Non-small cell lung cancer (NSCLC) is a serious threat to people’s health. This study aims to determine the possible effect of Gujin Xiaoliu Tang (GJXLT) on NSCLC, which is an empirical formula from Professor Dai-Han Zhou. In this study, chromatographic fingerprinting of GJXLT and A549 cell model in vitro and in vivo was established. We cultured A549 cells in vitro and found that GJXLT inhibited A549 cell growth and induced apoptosis. Compared with the control group, the expression of p-STAT3 and VEGF proteins in the GJXLT groups was decreased. Similar findings were also observed in vivo. First, GJXLT inhibited the growth of transplanted tumor and did not reduce the weight of the tumor-bearing mice in comparison with that of the control group. Then, the Ki-67 expression of transplanted tumor in the GJXLT groups was decreased. In addition, the apoptosis rate of transplanted tumor in the GJXLT groups was increased. Overall, our data showed that GJXLT inhibited A549 cell proliferation and induced apoptosis in vivo and in vitro. Furthermore, GJXLT inhibited the growth of lung cancer xenograft in nude mice model with no obvious side effects. The anti-tumor effect of GJXLT might also be related to the inhibition of p-STATS and VEGF expression in the JAK2/STAT3 pathway. Our results demonstrated the potential of GJXLT as a novel treatment for NSCLC.
Lung cancer, a common and severe disease of the respiratory system, ranks first in terms of mortality among all cancers [
Chinese herbs have been used to treat malignant tumor for hundreds of years. Modern research shows that they can inhibit tumor growth in various ways, such as inducing cell cycle arrest and attenuating the tumor-associated macrophage-stimulated proliferation [
Raw herbs were obtained from the Chinese pharmacy of The First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine. The eight herbs of GJXLT,
3
Human NSCLC A549 cells were purchased from the Cell Bank of the Chinese Academy of Sciences of Shanghai. Cells were cultured in RPMI1640 medium with 10% fetal bovine serum (FBS, Gemini, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin in a humidified atmosphere with 5% CO2 at 37°C (Thermo Fisher Science, MA, USA). The cells with 80% confluences were treated with different concentrations of GJXLT.
BALB/c female nude mice (4 weeks old, weighing 18–22 g) were maintained under specific pathogen-free conditions with constant temperature (23 ± 2°C) and controlled light (12 h light:12 h dark). The study was approved by the Institutional Animal Care and Use Committee (animal authorization reference number: SCXK2013-0034) at Guangzhou University of Chinese Medicine (Guangdong, China). Animal welfare and experimental procedures were strictly carried out in accordance with the Guide for the Care and Use of Laboratory Animals (The Ministry of Science and Technology of China, 2006). All efforts were made to minimize animals’ suffering and to reduce the number of animals used.
We used the Waters High-Performance Liquid Chromatography (HPLC) system to analyze the chemical composition of GJXLT. The system comprised a 626 pump, a 600 s controller, and a 996 photodiode array detector. A C18 column (250 mm × 4.0 mm, 5
MTT assay was used to measure cell proliferation. Briefly, A549 cell lines were seeded in 96-well culture plates at a density of 5 × 103 cells per well in complete medium, incubated overnight to allow attachment, and divided into different groups (n = 6). The cells in the control group were treated with culture medium, while others were treated with culture medium containing different concentrations of GJXTL (8.88–568.00
Annexin V-FITC/PI stained fluorescence-activated cell sorter (FACS) and Annexin V-FITC stained fluorescence microscopy were used to measure cell apoptosis. Briefly, A549 cell lines were seeded at a density of 2 × 104 cells/well overnight, divided into different groups (n = 3), and then treated with GJXLT at different concentrations for 24 h. All cells were harvested through trypsinization and washed twice with cold PBS (0.15 mol/L, pH 7.2). The cells were centrifuged at 1000 r/min for 5 min. Then, the supernatant was discarded and the pellet was resuspended in 1× binding buffer at a density of 1.0 × l06 cells/mL. A total of 100
The cells were treated with GJXTL at different concentrations for 24 h and lysed with RIPA buffer for 30 min on ice. Then, the cells were centrifuged at 12000 rpm for 15 min at 4°C, and the supernatant was collected. The protein concentration was measured by the BCA method. Equal amount of protein (80
A549 cells were cultured and collected by centrifugation (1000 rpm, 5 min) and washed twice with ice-cold PBS. Then, 7 × 106 A549 cells in 200
Paraffin-embedded tumor sections (3
The apoptotic cells in paraffin-embedded tumor sections were detected with an in situ cell death detection kit based on the labeling of DNA strand breaks. The tumor sections were labeled successively by Streptavidin-FITC and POD-conjugated Anti-FITC, followed by treatment with a DAB staining system (Merck Millipore, Billerica, MA, USA) and counterstaining with hematoxylin. The stained sections were imaged using a microscope (Olympus BX61, Tokyo, Japan).
