Enhancement of recombinant myricetin on the radiosensitivity of lung cancer A549 and H1299 cells

Abstract

Objective: Myricetin, a common dietary flavonoid is widely distributed in fruits and vegetables, and is used as a health food supplement based on its immune function, anti-oxidation, anti-tumor, and anti-inflammatory properties. The aim of this study was to investigate the effects of myricetin on combination with radiotherapy enhance radiosensitivity of lung cancer A549 and H1299 cells.

Methods: A549 cells and H1299 cells were exposed to X-ray with or without myricetin treatment. Colony formation assays, CCK-8 assay, flow cytometry and Caspase-3 level detection were used to evaluate the radiosensitization activity of myricetin on cell proliferation and apoptosis in vitro. Nude mouse tumor xenograft model was built to assessed radiosensitization effect of myricetin in vivo.

Results: Compared with the exposed group without myricetin treatment, the groups treated with myricetin showed significantly suppressed cell surviving fraction and proliferation, increased the cell apoptosis and increased Caspase-3 protein expression after X-ray exposure in vitro. And in vivo assay, growth speed of tumor xenografts was significantly decreased in irradiated mice treated with myricetin.

Conclusions: The study demonstrated both in vitro and in vivo evidence that combination of myricetin with radiotherapy can enhance tumor radiosensitivity of pulmonary carcinoma A549 and H1299 cells, and myricetin could be a potential radiosensitizer for lung cancer therapy.

Virtual slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5791518001210633.

Figure 1

Myricetin radiosensitizes cancer cells in vitro. (A) and (B) Myricetin alone on clonogenic survival in A549 and H1299 cells lines. Cells were treated with indicated doses of myricetin for 24 h and colony formation assays were conducted. Compared to the control, there was little effect on clonogenic survival treated with different doses myricetin (P < 0.05). (C) Radiosensitivity was measured by colony formation assay in the human lung cancer cell line A549 treated with control or 25 μM of myricetin (1 h before IR and maintained for 24 h), Compared to the control, clonogenic survival of A549 cells treated with myricetin were decreased significantly under different irradiation doses. (D) Radiosensitivity of the human lung cancer cell line H1299. Shown are averages of triplicate samples. Standard errors are shown by error bars.

Figure 2

Myricetin enhances radiosensitivity on inhibiting the tumor cell proliferation. (A) Radiosensitivity was measured by CCK-8 assay in the human lung cancer cell line A549 treated with 25 μM of myricetin alone or myricetin plus radiotherapy (1 h before IR and maintained for 24 h), Compared to the radiotherapy alone group, the proliferation of A549 cells were slower down significantly in radiotherapy combine with myricetin group. (B) Radiosensitivity of the human lung cancer cell line H1299, which results was same as the A549.

Figure 3

Radiosensitizing effect of myricetin on tumor cells apoptosis. (A) Myricetin increase the radiosensitivity of apoptosis rate in A549 and H1299 cells. The cells were stained by annexin V-FITC/PI, and cell apoptosis was analyzed by FCM. The data showed that apoptotic cells was statistically significant increased (*P < 0.05) in radiotherapy + myricetin combination group, compared to other three groups. (B) Western blot for Caspase-3 expression in control (c), myricetin-alone (M), radiotherapy-alone (R) and radiotherapy + myricetin combination (M + R) group. (C) Myricetin increase the Caspase-3 expression in occurred for radiotherapy in A549 and H1299 cells. The data aslo showed higher expression of Caspase-3 (*P < 0.05) in radiotherapy + myricetin combination group, compared to other three groups. Data are presented as the mean of triplicate experiments.

Figure 4

A549 cell xenograft tumor for detecting myricetin sensitizing tumors to irradiation in vivo. A549 cells were implanted subcutaneously in immunodeficient female BALB/C nude mice. Mice treated with myricetin alone (20 mg/kg, once daily for 14 days) or myricetin plus radiotherapy (myricetin was given 1 h before IR) at 2-Gy fractions for a total dose of 20 Gy. Shown are medium tumor volume of each group as function of time after implantation. The data curve showed that radiotherapy combine with myricetin could inhibit the tumor growth significantly, compared with other three groups.