Different inhibitory effect and mechanism of hydroxyapatite nanoparticles on normal cells and cancer cells in vitro and in vivo

Abstract

Hydroxyapatite (HAP), similar to inorganic phase in bones, shows good biocompatibility and bioactivity as bone defect repairing material. Recently, nanoscaled HAP shows the special properties differing from bulk HAP in physics, chemistry and biology. This paper demonstrates that HAP nanoparticle (nHAP) possesses the ability for inhibiting cancer cell growth in vitro and in vivo. In vitro, after treatment with nHAP for 3 days, proliferation of human cancer cells are inhibited by more than 65% and by less than 30% for human normal cells. In vivo, injection of nHAP in transplanted tumor results in significant reduction (about 50%) of tumor size. The anticancer effect of nHAP is mainly attributed to high amount by endocytosis in cancer cells and inhibition on protein synthesis in cells. The abundant nHAP internalized in cancer cells around endoplasmic reticulum may inhibit the protein synthesis by decreasing the binding of mRNA to ribosome due to its high adsorption capacity for ribosome and arrest cell cycle in G0/G1 phase. nHAP shows no ROS-involved cytotoxicity and low cytotoxicity to normal cells. These results strongly suggest that nHAP can inhibit cancer cell proliferation and have a potential application in cancer treatment.

Figure 1. Degree of inhibition of nHAP on cell proliferation.

Comparative effect of inhibition of nHAP and SCA on Bel-7402 and L-02 cells. Each column represents the mean of 3-5 separate experiments; bar represents SE.

Figure 2. Size and dosage effects of HAP particles on Bel-7402 cancer cell proliferation.

The average sizes of HAP particles were 60 nm (a), 170 nm (b), 290 nm (c), respectively. All the particles were applied at the concentrations of 0.14 g L⁻¹, 0.35 g L⁻¹ and 0.56 g L⁻¹, respectively.

Figure 3. Inhibitory effect of nHAP treatment on nude mice with transplanted tumor.

TEM images of (a) nHAP treated tumor tissues, (b) control. The scale bars represent 1 μm. (c) Tumor growth in volume.

Figure 4. CLSM observation of nHAP in Bel-7402 cells (a) and L-02 cells (b).

The small arrows show the particles around ER in the cells. The cells in the left-bottom frames are the magnification graphs.

Figure 5. Effect of size on the internalization of HAP particles by Bel-7402 cancer cells under TEM observation.

Bel-7402 cancer cells were cultured with different sized HAP particles, (a): 60 nm, (b): 170 nm, (c): 290 nm.

Figure 6. Effect of nHAP on the cell numbers in the different phases of cell cycle.

Each column represents the mean of 3 separate experiments with bar as SE. The significant levels of difference between control and treatment are indicated by asterisks ** for P < 0.01, respectively.

Figure 7. Inhibitory effect of nHAP on protein synthesis.

nHAP treatment inhibited (a) the synthesis of TR in vivo; (b) the synthesis of EGFP by cell-free protein synthesis system; and (c) the binding of mRNA with ribosome. Each column or circle represents the mean of 3–5 separate experiments with bar as SE.