Theabrownin Inhibits Cell Cycle Progression and Tumor Growth of Lung Carcinoma through c-myc-Related Mechanism

Abstract

Green tea, the fresh leaves of Camellia sinensis, is not only a health-promoting beverage but also a traditional Chinese medicine used for prevention or treatment of cancer, such as lung cancer. Theabrownin (TB) is the main fraction responsible for the medicinal effects of green tea, but whether it possesses anti-cancer effect is unknown yet. This study aimed to determine the in vitro and in vivo anti-lung cancer effect of TB and explore the underlying molecular mechanism, by using A549 cell line and Lewis lung carcinoma-bearing mice. In cellular experiment, MTT assay was performed to evaluate the inhibitory effect and IC50 values of TB, and flow cytometry was conducted to analyze the cell cycle progression affected by TB. In animal experiment, mice body mass, tumor incidence, tumor size and tumor weight were measured, and histopathological analysis on tumor was performed with Transferase dUTP nick-end labeling staining. Real time PCR and western blot assays were adopted to detect the expression of C-MYC associated genes and proteins for mechanism clarification. TB was found to inhibit A549 cell viability in a dose- and time-dependent manner and block A549 cell cycle at G0/G1 phase. Down-regulation of c-myc, cyclin A, cyclin D, cdk2, cdk4, proliferation of cell nuclear antigen and up-regulation of p21, p27, and phosphate and tension homolog in both gene and protein levels were observed with TB treatment. A c-myc-related mechanism was thereby proposed, since c-myc could transcriptionally regulate all other genes in its downstream region for G1/S transitions of cell cycle and proliferation of cancer cells. This is the first report regarding the anti-NSCLC effect and the underlying mechanism of TB on cell cycle progression and proliferation of A549 cells. The in vivo data verified the in vitro result that TB could significantly inhibit the lung cancer growth in mice and induce apoptosis on tumors in a dose-dependent manner. It provides a promising candidate of natural products for lung cancer therapy and new development of anti-cancer agent.

Keywords: TUNEL; c-myc; cell cycle; lung cancer; theabrownin.

FIGURE 1

Effect of Theabrownin (TB) and green tea extract on cell viability of A549 cells determined by MTT assay. Values were presented as mean ± SD (n = 5).

FIGURE 2

Effect of TB on cell cycle of A549 cells determined by flow cytometry. Values were presented as mean ± SD (n = 3). ^∗P < 0.05 and ^∗∗P < 0.01 for comparison between control group and TB group.

FIGURE 3

Relative mRNA expression of target genes in A549 cells with TB treatment at 200 μg/ml for 24, 48, and 72 h. Values are presented as mean ± SD of three replicates. Different symbols (a, ab, b, c, bc, d) indicate significant difference between groups [Fisher’s least significant difference (LSD), P < 0.05], and the values decrease with the order from a to d.

FIGURE 4

Protein expression of target genes in A549 cells with TB treatment at 200 μg/ml for 24, 48, and 72 h.

FIGURE 5

Mice body weight of all groups (n = 10).

FIGURE 6

Tumor volume of mice in all groups (n = 10). Different symbols (a, ab, bc, c) indicate significant difference between groups (Fisher’s LSD, P < 0.05), and the values decrease with the order from a to c. Bar = 1 cm.

FIGURE 7

Transferase dUTP nick-end labeling (TUNEL) and DAPI staining on mice tumors and the TUNEL-positive cell number comparison among all groups. Different symbols (a, b, c, d) indicate significant difference between groups (Fisher’s LSD, P < 0.05), and the values decrease with the order from a to d.

FIGURE 8

c-myc-related mechanism of TB on A549 cells.