Lung cancer is one of the most common cancers and the leading cause of cancer-related death worldwide [1
]. In China, lung cancer is the most common cancer with around 733,300 new cases and 610,200 deaths in 2015 [2
]. Although different strategies including chemotherapy, radiotherapy, targeted therapy and immunotherapy have been developed to treat lung cancer [3
], the overall five-year survival rate is still lower than 18% [6
]. Therefore, it is necessary to explore new drugs for the treatment of lung cancer.
Recently, a number of studies have been focused on the anti-tumor effects of natural products, especially traditional Chinese medicine [7
vegetables belong to the Brassicaceae family and are cultivated in many countries. They contain various bioactive components including glucosinolates, carotenoids, tocopherols, ascorbic acid and phenolic compounds, and show both antioxidant and antitumor activities [11
]. Brassica rapa
L. has been used in Uyghur folk medicine to treat coughs and asthma for a long time in the Xinjang Uygur Autonomous Region, China [15
]. More and more bioactive components including polysaccharides, phenolics, flavonoids and ascorbic acid have been isolated and identified from B. rapa
L., which show different biological functions such as immunostimulation, anti-inflammation, anti-allergy and antioxidant [15
]. However, the anti-tumor effects of B. rapa
L. are yet to be investigated. In this study, we prepared the n
-butanol subfraction of B. rapa
L. (BRBS) and investigated the anti-tumor effect on A549 lung cancer cells. Our results showed that BRBS could induce apoptosis and cell cycle arrest in A549 cells through ROS generation and the mitochondria-dependent pathway.
High dietary intake of Brassica
vegetables is associated with a reduced risk of cancer and various compounds including flavonoids, lectin and 1-cyano-2,3-epithiopropane have been isolated from Brassica
vegetables which can inhibit the growth of tumor cells through induction of apoptosis [30
]. In the present study, we found that BRBS dose- and time- dependently suppressed the proliferation of A549 cells through induction of cell cycle arrest at G0/G1 phase and mitochondria-dependent apoptosis. A549 cell migration was also significantly inhibited by BRBS treatment.
ROS play an essential role in the regulation of cell cycle and apoptosis [33
]. An active compound derived from Brassica
vegetables, 3,3′-Diindolylmethane, induces cell cycle arrest and apoptosis in cancer cells through up-regulation of ROS production [35
]. Similarly, we found that BRBS significantly increased intracellular ROS generation and decreased the intracellular GSH/GSSG ratio in A549 cells, which might mediate the induction of cell cycle arrest and apoptosis. ROS generation can activate c-Jun N-terminal kinase to promote and inhibit the expressions of pro-apoptotic and anti-apoptotic BCL-2 proteins respectively, which play a critical role in mitochondria-dependent apoptosis pathway through regulation of mitochondrial membrane integrity [34
]. Upon BRBS treatment, the expressions of Bax and Bcl-2 in A549 cells are up-regulated and down-regulated respectively, which cause further reduction in MMP and the release of cytochrome c. Consequently, cytochrome c activates the processing of caspase-3 and PARP observed in this study, which finally induces the apoptosis in A549 cells.
In conclusion, BRBS inhibits the proliferation of A549 cells through induction of cell cycle arrest and mitochondria-dependent apoptosis. These results indicate that B. rapa L. might be considered as a functional vegetable with potential role for the treatment of lung cancer.
4. Materials and Methods
4.1. N-Butanol Subfraction of B. rapa L. (BRBS)
B. rapa L. was collected from Turpan in the Xinjiang Uygur Autonomous Region, China. Homogenate of fresh root of B. rapa L. was made and extracted twice using 10 volumes of water for 2 h at 60 °C. The water extraction was collected and centrifuged at 6000 rpm for 15 min. The pellet was collected and extracted with 10 volumes of 95% ethanol at 60 °C for three times according to the following order of extracting time: 3 h, 2 h and 1.5 h. The ethanol extraction was centrifuged at 6000 rpm for 15 min and the supernatant was collected and concentrated using a rotary vacuum evaporator at 45 °C. Then, the concentrated liquid was extracted with an equal volume of water saturated n-butanol. Finally, the n-butanol extraction was collected and dried at room temperature (RT). The dry n-butanol subfraction was dissolved in dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO, USA) at a concentration of 100 mg/mL and filtered with a 0.22 μm filter.
4.2. Cell Culture
The A549 cells were obtained from the Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, Xinjiang University (Urumqi, Xinjiang, China) and maintained in GIBCO®1640 (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (MRC, Changzhou, China) and 1% Penicillin-Streptomycin (MRC) in a 5% CO2 atmosphere at 37 °C.
4.3. Cell Viability Assay
A549 cell (100 μL, 5 × 104/mL) were seeded in a 96-well plate, cultured overnight and then treated with different concentrations (200, 400 and 600 μg/mL) of BRBS for 24, 48 and 72 h. Cisplatin (20 μg/mL) was used as a positive control. After centrifugation at 1200 rpm for 7 min, supernatant was discarded and 100 μL of MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide] (Sigma) solution (0.5 mg/mL in PBS) was added to each well. After 4 h, 150 μL DMSO was added into each well to dissolve the formed formazan crystals. The optical density at 490 nm was measured by a 96-well microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).
