP is unstable in aqueous or protein-rich environments, which reduces the activity of BP. Therefore, we used an LPPC (Lipo-PEG-PEI complex) that can encapsulate both hydrophobic and hydrophilic compounds to improve the limitation of BP. The purpose of this study is to investigate the anti-tumor mechanisms of BP and BP/LPPC and further test the efficacy of BP encapsulated by LPPC on SK-BR-3 cells. BP inhibited breast cancer cell growth, and LPPC encapsulation (BP/LPPC complex) enhanced the cytotoxicity on breast cancer by stabilizing the BP activity and offering endocytic pathways. Additionally, BP and LPPC-encapsulated BP induced cell cycle arrest at the G₀/G₁ phase and might trigger both extrinsic as well as intrinsic cell apoptosis pathway, resulting in cell death. Moreover, the BP/LPPC complex had a synergistic effect with doxorubicin of enhancing the inhibitory effect on breast cancer cells. Consequently, LPPC-encapsulated BP could improve the anti-cancer effects on breast cancer in vitro. In conclusion, BP exhibited an anti-cancer effect on breast cancer cells, and LPPC encapsulation efficiently improved the cytotoxicity of BP via an acceleration of entrapment efficiency to induce cell cycle block and apoptosis. Furthermore, BP/LPPC exhibited a synergistic effect in combination with doxorubicin.

Keywords: Cell apoptosis; Cell cycle; Polycationic liposome containing PEI and polyethylene glycol complex (LPPC); Synergistic effect; n-Butylidenephthalide (BP).

Figure 1

BP inhibited SK-BR-3 and MDA-MB-231 cells. SK-BR-3 and MDA-MB-231 cells were separately exposed to BP (0-200 μg/ml) for 24 and 48 hrs. After that, an MTT assay was performed to calculate cell viability; absorbance was measured at 550 nm. (A) SK-BR-3 cells; (B) MDA-MB-231 cells.

Figure 2

LPPC protected the activity of BP in different temperatures, as well as in a protein-rich environment. SK-BR-3 cells were exposed to BP (storage at 4 °C), BP (storage in 10% FBS at 37 °C), BP/LPPC (storage at 4 °C) or BP/LPPC (storage in 10% FBS at 37 °C), and the concentration of 50% inhibition was calculated by MTT assay. *: Indicated a significant difference between BP treatment and BP/LPPC treatment (storage at 4 °C, P < 0.05). #: Indicated a significant difference between BP and BP/LPPC (storage in 10% FBS at 37 °C, P < 0.05).

Figure 3

LPPC encapsulation improved the anti-proliferative ability of BP in breast cancer cell lines. SK-BR-3 and MDA-MB-231 cells were separately exposed to BP/LPPC (0-100 µg/ml) for 24 and 48 hrs. After that, an MTT assay was performed to calculate cell viability; absorbance was measured at 550 nm. (A, B) SK-BR-3 cells; (C, D) MDA-MB-231 cells. *: Indicated a significant difference between the IC₅₀ of BP and the IC₅₀ of BP/LPPC (P < 0.05).

Figure 4

The amount of BP/LPPC showed rapid uptake by breast cancer cells. Cells were exposed to BP or BP/LPPC (50 μg/ml) for the indicated time points, and then the fluorescence of BP was observed under microscopy and the content of BP was extracted from phenol-chloroform, followed by detection with absorbance at 350 nm. (A) The amount of BP uptake; (B) The content of BP extracted from cells. *: Indicated a significant difference between BP/LPPC and BP treatment (P < 0.05).

Figure 5

LPPC encapsulation induced breast cancer cell activation of cell endocytic pathways. Cells (2.5×10⁵) were pretreated with inhibitors (AHH, FIII and CPZ) for 1 hr. The inhibitors were discarded and cells were exposed to BP/LPPC (50 µg/ml) for the indicated time points, then BP was extracted from phenol-chloroform, followed by detection with absorbance at 350 nm. *, #, †: Indicated a significant difference compared to AHH (*), FIII (#), and CPZ (†) treatment, respectively, with no inhibitors (P < 0.05).

Figure 6

BP induced cell cycle arrest at G₀/G₁ phase in breast cancer cells. Cells (2×10⁶) were exposed to BP (60, 90 and 120 μg/ml) for different time points and then harvested and analyzed using flow cytometry. (A) The peak of cell cycle distribution; (B) Quantifiable cell cycle distribution with different doses of BP; (C) Quantifiable cell cycle distribution with different time points of BP. *: Indicated a significant increase between BP/LPPC or BP and control (P < 0.05). #: Indicated a significant decrease between BP/LPPC or BP and control (P < 0.05).

Figure 7

BP/LPPC induced cell cycle arrest at G₀/G₁ phase in breast cancer cells. Cells (2×10⁶) were exposed to BP/LPPC (20, 40 and 60 μg/ml) for different time points and then harvested and analyzed using flow cytometry. (A) The peak of cell cycle distribution; (B) Quantifiable cell cycle distribution with different doses of BP/LPPC; (C) Quantifiable cell cycle distribution with different time points of BP/LPPC. *: Indicated a significant increase between BP/LPPC or BP and control (P < 0.05). #: Indicated a significant decrease between BP/LPPC or BP and control (P < 0.05).

Figure 8

BP/LPPC induced cell apoptosis in breast cancer cells. Cells were exposed to different concentrations of BP or BP/LPPC for the indicated time points. (A, B) After treatment, cells were harvested and the sub-G₁ phase was analyzed using flow cytometry. (C) Cells were harvested and stained with TUNEL (green) and PI (red) solution, and the cell death morphology was observed under microscopy in x400 field. Red arrow: apoptotic bodies; yellow arrow: DNA fragments; blue arrow: chromatin condensation; white arrow: anoikis. *: Indicated a significant difference between treatment and control groups (P < 0.05). *, #: Indicated a significant difference between BP (*) or BP/LPPC (#) treatment, respectively, and the control group (P < 0.05).

Figure 9

BP and BP/LPPC induced cell cycle-related proteins and activated apoptosis proteins in breast cancer cells. Proteins were extracted by RIPA lysis, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto PVDF. Afterward, membranes incubated primary antibodies overnight, second antibodies for 2 hrs and HRP at RT for 2 hrs, separately. The membranes were detected by a Luminescent Image Analyzer. (A) The cell cycle regulators and apoptosis-related proteins of BP; (B) The cell cycle regulators and apoptosis-related proteins of BP/LPPC.

Figure 10

BP/LPPC combined with doxorubicin had a synergistic effect on breast cancer cells. SK-BR-3 and MDA-MB-231 cells were treated with BP/LPPC combined with doxorubicin. (A, B) SK-BR-3 cells. (C, D) MDA-MB-231 cells. *: Indicated a significant difference between the combination of drugs and drug alone (black bar) (P < 0.05).