Effects And Mechanisms Of Platycodin-D On NSCLC Cell Lines H460 And A549

Objective:Non-small cell lung cancer (NSCLC) accounts for approximately 80% to 85% of lung cancer, which has high degree of malignancy, and the five-year survival rate of its patients does not exceed 15%. Furthermore, about 80% to 90% of NSCLC patients have died of the metastasis of lung cancer eventually. Thus, more effective anti-NSCLC drugs need to be developed for inhibiting the growth and metastasis of NSCLC, so as to improve the survival rate of its patients. Recently, more than 60% of anti-cancer drugs are directly or indirectly derived from natural sources, especially the monomer extracted from the traditional Chinese medicine plays a vital role in the treatment and prevention of cancer. Platycodon grandiflorum has been widely used in traditional Chinese medicine treatment against various pulmonary diseases and respiratory disorders. Platycodin-D (PD), a triterpenoid saponin extracted from Platycodon grandiflorum, is the main effective component of anti-cancer effects of Platycodon grandiflorum. Our preliminary studies have demonstrated that PD had an effective inhibition of NSCLC cell lines H460 and A549 in vitro through high-throughput screening. In this paper, we aimed to investigate its underlying mechanisms focusing on the four aspects including the arrest of cancer cell cycle progression, the induction of cancer cell apoptosis and autophagy, and the inhibition of cancer cell metastasis, in order to provide experimental basis and foundation for further drug development and clinical application of PD.Methods:MTT assay was used to detect the rate of cell proliferation inhibition; Hoechst-33258 fluorescent staining assay was used to observe the nuclear morphological changes of cells; the ultrastructure changes of cells were observed by transmission electron microscopy; Flow cytometry assay was used to analyze the cell cycle distribution and the early apoptosis-induction; Annexin-V/PI/Hoechst-33258 fluorescence staining combined with high content screening was used to analyze cell apoptosis; the levels of LC3-Ⅱ, MMP-2 and MMP-9 mRNA was determined by RT-PCR; cell adhesion assay, wound-healing assay and Transwell chamber migration assay were used to detect the abilities of cell adhesion, migration and invasion respectively; Western-blot assay was performed to determine related protein expression levels of cell cycle, mitochondrial apoptosis pathway, MAPK signaling pathways, PI3K/Akt/mTOR signaling pathway, MMP-2 and MMP-9.Results:1. MTT assay indicated that the cell viability and proliferation was inhibited by PD in H460 and A549 cells in a time-and dose-dependent manner respectively. Flow cytometry results showed, compared with the control group, the number of cell cycle distribution of G2/M and S phase was significantly increased after PD treatment in H460 cells (P< 0.05); the number of cell cycle distribution of G0/G1 phase was significantly increased after PD treatment in A549 cells (P<0.05). The changes of cell cycle related proteins were further detected by Western-blot assay after PD treatment. The results found that the expression levels of G2/M and S phase-related protein Cyclin-E2, CDK2, Cyclin-B1 in H460 cells were down-regulated, and the expression level of cyclin-dependent kinase inhibitor P27 was up-regulated; however, the expression levels of G0/G1 phase-related protein cyclin-D1, cyclin-D3, CDK4 and CDK6 in A549 cells were down-regulated, and the expression level of cyclin-dependent kinase inhibitor PI8 was up-regulated. Meanwhile, PD inhibited the expression levels of E2F1 and p-Rb (Ser807 and Ser795), the downstream cell cycle regulators.2. Hoechst-33258 fluorescent staining assay found the nuclear morphological changes of apoptotic characteristics, such as nuclear shrinkage, chromatin fragmentation, forming a plurality of micronucleus, were observed after PD treatment in H460 and A549 cells, and these changes became more obvious with the increasing concentrations of PD. Flow cytometry detection found that PD increased apoptosis rate of both cell lines in a dose-dependent manner (P<0.05 or P< 0.01). High content screening analysis indicated that average fluorescence intensities of Hoechst-33258, Annexin-V and PI in H460 and A549 cells were increased with the increasing doses of PD (P<0.05), suggesting PD induced apoptosis in both cell lines in a dose-depedent manner. Further detection by Western-blot assay showed that PD induced mitochondrial apoptosis in H460 and A549 cells via increasing the expression levels of Bax, Bim and cleaved-Caspase-3 and decreasing the expression levels of Bcl-2 and Casepase-3 in H460 and A549 cells; moreover, PD inhibited the expression levels of Ras, p-c-Raf and p-Erk protein in the Erk signaling pathway, and this effect may be related to PD-induced apoptosis in H460 and A549.3. After PD treatment the autophagosomes of double-layered membranes were found in H460 and A549 cells by transmission electron microscopy. PD could induce autophagy in H460 and A549 cells via increasing the conversion of LC3-Ⅰ to LC3-Ⅱ and accumulation of Atg-3, Atg-7 and Beclin-1, which was detected by Western-blot assay. Moreover, the expression of LC3-Ⅱ, an ultimate biomarker of cell autophagy, in mRNA level was determined by RT-PCR, and found that PD contributed to up-regulate the level of LC3-Ⅱ mRNA in both cell lines (P< 0.05). We further investigated the effect of PD on the PI3K/Akt/mTOR signaling pathway, and found the expression levels of p-Akt (Ser473), p-p70S6K (Thr389), p-4EBP1 (Thr37/46) in both cell lines were decreased. Furthermore, the data demonstrated that PD-induced autophagy in H460 and A549 was related to the inhibition of PI3K/Akt/mTOR signaling pathway through using insulin (an activator of PI3K/Akt/mTOR signaling pathway), LY294002 (an inhibitor of PI3K) and RAP (an inhibitor of mTOR). We detected the effect of the PD on MAPK signaling pathways, and found p-p38 MAPK and p-JNK were activated by PD in a dose- and time-dependent manner, however, p-Erk1/2 was inhibited by PD. Additionally, we clarified these effects with U0126 (Erk1/2 kinase inhibitor), SP600125 (JNK kinase inhibitor) and SB203580 (p38 MAPK kinase inhibitor), and suggesting PD-induced autophagy in H460 and A549 cells through the activation of p-p38 MAPK and p-JNK, while the inhibition of p-Erk1/2 was not responsible for that.4. PD effectively inhibited the abilities of cell adhesion, invasion and migration in H460 and A549 cells in dose-dependent manner (P<0.05). PD reduced the levels of MMP-2 and MMP-9 mRNA in H460 and A549 cells (P<0.01); meanwhile, PD down-regulated the expression levels of MMP-2 and MMP-9, and inhibited the expression of its upstream proteins Ras, p-c-Raf, p-Erkl/2 and p-Akt in a time-and dose-dependent manner.Conclusion:1. PD inhibited the cell proliferation and viability effectively. In addition, PD triggered G2/M and S phase arrest in H460 cells, as well as GO/Gl phase arrest in A549 cells.2. PD induced apoptosis through the mitochondrial apoptotic pathway, and the PD-induced apoptosis may be related to the inhibition of Erk signaling pathway in H460 and A549 cells.3. PD induced autophagy in H460 and A549 cells through inhibiting PI3K/Akt/mTOR signaling pathway and activating JNK and p38 MAPK signaling pathways.4. PD inhibited cell adhesion, invasion and migration in NSCLC cells, and these effects were related to down-regulate the expression of MMP-2 and MMP-9 both in mRNA and protein levels, and inhibite its upstream expression of Erk signaling pathway and p-Akt.