Zi Shen Decoction Inhibits Growth and Metastasis of Lung Cancer via Regulating the AKT/GSK-3 β/ β-Catenin Pathway

Abstract

Lung cancer has become the leading cause of cancer-related death worldwide. Oxidative stress plays important roles in the pathogenesis of lung cancer. Many natural products show antioxidative activities in cancer treatment. Zi Shen decoction (ZSD) is a classic prescription for the treatment of lung disease. However, its effect on lung cancer lacks evidence-based efficacy. In this study, we investigated the anticancer effects of ZSD on lung cancer in vivo and in vitro. Our results showed that oral administration of ZSD suppressed the Lewis lung cancer (LLC) growth in a subcutaneous allograft model and promoted necrosis and inflammatory cell infiltration in the tumor tissues. Furthermore, ZSD not only inhibited tumor cell proliferation and migration but also induced cell apoptosis in lung cancer cells. PI3K/AKT signaling is well characterized in response to oxidative stress. The bioinformatics analysis and western blot assays suggested that ZSD decreased the enzyme activity of PI3K and AKT in vivo and in vitro. We also found that the AKT/GSK-3β/β-catenin pathway medicated anticancer effect of ZSD in lung cancer cells. In conclusion, we demonstrate for the first time that ZSD possesses antitumor properties, highlighting its potential use as an alternative strategy or adjuvant treatment for lung cancer therapy.

Figure 1

The network pharmacology analysis of ZSD. (a) The flowchart of the network pharmacology. (b) Compound-target network: the red diamond nodes represent compounds of ZSD. The blue circle nodes represent corresponding targets. (c) GO enrichment analysis of the candidate targets of ZSD. (d) KEGG pathway enrichment analysis of candidate targets of ZSD. (e) Molecular docking process of 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-6-(3-methylbut-2-enyl) chromone-4-one with AKT1. (f) Docking process of quercetin with AKT1. (g) Docking process of quercetin with EGFR. (h) Docking process of quercetin with ALK.

Figure 2

ZSD suppressed growth of LLC-allograft tumors. (a) The body weight of mice in each group. (b) Images of mice injected subcutaneously with LLC cells and tumor tissues from LLC-allograft mice treated with NS, HSOL, and ZSD. (c) Tumor volumes were recorded every two days during the treatment. (d) Mice were sacrificed after 21 days of treatment, and representative tumors and the weight in each group were showed. (e) H&E staining of tumor tissues from the model and high dose of ZSD group. Scale bar: 200 μm.

Figure 3

ZSD inhibited the proliferation of lung cancer cells in vitro. (a) Calculated IC50 of different cancer cells and normal human BEAS-2B cells. The cells were treated with various concentrations of ZSD (0.25, 0.5, 1, 2, and 4 mg/mL) for 48 h and IC50 was calculated. (b–e) ZSD inhibited the viability of BEAS-2B, H1299, H1975, A549 cells in a time and dose-dependent manner. BEAS-2B and lung cancer cells were treated with different concentrations of ZSD for 12 h, 24 h, and 48 h. The percentages of viable cells were determined using MTT assay. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 vs. the control group. (f) 4 mg/mL of ZSD exhibited more prominent inhibitory effect on lung cancer cells than BEAS-2B cells. (g) ZSD reduced colony formation of lung cancer cells. Cells were treated with indicated concentrations of ZSD and cultured for 14 days to form colonies. Colonies were stained with crystal violet and then counted. (h) Quantitation analysis of colony formation numbers. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 vs. the control group.

Figure 4

ZSD inhibited the migration and invasion of lung cancer cells. (a, b) ZSD inhibited mobility of lung cancer cells and had no effect on BEAS-2B cells in wound-healing assay. Scale bar: 200 μm. (c, d) ZSD inhibited migration of lung cancer cells in a dose-dependent manner and showed no effect on BEAS-2B cells. Scale bar: 100 μm. (e, f) ZSD inhibited invasion of lung cancer cells in a dose-dependent manner and showed no effect on BEAS-2B cells. Scale bar: 100 μm. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 vs. the control group.

Figure 5

ZSD induced the apoptosis of H1299 cells partially by regulating AKT/GSK-3β/β-catenin signaling. (a) ZSD induced the apoptosis of H1299 cells after treatment with ZSD (4 mg/mL) for 12 h, 24 h, and 48 h. (b) ZSD induced apoptosis of H1299 cells in a dose-dependent manner. (c) Pathway enrichment analysis of the genes in Human Signal Transduction Pathway Finder PCR Array. (d) The mRNA expression of AKT/GSK-3β/β-catenin signal cascades in H1299 cells after treatment with ZSD (4 mg/mL). ^∗p < 0.05 and ^∗∗p < 0.01 vs. the control group.

Figure 6

ZSD inhibited the AKT/GSK-3β/β-catenin pathway in lung cancer cells and tumor tissues. (a) Western blot analysis was used to assess the expression of multiple proteins involved in the AKT/GSK-3β/β-catenin pathway in lung cancer cells after ZSD treatment for 12 h, 24 h, and 48 h. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 vs. the control group. (b) Western blots showing the protein expression of AKT, p-AKT, GSK-3β, p-GSK-3β, β-catenin, cleaved caspase-3, and Bax and Bcl-2 in isolated tumor tissues. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 vs. the model group.

Figure 7

The AKT/GSK-3β/β-catenin pathway medicated anticancer effect of ZSD in lung cancer cells. H1299 cells were treated with ZSD and/or SC-79 for 24 h. (a) Representative western blots and quantitative analysis of the AKT/GSK-3β/β-catenin pathway related proteins. (b) The mobility of H1299 cells was detected by wound-healing assay. (c) The migration and invasion abilities of H1299 cells were detected by transwell assays. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 vs. the control group.