4.4. Observation of Cell Morphology
A549 cells were seeded in 96-well plates and incubated for 24 h at 37 °C. The cells were treated with different concentrations (200, 400 and 600 μg/mL) of BRBS for 24 h. After treatment, cell morphological changes were observed and photographed by inverted fluorescence microscopy (Nikon Eclipse Ti-E, Tokyo, Japan).
4.5. Determination of Intracellular Reactive Oxygen Species (ROS) and GSH/GSSG Ratio
A549 cells were treated with different concentrations (200, 400 and 600 μg/mL) of BRBS for 24 h or treated with 600 μg/mL of BRBS for 2, 4, 8, 12 and 24 h. After treatment, the level of intracellular ROS was measured by a ROS assay kit according to the manufacturer’s instructions (Beyotime, Shanghai, China). Briefly, cells were harvested and incubated with 2′,7′-dichlorofluorescein diacetate (DCFH-DA) for 20 min at RT. After washing with GIBCO® 1640 3 times, the fluorescence intensity was determined by flow cytometry (BD FACSCalibur, San Jose, CA, USA) or inverted fluorescence microscopy.
After BRBS treatment for 24 h, GSH/GSSG ratios in A549 cells were spectrophotometrically measured using a GSH/GSSG ratio kit (Beyotime) according to manufacturer’s instructions.
4.6. Analysis of Apoptosis and Cell Cycle
A549 cells were treated with different concentrations (200, 400 and 600 μg/mL) of BRBS. After 24 h, cells were harvested, washed with phosphate-buffered saline (PBS) and stained with an Annexin V-FITC/propidium iodide (PI) Apoptosis Detection Kit (YEASEN, Shanghai, China) according to manufacturer’s instructions. For analysis of the cell cycle distribution, cells were harvested after BRBS treatment and fixed in cold 70% ethanol at 4 °C for 30 min. Then, cells were stained with PI for 30 min at 37 °C. All samples were analyzed by flow cytometry.
4.7. Detection of Ki-67
A549 cells were treated with different concentrations (200, 400 and 600 μg/mL) of BRBS. After 24 h, cells were harvested and washed with PBS. After washing, cells were fixed and permeabilized with Foxp3 Staining Buffer Set (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. Intracellular staining was performed using FITC conjugated Ki-67 antibody (BD Biosciences, San Jose, CA, USA) for 15 min at RT. The samples were analyzed by flow cytometry.
4.8. Determination of Mitochondrial Membrane Potential
Mitochondrial membrane potential was determined by membrane-permeable JC-1 dye (Beyotime). Briefly, A549 cells were treated with BRBS, washed with PBS and stained with the JC-1 fluorescent probe according to manufacturer’s instruction for 20 min at RT. Samples were analyzed by flow cytometry and inverted fluorescence microscope.
4.9. Wound Healing Assay
A549 cells were seeded in 24-well plates at the concentration of 2 × 106 cells per well and incubated at 37 °C overnight. Cell monolayers that converged almost 100% were wounded with a sterile 200 µL pipette tip. Detached cells were removed from the plates carefully with PBS and completed GIBCO® 1640 was added. The cells were left either untreated or treated with different doses (200, 400 and 600 μg/mL) of BRBS. After 24 h, the medium was replaced with PBS and the scratched areas were photographed using inverted fluorescence microscopy. The scratch areas of samples were analyzed by Image J at the indicated time points. The percentage of wound healing was calculated by the equation: wound healing(%) = (1 − scratch area at indicated time point/scratch area at 0 h) × 100%.
4.10. Hoechst 33258 Staining
The morphological changes of A549 cell nuclei were analyzed by membrane-permeable DNA-binding dye Hoechst 33258. A549 cells were seeded into a 6-well plate at the concentration of 1 × 105 cells/well and incubated overnight. The cells were treated with 400 and 600 μg/mL of BRBS for 24 h and fixed with 4% ice-cold paraformaldehyde at 4 °C for 10 min. After washing 3 times with PBS, cells were stained with Hoechst 33258 (Beyotime) at 4 °C for 10 min. Samples were observed by inverted fluorescence microscope.
4.11. Western Blot
After BRBS treatment, all adherent and floating cells were collected and whole cell lysates were prepared using RIPA Lysis Buffer (Beijing ComWin BiotechCo., Ltd., Beijing, China) on ice for 30 min. The protein concentration was determined using a bicinchoninic acid assay (BCA) Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Each sample contained 35 µg protein was separated on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane. After blocking with 5% skim milk, the membranes were incubated with respective primary antibodies (Cell Signaling Technology, Danvers, MA, USA) for 2 h at 37 °C. After washing with PBST solution (PBS with 0.05% Tween-20), the membranes were incubated with horse radish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology) for 1 h at 37 °C. After washing 3 times with PBST, the target proteins were detected using an enhanced chemiluminescence (ECL) assay kit (Beyotime).
4.12. Statistical Analysis
All data were expressed as mean ± standard error of the mean (SEM). Statistical analysis was conducted using one-way analysis of variance (ANOVA). p < 0.05 was considered statistically significant.