Apoptosis Induced By Pseudo-G-Rh2 In Human Lung Adenocarcinoma A549 Cells Based On The Ras/Raf/ERK/p53 Signal Pathway

Pseudo-G-Rh2(P-G-Rh2) is a new compound which Protopanaxadiol transform into, its structure is similar to Ginsenoside Rh2. Previous studies in our laboratory found that P-G-Rh2 inhibites thw growth of many human tumor cells a, it can active the cascade of Caspase family with mitochondrial pathway activation and induce apoptosis of human gastric cancer SGC cells, but the inhibitory effect on human lung adenocarcinoma and its mechanism has not been reported.Object:To investigate the molecular mechanism of P-G-Rh2 inducing human breast cancer A549 cells apoptosis based on Ras/Raf/ERK/p53 signaling pathway.Methods : A549 cells were incubated with different concentrations of pseudo-G-Rh2(the final concentration of was 25, 35, 45, 55, 65μg/ml) and cytotoxicity was evaluated by MTT assay. Occurrence of apoptosis was detected by DAPI and Annexin V-FITC/PI double staining. The apoptotic ratio, mitochondrial transmembrane potential and ROS in P-G-Rh2 treated A549 cells were measured by flow cytometry. Expression of the Bcl-2, Bax, Caspase family protein, expression of Ras/Raf/ERK/p53 signaling pathway relative protein Ras, p-Raf, p53 and expression of the MAPK protein ERK, p38, JNK were determined by Western blot analysis.Results:1. P-G-Rh2 dose-dependently inhibited cell proliferation in A549 cells, with IC50 values of 46.56 μg·m L-1, 40.79 μg·m L-1 and 37.75μg·m L-1 at 24 h, 48 h and 72 h;2. A549 cells treated with P-G-Rh2 presented typical apoptosis, as observed by morphological analysis in cell stained with DAPI;3. The percentages of Annexin V-FITC positive cells were 2.65%, 4.44%, 14.16% and 48.56% in A549 cells treated with 0, 15, 30 and 60 μg·m L-1 of P-G-Rh2, respectively;4. P-G-Rh2 could induce an increase of intracellular ROS level and mitochondrial membrane potential loss, from(91.45±1.67) % to(74.62±2.86) %;5. P-G-Rh2 could up-regulate Bax expression and down-regulate Bcl-2 expression. These events paralleled activation of caspase-9,-3;6. Induction of apoptosis was also accompanied by sustained phosphorylation and activation of Ras, Raf, ERK and p53.Conclusion:1. P-G-Rh2 significantly inhibits A549 cells proliferation and induced apoptosis in a dose-dependent manner;2. P-G-Rh2 decreases the level of Bcl-2 expression, increases the level of Bax expression, regulates Bax/Bcl-2 ratio to decrease mitochondrial membrane potential, activates Caspase protein family, and induces A539 cells apoptosis through mitochondrial pathway at protein level;3. P-G-Rh2 could induce an increase of intracellular ROS level, mitochondrial membrane potential loss;4. P-G-Rh2 induces A549 cells apoptosis by activiting the Ras/Raf/ERK/p53 signal pathway.