Increasing the effect of annonacin using nanodiamonds to inhibit breast cancer cells growth in rats (Rattus norvegicus)-Induced breast cancer

Abstract

Background: Annonaceous acetogenins have been reported to have anti-cancer properties but low viability. In this study, we aimed to investigate the potency of nanodiamonds to be employed as a carrier of annonacin to help increase its viability and inhibit the growth of breast cancer cells.

Methods: The annonacin was coupled with nanodiamond and characterized using UV-Vis spectrophotometer, FTIR, SEM, and PSA, and determined their stability and drug release. A cell growth inhibition assay and cell migration assay was performed using the breast cancer MCF7 and T747D cell lines, and in vivo analysis was performed in rats (Rattus norvegicus). MCF7 and T747D cells were treated with 12.5 μg/mL annonacin coupled with nanodiamonds for 24 and 48 h and further analyzed by MTT, cell migration, and reactive oxygen species (ROS) assays. Twenty-five female rats were divided into five groups. Breast cancer was induced using two intraperitoneal doses of N-nitroso-N-methylurea (NMU) (50 and 30 mg/kg body weight). Annonacin coupled with nanodiamonds was administered by intraperitoneal injection (17.5 mg/kg body weight) for 5 weeks, one injection per 3 days.

Results: Administration of annonacin coupled with nanodiamonds significantly reduced MCF7 cell growth and reactive oxygen species (ROS) levels. The in vivo study showed that administration of annonacin coupled with nanodiamonds significantly reduced PI3KCA levels and increased p53 expression, reduced cancer antigen-15-3 (CA-15-3) levels in serum, increased caspase-3 expression, reduced Ki-67 levels, and reduced the thickness of the mammary ductal epithelium.

Conclusions: Collectively, this study demonstrated the effectiveness of nanodiamonds as a carrier of annonacin to inhibit breast cancer cell growth through inhibition of the PI3K/Akt signaling pathway.

Keywords: Annonacin; Breast cancer; Nanodiamonds; PI3K; ROS.

Figure 1

(A) Imaging of annonacin-nanodiamond complex by using SEM (magnification 40.000x). (B) Uv-vis of annonacin before and after adsorption. (C) Fourier transform infrared spectroscopy of annonacin, nanodiamond, and annonacin nanodiamond.

Figure 2

In vitro annonacin release from nanodiamond complex.

Figure 3

IC₅₀ of free annonacin and annonacin coupled with nanodiamond in MCF7 (A–B) and T47D cells (C–D).

Figure 4

Cell migration assay of MCF-7 and T47D cells administered with 12.5 and 75 μg/mL of annonacin coupled with nanodiamond for 48 h. Mean values with the different signs are significantly different compared to the control group.

Figure 5

The ROS level in MCF-7 cells administered with 12.5 μg/mL of annonacin coupled with nanodiamond for 24 h. Mean values with the different signs are significantly different at P < 0.05.

Figure 6

The serum levels of MDA in breast cancer-induced rats following treatment with 17.5 mg/kg body weight of annonacin coupled with nanodiamond for 5 weeks. Mean values with the different signs are significantly different at P < 0.05. Each bar represents mean ± SD (n = 5).

Figure 7

The ERBB2, PI3KCA, and P53 expression in breast cancer-induced rats following treatment with 17.5 mg/kg body weight of annonacin coupled with nanodiamond for 5 weeks. Mean values with the different signs are significantly different at P < 0.05. Each bar represents mean ± SD (n = 5).

Figure 8

The serum levels of CA-15-3 in breast cancer-induced rats following treatment with 17.5 mg/kg body weight of annonacin coupled with nanodiamond for 5 weeks. Mean values with the different signs are significantly different at P < 0.05. Each bar represents mean ± SD (n = 5).

Figure 9

Cells with positive Ki-67 staining in breast cancer-induced rats following treatment with 17.5 mg/kg body weight of annonacin coupled with nanodiamond for 5 weeks (Scale bar: 40 μm). Mean values with the different signs are significantly different at P < 0.05. Each bar represents mean ± SD (n = 5).

Figure 10

Cells with positive Caspase-3 staining in breast cancer-induced rats following treatment with 17.5 mg/kg body weight of annonacin coupled with nanodiamond for 5 weeks (Scale bar: 40 μm). Mean values with the different signs are significantly different at P < 0.05. Each bar represents mean ± SD (n = 5).

Figure 11

The thickness of mammary ductal epithelium in breast cancer-induced rats following treatment with 17.5 mg/kg body weight of annonacin coupled with nanodiamond for 5 weeks (Scale bar: 10 μm). Mean values with the different signs are significantly different at P < 0.05. Each bar represents mean ± SD (n = 5).

Suppl. Fig. 1