Objective: To research the inhibitory effect on SGC-7901 cells of α-pinene, and the related mechanism of α-pinene. Methods: Used the MTT method to detect inhibition rate and western blotting to detect the influence on expression of ATM, Phos-S1981ATM, H2AX, γH2AX, CHK2 and p-CHK2, p53 and phos-p53 cell cycle related protein in SGC-7901 cells. Results: The research found α-pinene could inhibit the proliferation of SGC-7901 cells observably in vitro, and the inhibition rate assumes the dependence on concentration; and western blotting results showed that, α-pinene could activate phospho-ATM, increase the amount of γH2AX (p < 0.05, p < 0.05); increase the expression of p-CHK2, p53 and phos-p53 (p < 0.05, p < 0.05, p < 0.05); But there is no significant effect on expression of CHK2 (p > 0.05). Conclusions: α-pinene could inhibit the proliferation of SGC-7901 cells, and by inducing ATM (Ataxia Telangiectasia-Mutated) kinase signal pathway in DNA damage response, activating cell cycle checkpoint, making the cell cycle arrest then exerts its anti-tumor effects.
Gastric cancer is a malignant disease which has had a serious impact on human health. Finding highly efficient and low or non-toxic compounds from within Chinese medicine to treat gastric cancer has been a topic of interest in Eastern oncology. From within Chinese medicine, it has been long known that the crude extract of pine needle oil has the potential to delay aging by dissolving compounds that accumulate as the body ages, as well as generally promote body function, and immunity [
Gastric cancer SGC-7901 cells, α-pinene and PTX (paclitaxel), all presented by the basic school of university of JiaMuSi; MTT (Methyl thiazolyl tetrazolium), antibodies, all purchased by cell signaling company; And prepared culture medium diluted to the required concentration; Heracell incubator, Thermo Forma Corp; Inverted microscope, Leica Corp; Enzyme-linked Immuno-metric meter, Bio-Rad Corp; Level-perpendicular plate electrophoresis and electrotransfer devices, Bio-Rad Corp; DU800 protein-nuclenic acid analysis meter, Beckman Corp.
SGC-7901 cells were serially cultured with RPMI-1640 medium which containing 10% FBS, 1% penicillin streptomycin combination under the condition of 5% CO2, 37˚C. A well growing SGC-7901 cell was chosen and digested using 0.25% trypsin, adjusting the density 1 × 105 cells/ml with RPMI-1640 medium that contain 10% FBS, inoculating to 96 well cell culture plate, and 0.1 ml per well, added different levels of α-pinene and PTX which prepared with RPMI-1640 medium, the final concentration were 0, 0.2, 0.4, 0.6, 0.8 and 1.0 μg/ml, respectively, and eight repeated well every level. A group was set up control group (not contained drug) and DMSO group was blank group (0.05% DMSO, medium). All cultures continued to cultivate for 24 hours, α-pinene group and PTX group supposed to be B group and C group, and PTX group was positive group, to test SGC-7901 cells proliferating inhibition after treated with drugs by MTT assay. Using enzyme-linked immunometric meter to determinate the light absorbance value (A) at 490 nm wavelength, the cell inhibition ratio was calculated with the following:
Making the logarithmic phase SGC-7901 cell digesting with 0.25% trypsin, then adjusting the density 1 × 105 cells/ml with medium that contain 10% FBS, and then inoculating to 6 well cell culture plate, 2 ml per well, After 24 hours, adding different levels of α-pinene which prepared with RPMI-1640 medium, the final concentration were 0, 0.2, 0.4, 0.6 and 0.8 μg/ml, respectively. Continuing to culture cells 24 hours, collecting each group’s cells, cells were lysed in a buffer 30 μg of total cell lysate, based on the standard curve to calculate the sample volume required. Be separated on SDS-PAGE gel and transferred onto nitrocellulose Membranes. Membranes were blocked with 5% skim milk and then incubated with the indicated rabbit primary antibodies at 4˚C for overnight. The goat-anti-rabbit second antibody incubating 1 hour at room temperature. Signals were detected using an ECL Western Blotting Kit. Primary antibodies used were: anti-ATM, anti-phospho-ATM (S1981), anti-p53, anti-phospho-p53(S15), anti-H2AX, anti-γH2AX (S139), anti-CHK2, anti-phospho-CHK2 (S345) and anti-actin. Adopted Quantity One 4.6.2 software analysis band signals.
