Gallic acid induces the apoptosis of human osteosarcoma cells in vitro and in vivo via the regulation of mitogen-activated protein kinase pathways

Abstract

To examine the antitumor effects of gallic acid (GA) on osteosarcoma, two human osteosarcoma cell lines U-2OS and MNNG/HOS were treated by GA and subjected to cell proliferation and apoptosis assays. In addition, MNNG/HOS xenograft tumors were established in nude BALB/c mice to evaluate the anticancer capacity of GA in vivo. The results showed that GA inhibited the proliferation and induced the apoptosis of osteosarcoma cells, accompanied by the upregulation of p-38 activation and the downregulation of c-Jun N-terminal kinase (JNK) and extracellular signal regulated kinase (ERK1/2) activation. Additionally, p38 MAPK inhibitor abrogated GA-induced growth inhibition of osteosarcoma cells, whereas JNK or ERK1/2 inhibitors sensitized osteosarcoma cells to GA-induced growth inhibition. In vivo studies further showed that GA administration decreased xenograft tumor growth in a dose-dependent manner. Immunohistochemistry analysis demonstrated the downregulation of PCNA and CD31 expression and upregulation of apoptosis in MNNG/HOS tumor tissues following GA treatment. This study demonstrates the antitumor efficacy of GA for osteosarcoma that is mediated by the modulation of cell proliferation, apoptosis, and angiogenesis. Our findings suggest that GA could be a potent agent for osteosarcoma intervention.

FIG. 1.

GA inhibits the proliferation of human osteosarcoma cells in a dose- and time-dependent manner. (A, B) Cells were exposed to GA with different concentrations for various time and the OD values were obtained through reading plate at 570 nm with 96-well microtest spectrophotometer by MTT assay. (C, D) After exposing to GA with different concentrations for various time, cell proliferation was analyzed by BrdU incorporation assay. The viability rate was expressed as the percentage of cell viability rate compared with the control. The data were expressed as mean±SD obtained from triplicate samples. GA, gallic acid.

FIG. 2.

GA induces apoptosis of human osteosarcoma cells. (A) Hoechst staining of osteosarcoma cells treated with 25, 50, and 75 μM for 24 hours, respectively. The nuclei were stained by Hoechst 33258 and visualized under fluorescence microscope (magnification, 200×; scale bar=100 μm). (B) The apoptotic ultrastructure of osteosarcoma cells treated by GA. The cells were examined under a transmission electron microscope (×3700 power). In the 50 μM GA group, typical apoptotic cells were observed in U-2OS and MNNG/HOS; scale bar=2 μm. (C) Annexin V-FITC/PI staining of osteosarcoma cells treated by GA. (a) control group; (b) 25 μM GA; (c) 50 μM GA; (d) 75 μM GA. The data were representative of three independent experiments. (D) GA induces the activation of caspase-3 and -9 while does not significantly influence the activation of caspase-8. Lysate was prepared from cells grown in the absence or presence of GA (25, 50, and 75 μM) for 24 hours and tested for activities. Data represented the mean of three measurements±SD. **p<0.01 versus control group; ^#p<0.001 versus control group.

FIG. 3.

GA upregulates p-38 activation and downregulates JNK and ERK1/2 activation in human osteosarcoma cells in a dose-dependent manner. (A–C) Western blotting analysis of p-38 and p-p-38, JNK and p-JNK, ERK1/2, and p-ERK1/2 in human osteosarcoma cells treated with 25, 50, and 75 μM GA for 24 hours, respectively. The density of target proteins was analyzed by Gel-Pro-analyzer (Media Cybernetics). β-Actin served as loading control. (D–F) Real-time RT-PCR analysis for quantitative evaluation of the mRNA expression of target genes. (G) The effects of p38, JNK, and ERK1/2 MAPK inhibitors on GA-induced growth inhibition. Data were expressed as mean±SD of three samples. *p<0.05 versus control group; **p<0.01 versus control group; ^#p<0.001 versus control group. ^○p<0.05 GA group versus MAPK inhibitors group. ^□p<0.01 GA group versus MAPK inhibitors group. JNK, c-Jun N-terminal kinase; ERK1/2, extracellular signal regulated kinase; MAPK, mitogen-activated protein kinase.

FIG. 4.

GA inhibits the growth of MNNG/HOS xenograft in athymic nude mice. Approximately 5×10⁶ MNNG/HOS cells were injected subcutaneously into the right axillary fossa of each nude mouse under aseptic conditions. After 24 hours, mice were fed plain water (control group) or 0.3% and 1% (w/v) dose of GA in water for 5 days/week for 5 weeks. (A) Average tumor volume (mm³) was plotted as a function of week of GA feeding. At the end of 5 weeks of xenograft studies, tumors were excised and processed for H&E analysis (B) and immunohistochemical staining for TUNEL (C), PCNA (D), and CD31 (E). The representative pictures were taken at 400×magnification of microscopic field from each group. Bar diagrams represented quantitative analysis (mean area±SD) of TUNEL (F), PCNA (G), and CD31 (H) positive cells. Data were shown as mean±SD. **p<0.01 versus control group; ^#p<0.001 versus control group. Scale bar=50 μm.