Weight of the mice was measured before the experiment. At the end of the experiment, the weight increase rate of every group was calculated as follows: weight increase rate (%) = [average weight (after the experiment) − average weight (before the experiment)]/average weight (before the experiment) × 100%.
After the treatment, all mice were anesthetized, and the livers and kidneys of mice were removed, cut at 5
Blood samples were collected from mice under terminal anesthesia through cardiac punctures. Clear blood samples were prepared, and blood cells were measured with a three-classification blood cell analyzer (pocH-100i, SYSMEX, Japan). Clear serum samples were prepared and measured with an automatic clinical biochemistry analyzer (ADVIA 1800, SIEMENS, Germany).
All data are presented as the mean ± SEM (standard error of mean) and obtained from at least three independent experiments. The Mann-Whitney U test was used to determine the significance of between-group differences. Statistical significance was set at
GJXLT extract was isolated with the HPLC system, and its PDA polychromatic spectrogram was established as shown in Figure
Chromatographic fingerprinting of GJXTL. (a) PDA polychromatic spectrogram of GJXTL. A total of 74 peaks were identified as the characteristic profile of GJXTL extract. (b) Simplified chromatographic fingerprinting of GJXTL.
MTT assays were performed with the NSCLC cell line A549 after treatment with different concentrations of GJXLT (8.88–568.00
Cytotoxicity of different concentrations of GJXTL against A549 cells at different time points.
GJXLT |
n | OD value | ||
---|---|---|---|---|
12h | 24h | 36h | ||
0 | 6 | 0.8467±0.0102 | 1.0446±0.1838 | 1.4629±0.1468 |
8.88 | 6 | 0.8293±0.1634 | 0.9285±0.0765 | 0.8423±0.0826 |
17.55 | 6 | 0.6568±0.0711 |
0.8687±0.0209 |
0.7755±0.1327 |
35.50 | 6 | 0.5409±0.0646 |
0.6798±0.0677 |
0.6658±0.1141 |
71.00 | 6 | 0.4765±0.0246 |
0.5897±0.0930 |
0.5983±0.0497 |
142.00 | 6 | 0.3139±0.0205 |
0.5653±0.1334 |
0.4321±0.0327 |
284.00 | 6 | 0.2788±0.0292 |
0.4621±0.0081 |
0.3836±0.0498 |
568.00 | 6 | 0.2705±0.0542 |
0.3648±0.0268 |
0.3803±0.0581 |
Data represent mean ± SD.
Inhibition rates of different concentrations of GJXTL. (a) Inhibition rates of different concentrations of GJXTL at different time points. (b) Photograph of the 96-well culture plates, in which A549 cells were treated with complete medium containing different concentrations of GJXTL for 24 h, incubated with MTT, and dissolved in DMSO. (c) Scatter plot of inhibitory rate of different concentrations of GJXTL on A549 cells for 24 h. Data are presented as the mean ± SD obtained from at least three independent experiments.
By staining cells with Annexin V-FITC and PI, FACS was used to distinguish and quantitatively determine the percentage of dead, viable, apoptotic, and necrotic cells after treatment with GJXTL at different concentrations for 24 h (Table
Apoptotic rate of A549 cells treated with different concentrations of GJXTL for 24 h.
GJXTL (ug/mL) | n | Viable cells (%) | Early apoptotic cells (%) | Advanced apoptotic cells (%) |
---|---|---|---|---|
0.00 | 3 | 95.1400±0.6161 | 0.4066±0.1950 | 1.8600±0.2821 |
142.00 | 3 | 83.5933±2.9206 | 1.4467±0.3465 | 4.6900±0.1931 |
284.00 | 3 | 80.0533±2.8002 | 2.5667±0.8159 | 7.9867±1.8336 |
568.00 | 3 | 71.8667±2.2550 | 5.28±1.31 |
12.2633±1.9886 |
Data represent the mean ± SD, n = 3,
Early and late apoptosis induction of GJXTL against A549 cells for 24 h. Data represent the mean ± SD, n = 3,
To determine whether GJXLT can suppress JAK2/STAT3 pathway activation, Western blot was used to examine STAT3, p-STAT3, and VEGF protein activity changes in the JAK2/STAT3 pathway after treatment with GJXTL at different concentrations for 24 h (Figure
Effects of GJXTL on relative expression of STAT3, p-STAT3, and VEGF protein activity in the JAK2/STAT3 signal pathway. Data represent the mean ± SD, n = 3,
We evaluated the anticancer effect of GJXLT on female nude mice bearing A549 tumor. After treatment for four weeks, all mice were anesthetized, and the tumors were removed. DDP (2 mg/kg) and middle and high concentration of GJXLT decreased tumor volume to some extent, and a statistical difference was observed in comparison with the control group. Low concentration of GJXLT slightly decreased tumor volume, and no statistical difference was observed in comparison with the control group. In A549 xenograft mice, the tumor volume was decreased by GJXLT dose-dependently (Figure
Tumor inhibitory effect of GJXTL in vivo. (a) The tumor was excised from animals after treatment. (b) The tumor volumes were measured once every four days. (c) The comparison of stripped tumor volume of five groups. The minimum and maximum values should be excluded for the calculation of average tumor volumes. Data represent the mean ± SD,
GJXLT decreased the protein expression of Ki-67 in A549 tumor tissue in a dose-dependent manner (Table
GJXLT reduces Ki-67 protein expression in vivo.