Test results were represented by means ± standard errors, data was analyzed using the SPSS17.0 software, statistical methods such as analysis of variance, paired sample t test and t-test for independent samples were used for testing and analysis, p < 0.05 represented statistical significance.
Firstly, each group SGC-7901 cells’ morphological changes after treated with different concentrations of α-pi- nene after 24 hours was observed. A group was as normal as control group and cells grew normally in this group. B group was treated by α-pinene. SGC-7901 cells morphological changes could be observed when the concentration of 0.4 - 0.8 μg/ml, cells became smaller, Cytoplasmic shrinkage, round and the amount of cells decreased significantly; C group as positive control (PTX group), was almost the same as α-pinene group (
Detection of inhibiting ratio of SGC-7901 cells after treated by α-pinene, the inhibition ratio gradually increased with the increasing of concentration of α-pinene, while the concentration of α-pinene was 0.4 μg/ml, compared with the A group (no drug treatment) (p < 0.05); with the increasing of concentration of α-pinene, the inhibition ratio was increased, when concentration of α-pinene was 0.8 μg/ml reached the highest ratio, that illustrated the inhibition ratio assumed a dose-dependent manner when concentration of α-pinene between 0.4 μg/ml and 0.8 μg/ml. Then the ratio started to level out, the inhibition of trends the same as the positive control group, There was no statistical difference between the two groups (p > 0.05) (
Within the SGC-7901 cells that were given α-pinene of concentrations are 0, 0.2, 0.4, 0.6 and 0.8 μg/ml, and detected of the contents of ATM, Phos-S1981ATM, H2AX, γH2AX, Chk2, p-Chk2, p53 and phos-p53 after 24 hours. As results displayed, while treated SGC-7901 cells with α-pinene 24 hours, with the drug concentration rised, the damage signals phosphorylate ATM to Phos-S1981ATM were stronger; increased the expression of γH2AX. Compared with the negative groups, these differe-nces would be statistically significant (p < 0.05, p < 0.05) (
DNA damage checkpoints [
ATM participates in regulating cell cycle checkpoints and the signal transduction pathway of DNA damage repair [
DNA at specific checkpoints in the cell cycle and repair it. When various kinds of causes lead to the function of ATM impaired, and cells would lose this function and could not repair the damaged DNA timely. The damaged DNA is prone to enter next generation cells along cell division and generate tumor ultimately. After DNA damaged [
This experiment adopted that different concentration α-pinene impacted on gastric cancer SGC-7901 cells after 24 hours, the inhibition to the cells would grow depending on the concentration within certain range. Then, detecting the content of cell cycle related proteins, such as ATM, Phos-S1981ATM, H2AX, γH2AX, Chk2, p-Chk2, p53 and phos-p53. Our results have shown that, comparing with the control group, the CHK2 phosphorylation content depended on the concentration, but the amount of CHK2 did not be affected. p53 is another key molecule in DNA damage signal pathway, the results indicated that α-pinene could up regulated p53 and
phosphorylate p53 content, it illustrated that there was one signal pathway which rely on p53 in DNA damage response.ATM could phosphorylate and activate p53, then, signal transduced to downstream target protein and lead to cell cycle blocked eventually. Our research demonstrated that, the checkpoint function of cell cycle disturbed when the expression of ATM deficiency or decreased, and the damaged DNA could not be repaired in time, cell cycle function-blocking weakened. And then, the tumor cells proliferation would enhance as well. The expression of ATM would rise when the concentration of drugs increased, then phosphorylation content of ATM would rise with it and cell cycle blocked. That would make cells having enough time to be repaired and avoid harmful gene spread to next generation. This result indicated that α-pinene induced cell cycle arrest might be related to “ATM-p53-p21-CDK1 pathway” [
To conclude the study, we could affirm that the inhibiting effect of α-pinene on SGC-7901 cells could be mediated by ATM signal pathway preliminary, but some mechanisms for further study are suggested, our researches provide experimental basic for future studies on α-pinene that induce the cell cycle arrest of tumor cells.
Innovation Fund of National Science Foundation of China (Project Number: 81201568).