Lung cancer, as one of the most malignant tumors, has a huge social and economic impact on human health in China and the world (
Natural chemicals have much more chemical diversity than synthetic ones, and have long been recognized as privileged scaffolds to develop drugs due to their evolved biological target specificities, and their proven biological targets are predominantly diverse functional proteins of organisms (
Medicinal plant materials were acquired from the wild in Kunming (Yunnan, China) during the summer of 2014 to prepare a phytochemical extract library, which was identified by Dr. Haizhou Li from the Faculty of Life Science and Technology of Kunming University of Science and Technology (Kunming, China). For the preparation of the phytochemical extracts, the plant materials, including branches and leaves, were washed, dried, and finely chopped and grinded. The samples were first extracted with 95% ethanol by an ultrasonic method (
Human non-small cell lung cancer A549 cell line was purchased from the Kunming Institute of Zoology, Chinese Academy of Sciences (Kunming, China). A549 cells were maintained in RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum (ScienCell Research Laboratories, Inc., Carlsbad, CA, USA) and 100 U/ml penicillin and streptomycin (Solarbio Science & Technology Co., Ltd., Beijing, China), asnd were incubated at 37°C in a humidified incubator (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 5% CO2 supplementation.
A549 cells in 100 µl medium were seeded in a 96-well plate at a density of 5×103 cells/well. Following 24 h, the cells were either treated with phytochemical extracts at different concentrations (3.91, 7.81, 15.63, 31.5, 62.5,125, 250 and 500 µg/ml) for 24, 48 and 72 h, respectively, or treated with 0.5% DMSO as controls. Subsequently, 5 mg/ml MTT (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) solution was added into each well and incubated for 4 h. Following this, the supernatant in each well was discarded and 100 µl DMSO was added. Optical density of each culture was measured at 490 nm using a microplate reader (Infinte-M200 Pro; Thermo Fisher Scientific, Inc.). The percentage of cell growth inhibition was calculated using the following formula: Percentage of cell growth inhibition = (C-T) / C × 100, where C denotes absorbance of control cells and T denotes absorbance of treatment cells. Data were presented in percentages of cell inhibition relative to the control. Percentage of cell growth inhibition was used to determine the IC50 values of the anticancer activity of phytochemical extracts using Probit analysis with GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA, USA).
A549 cells were plated in 6-well plates at a density of 200 cells/well. Each culture was mixed with a PE extract at concentrations of 0, 16.5, 31.5, 62.5, 125 and 250 µg/ml respectively. Following 12 days of incubation, the cell colonies formed in each well were stained with crystal violet (Beyotime Institute of Biotechnology) after fixation with formaldehyde, and the number of colony formed in each well was manually counted.
Morphology of A549 cells treated with a PE extract concentrations of 62.5 or 31.25 µg/ml, or with 0.5% DMSO control for 72 h was observed under a bright field using an inverted fluorescence microscope (Olympus Corp., Tokyo, Japan) at ×200 magnification.
Briefly, A549 cells were cultured in 24-well plates at 1×104 cells/well and were analyzed following 24-h treatment with PE extract (31.25 and 62.5 µg/ml, respectively). Treated cells were fixed with cold 4.0% formaldehyde for 10 min, washed with phosphate-buffered saline (PBS), and incubated with 10 µM Hoechst 33342 (Sigma-Aldrich; Merck Millipore) at 37°C for 15 min. Subsequently, the cells were washed with PBS and the cell nuclei were observed under a fluorescence microscope (Olympus Corp.).
A549 cells at 50–60% confluence were treated with a phytochemical extract at concentrations of 31.25 and 62.5 µg/ml, respectively, for 24 h. Cells were subsequently harvested by trypsinization and washed twice with PBS. Afterwards, the cells were fixed with cold 70% ethanol for 24 h at 4°C and centrifuged at 1,000 ×
Prepared A549 cells were cultured in 6-well plates at a density of 5×105 cells/ml and were treated with a PE extract at concentrations of 31.25 and 62.5 µg/ml, respectively, for 24 h. Following treatment, the cells were collected and washed with 1 ml cold PBS, and were resuspended with 250 µl staining buffer (Beyotime Institute of Biotechnology) with Annexin V/fluorescein isothiocyanate (5 µl) and PI (10 µl, 20 µg/ml). Cells were incubated at 37°C in the dark for 15 min. Finally, the stained cells were analyzed using a flow cytometer. Data were analyzed by FlowJo 7.6 software.
All data were presented as the mean ± standard deviation. Student's t-tests were performed to analyze the significant difference between treatment and control data. P<0.05 was considered to indicate a statistically significant difference.
Cytotoxic activities against A549 cell growth following treatment with phytochemical extracts of PE, chloroform, ethyl acetate, and n-butyl alcohol of
The capability of cell colony formation may represent cell viability after cell inoculation and indicate how cell growth depends on the cell population and the ability of cell propagation. When A549 cells were treated with increasing concentrations of the PE extract of
When comparing the morphological properties of control A549 cells treated with 0.5% DMSO and the PE-treated A549 cells, the morphologies of A549 cells treated with the PE extract of
To investigate the mechanism of the cell growth inhibitory effect induced by the PE extract on A549, the cell cycle of A549 cells was assessed following treatment with 31.25 and 62.5 µg/ml PE extract for 24 h. The results showed that these phytochemical treatments significantly increased the ratio of the G1 population of the cells (
In addition, the effect of PE extract of
Chinese medicinal herbs have been widely been used as a folk medicine for centuries in China and southeast Asia (
Cancer cells generally evade the programmed cell death regulatory pathways of normal tissues to support their malignant growth (
Cell cycle progress is crucial for cell proliferation (
At present, herbal medicines have been shown to be a promising approach for curing lung cancer (
In conclusion, the present study, for the first time, screened different phytochemical extracts from
The present study was supported by the Key Subject Project Foundation for Natural Product and New Drug Research of Kunming University of Science and Technology (grant no. 14078183), the Personnel Training Project of Yunnan Province (grant nos. KKSY201226096 and KKSY201226097).
Growth inhibitory effects of the PE extract of
Inhibitory effects of the PE extract of
Morphological changes of A549 cells treated with the PE extract of
Morphological changes of the nucleus of A549 cells treated by the PE extract of
Cell cycle analysis of A549 cells cultured with (A) 0.5% DMSO (control), and (B) 31.25 µg/ml and (C) 62.5 µg/ml of petroleum ether extract of
Apoptosis analysis of A549 cells treated with (A) 0.5% DMSO (as control), and (B) 31.25 µg/ml and (C) 62.5 µg/ml petroleum ether extract from
IC50values of extracts of
Samples | IC50 values (µg/ml) |
---|---|
Petroleum ether extract | 33.31±2.79 |
Chloroform extract | 84.96±5.43 |
Ethyl acetate extract | 304.79±3.92 |
N-butyl alcohol extract | ND |
Gemcitabine | 0.45±1.28 |
IC50 values of each extracts of