Theabrownins Produced via Chemical Oxidation of Tea Polyphenols Inhibit Human Lung Cancer Cells in vivo and in vitro by Suppressing the PI3K/AKT/mTOR Pathway Activation and Promoting Autophagy

Abstract

During the fermentation of dark tea, theabrownins (TBs), carbohydrates, and other substances get irreversibly complex. Recent research on the biological activity of TBs is not based on free TBs. In the present study, some brown polyphenol oxidized polymers, the generalized TBs (TBs-C), were prepared via alkali oxidation from tea polyphenols (TP). We also investigated the inhibitory mechanism of TBs-C on non-small-cell-lung cancer (NSCLC). TBs-C demonstrated a stronger inhibition than TP on the NSCLC cell lines A549, H2030, HCC827, H1975, and PC9. Next, A549 and H2030 cell lines were selected as subjects to explore this mechanism. TBs-C was found to inhibit proliferation, promote apoptosis, and induce G1 cell-cycle arrest in the cells. In addition, TBs-C increased autophagic flux, which in turn promoted the death of lung cancer cells. Moreover, TBs-C suppressed the PI3K/AKT/mTOR pathway activation, promoted autophagy, and increased the expression of p21 downstream of AKT, which resulted in G1 cell-cycle arrest. In xenotransplanted NSCLC nude mice derived from A549 cells, TBs-C could significantly suppress tumor growth by inhibiting the PI3K/AKT/mTOR pathway without causing hepatotoxicity, brain toxicity, or nephrotoxicity. We believe that our present findings would facilitate advancement in the research and industrialization of TBs.

Keywords: chemical oxidation; human lung cancer; in vivo and in vitro; tea polyphenols; theabrownins.

FIGURE 1

Comparison of the anti-tumor effects of TBs-C and TP on NSCLC cells. (A,B) The experimental cells were treated with different concentrations (0, 25, 50, 75, 100, 125, 150, 175, and 200 μg/mL) of TBs-C and TP for 48 h, and the cell viability was determined by the CCK-8 assay, with the calculation of the IC50 values. (C) The comparison of the IC50 values between TP and TBs-C in different NSCLC cell lines are presented, *p < 0.05.

FIGURE 2

Effects of TBs-C on lung cancer cell proliferation, apoptosis, and cell cycle in vitro. (A) The CCK-8 assay was performed to determine the viability of A549 and H2030 cell lines after 24 and 48 h of treatment with different doses of TBs-C. (B) The capacity of A549 and H2030 cell lines to form clones was examined at varying concentrations of TBs-C. (C) The analysis of the cell cycle by flow cytometry after a 24 h treatment with TBs-C is depicted. (D) Quantitative results of the cell cycle analysis. (E) Evaluation of the apoptosis of cells treated for 24 h of TBs-C by AnnexinV-7AAD flow cytometry. (F) Quantitative analysis of cell apoptosis, *p < 0.05, **p < 0.01.

FIGURE 3

Accumulation of autophagosomes in TBs-C treated lung cancer cells. (A) Western blotting results of the LC3 and p62 protein expression in TBs-C treated and untreated cells. (B) The densitometric analysis of the changes in the abundance of LC3-II normalized to the β-actin level. (C) The indicated cells were treated with TBs-C (60 μg/mL) or untreated for 24 h. The cells were labeled with CYTO-ID^® green detection reagent at the end of the treatment period to detect the autophagosomes. As a positive control for autophagosome accumulation, the cells were treated with HBSS in the presence of CQ (60 M) for 3 h. The puncta areas per cell are given in three randomly selected images for each culture condition, *p < 0.05, **p < 0.01.

FIGURE 4

Treatment with TBs-C promotes the autophagic flux in NSCLC cells. (A) After 24 h of treatment with different concentrations of TBs-C, the cells were treated with or without baf A1 (100 nmol/mL for 3 h), and the LC3 expression was evaluated by Western blotting. (B) Densitometric analysis of the changes in LC3-II abundance normalized to the β-actin level. (C,D) The indicated cells were transfected with the RFP-GFP-LC3B Premo™ Autophagy Tandem Sensor. Fluorescence microscopy was used to count the red and yellow puncta (Scale bar = 20 m). The number of yellow and red puncta in each cell was calculated from at least 20 cells from each group, *p < 0.05, ^**p < 0.01.

FIGURE 5

Inhibition of autophagy overcomes the antitumor effect of TBs-C in NSCLC cells. (A) Cells pretreated with or without 3-MA were treated with TBs-C (60 μg/mL) for 24 h, and the protein levels of LC3 and p62 were detected by Western blotting. (B) Cells pretreated with or without 3-MA were treated with TBs-C for 24 h, and the cell viabilities were determined by the CCK-8 assay. (C) Cells transfected with si-NC and si-ATG5 were treated with TBs-C (60 μg/mL) for 24 h, and the protein levels of LC3 and p62 were detected by Western blotting. (D) Cells transfected with si-NC and si-ATG5 were treated with TBs-C for 24 h, and the cell viabilities were determined by CCK-8 assay, *p < 0.05, **p < 0.01.

FIGURE 6

TBs-C inhibits the PI3K/AKT/mTOR pathway in NSCLC cells. The expression of major proteins in the PI3K/AKT/mTOR pathway and the AKT/p21 signal axis was detected by Western blotting in cells treated with different concentrations of TBs-C, with β-actin was used as a reference.

FIGURE 7

TBs-C induces autophagy and the G1 cell-cycle arrest in NSCLC cells by inhibiting the PI3K/AKT/mTOR pathway. (A) The expression of AKT, p-AKT, mTOR, p-mTOR, p21, LC3, cyclin D, and cyclin E in the indicated cells was detected by Western blotting. (B) The cell viability of indicated cells were measured by the CCK-8 assay. (C) The cell cycle distribution of each group was determined by flow cytometry. (D) Analysis of cell cycle distribution by histogram, *p < 0.05, **p < 0.01.

FIGURE 8

TBs-C inhibits the growth of A549 xenograft tumors in vivo. (A) Tumorigenesis of A549 cells of the 3 study groups on day 28. (B) Representative size of the 3 groups of tumors. (C) Tumor volumes (measured every 3 days). (D) Tumor weight in the control group, low-dose group, and high-dose group. (E) The weight of nude mice was recorded every 3 days. (F) H&E stained the heart, brain, and kidney of mice from each group. TBs-C (L), TBs-C low-dose group, TBs-C (H), TBs-C high-dose group, *p < 0.05, **p < 0.01.

FIGURE 9

TBs-C inhibits the PI3K/AKT/mTOR pathway in vitro. (A) The protein was extracted from tumors in each experimental group, and the expression of p-PI3K, p-AKT, p-mTOR, p21, and LC3 were detected by Western blotting. (B) Density analysis of Western blotting results among the study groups. TBs-C (L), TBs-C low-dose group, TBs-C (H), TBs-C high-dose group, *p < 0.05, **p < 0.01.

FIGURE 10

Schematic diagram of TBs-C inhibiting human NSCLC cells in vitro and in vivo. In vitro, TBs-C is capable of increasing autophagic flux, inhibiting cell proliferation, enhancing apoptosis, and inducing G1 cell cycle arrest via the PI3K/AKT/mTOR pathway and AKT/p21 axis. In vivo, TBs-C enhances autophagy and inhibits tumor growth by downregulating the PI3K/AKT/mTOR pathway without causing cardiac, brain, or kidney toxicity.