The Rearch Of 20(S)-Protopanaxadiol Induced Apoptosis And Inhibited P13K/AKT Pathway In NSCLC A549 Cells

In present, there is a trend that morbidity and mortality of lung cancer were increased. Non-small cell lung cancer(NSCLC) accounts for appoximately 80%~85% of all lung cancers. And the 5-year survival rate of patients with NSCLC was only 14%. The majority of the dignosed patients with NSCLC died of side effect of radiotherapy and chemotherapy. Therefore, there is an important strategy in searching for safe, effective and low-toxical anti-cancer drug from natural compound.Increasing evidence demonstrated that 20(S)-Protopanaxadiol (PPD), a saponin ginsenosides, isolated from Panax quinquefolium L, exerted cytotoxic effects in broad-spectrum anti-cancer. The study was to invesigated that PPD has a significant anti-cancer effect in vivo provided by nation 95 tackle key problems in science and technology(contract numbers: 96-901-01-83). And the results have gained invention patent: An anti-cancer drugs and application of effective constituent of 20(S)-Protopanaxadiol(ZL02146549.5), and alienated to Hainan Asia Pharmaceutical Sciences Company Limited, called Yijinsheng Capsule against malignant tumor. In present, more and more investigations were shown that PPD could induce many tumor cells apoptosis derived from human, especially for NSCLC A549 cells. However, the detailed mechanism of PPD induced apoptosis has no been reported to this day. The disproportion of PI3K/AKT pathway has intimated related with occurrence, development, metastasis and prognosis. Because appoximately 80% of NSCLC has a feature of abnormal AKT activation, there was a important and worthy study that the mechanism of PPD induced A549 cells apoptosis and inhibited PI3K/AKT pathway. The study provided not only a new route to anti-tumor mechanism but also a new target to therapy of lung cancer.Purpose:This study was to investigate that PPD induced the apoptotic mechanism and inhibited PI3K/AKT pathway.The main methods of this study were as follows:(1) The phase-contrast microscope, Hoechst 33342 and AO/EB fluorescence stain were performed to identify morphology of A549 cells apoptosis and apoptotic body. And DNA agarose gel electrophoresis was determined to DNA gragmentation and identify the specificial biochemistry change of A549 cells apoptosis.(2) MTT assay, PI single stain and Annexin V-PI double stain were performed to determined IC50 value, apoptosis rate, mitochondrial membrane potential and reactive oxygen species.(3) Western blot and QPCR method were determined the rate of Bax / Bcl-2, the release of Smac, Cyt c and AIF, and the cleave of Caspase-3, -9 and PARP fragment.The spectrophotometric method was determined activity of Caspase-3 and Caspase-9.(4) AKT phosphorylation, AKT upstream of PTEN and PDK1, and AKT downstream of GSK-3βand c-Raf phosphorylation were detected byWestern blot.(5) A549 cells treated with PPD and LY294002, survival rate and apoptosis were detected by MTT assay and PI single stain.(6) A549 cells were transiently transfected with constitutively active Akt, survival rate and apoptosis were detected by MTT assay and flow cytometric analysis.(7) p44/42, JNK/SAPK and p38MAPK phosphorylation were detected by Western blot.The main results of this study were as follows:(1) PPD could induce NSCLC A549 cells apoptosis in a dose- and time-dependent manner. IC50 value was 20.15μM. (2) The side cells were increased, cell nucleus were high pyknosis and agglutination, and apoptotic bodies were appeared by Hoechst 33342 fluorescent staining assay. The capacity of cells became smaller. The apoptotic cells had a unregular nuclear with yellow green fluorimetric stain, and side cells increased, the brink of cellular membrane became rough. In summary, PPD could induce A549 cells apoptosis in morphology.(3) PPD treatment significantly increased the apoptotic sub-G1 fraction in dose- and time-dependent manner (P<0.01) by PI single stain. And PPD could induce G0/G1 block. Annexin V and PI double stain was shown that PPD induced cell apoptosis owe to earlier apoptosis.(4) PPD induced cells DNA gragmentation, and DNA agarose gel electrophoresis was shown trapezoid ecletrophoretogram.(5) PPD could decrease the mitochondrial membrane potential, the retention signal intensity was weakened in time dependent manner, and the reactive oxygen species was increase (P<0.01).(6) Caspase-3 and Caspase-9 activity were markedly increased in A549 cells after exposure to Ppd in time- and dose-dependent manner.(7) PPD induced A549 cells apoptosis accompany with activation of Caspase family in turn. Meanwhile, we determined that Ppd activated caspase-3 resulting in PARP cleavage fragment, accord with activity of Caspase.(8) PPD could down-regulate the rate of Bcl-2/Bax, increase mitochondrial membrane permeability, and released Cyt c, Smac and AIF from mitochondrial membrane to cytoplasm.(9) Treatment with PPD caused a initial dose- and time-dependent down-regulation in the levels of the phosphorylated form of Akt (Ser 473). The level of Akt was not affected by the PPD treatment.(10) AKT upstream of PDK1 and AKT downstream of GSK-3βwere inhibited by PPD. And PPD could weaken inhibition of GSK-3β. However, PTEN phosphorylation was not detected in whole test. These results showed that A549 cells have many features, include PTEN deletion, mutation, inherent silence and abnormal AKT activation.(11) Treatment with PPD and LY294002 significant promoted the cells growth inhibition and apoptotic sub-G1 fraction, compare with treatment with PPD or LY294002 alone. The result showed that inhibition of PI3K can enhance anti-cancer effect of PPD.(12) PPD-induced cells apoptpsis was markedly inhibited in A549 cells transfected with pECE-Myr-HA-AKT. The result showed that PPD induced cells apoptosis by inhibiting PI3K/AKT signal transduction pathway.(13) The signal transduction pathway of p44/42 and JNK MAPK could take part in the process of PPD induced cells apoptosis.The main conclusion of this study was as follows:(1) PPD could inhibit A549 cells growth and induce cells apoptosis in dose- and time-dependent manner.(2) PPD could decrease mitochondrial membrane potential by down-regulate the rate of Bcl-2/Bax. Then Cyt c, Smac and AIF were release from mitochondrial membrane to cytoplasm and promote the activation of Caspase family in turn. The mechanism of PPD induced A549 cells apoptosis could relate with increase of reactive oxygen and dependent mitochondria.(3) PPD induced A549 cells apoptosis by inhibiting of PI3K/AKT signal transduction pathway.(4) The signal transduction pathway of p44/42 and JNK MAPK could take part in the process of PPD induced cells apoptosis.