Group | n | The positive expression rates of Ki-67 (%) |
---|---|---|
Control | 5 | 66.25±9.06 |
DDP | 5 | 65.28±8.14 |
GJXLT-low | 5 | 56.62±7.71 |
GJXLT-middle | 5 | 44.61± |
GJXLT-high | 5 | 29.94± |
Data represent the mean ± SD, n = 5,
GJXLT reduces Ki-67 protein expression in vivo. (a) Representative IHC staining of Ki-67 (IMC, 400×). (b) Comparison of the positive expression rates of Ki-67 protein of five groups. Data represent the mean ± SD, n = 5,
TUNEL assay was used to examine the situation of cell apoptosis in stripped tumor after treatment. The apoptotic cells of A549 in GJXLT groups were increased in a dose-dependent manner (Figure
GJXTL induced A549 cell apoptosis in vivo. Compared with the control group, the apoptotic cells in GJXLT groups increased in a dose-dependent manner (200×).
After treatment for four weeks, GJXTL increased the body weight of A549 xenograft mice dose-dependently. As shown in Figure
GJXTL caused no significant side effects in vivo. (a) GJXTL increased the body weight of A549 xenograft mice. (b) Representative HE staining of hepatic tissue (HE, 400×). Compared with the control group, the liver cells in the DDP group had mildly balloon-like change and edema, but the liver cells in the GJXTL group had no pathological changes. (c) Representative HE staining of nephridial tissue (HE, 400×). Compared with the control group, the nephridial tissue of the other groups had no pathological changes. (d) GJXTL did not decrease the number of blood cells of A549 xenograft mice. (e) GJXLT caused no change in liver and kidney function in A549 xenograft mice. Data represent the mean ± SD, n = 5,
Traditional Chinese medicine (TCM) has potential anticancer effects worthy of study. However, rigorous and systematic investigation is necessary to ensure the efficacy of evidence-based herbal formulas and transform traditional herbal practices into science-based medicines [
Fingerprinting of the formula was established to control the quality of GJXLT (Figure
Apoptosis is an important regulatory factor in the development process, maintenance of homeostasis, and elimination of damaged cells. It is the result of complex interaction between apoptotic and anti-apoptotic molecules [
Tumor angiogenesis is crucial in tumor growth and metastasis. The vascular endothelial growth factor (VEGF) is an important regulatory factor for tumor angiogenesis and has become a target for cancer treatment [
The JAK2/STAT3 signaling pathway is closely related to tumor development. In many cases, TAM-derived IL-6 and other cytokines activate STAT3 to promote tumor development by inducing proliferation and inhibiting apoptosis [
This study confirmed the anti-tumor effect of GJXLT and preliminarily revealed the anticancer mechanism of GJXLT. GJXLT can inhibit A549 cell proliferation and induce apoptosis in vivo and in vitro. Moreover, it also inhibits the growth of lung cancer xenograft in nude mice model with no obvious side effects. The anti-tumor effect of GJXLT might be related to the inhibition of p-STATS and VEGF expression in the JAK2/STAT3 pathway.
All relevant data are within the paper and its Supporting Information file.
The authors declare that they have no competing interests.
This study was funded by the National Natural Science Foundation of China (No. 81573918), Natural Science Foundation of Guangdong Province (No. 2014A030313413), “High Level University Construction” Project of Guangzhou University of Chinese Medicine (No. 201611), and “Chuangxin Qiangyuan” Project of the First Affiliated Hospital of Guangzhou University of Chinese Medicine (No. 2016LP03).
The Supplementary Material includes the raw date of the experiment, in which the blood routine biochemical results of mouse experiment and the data related to the molecular cell animal experiments were provided respectively.