Inhibition Of Ophiopogon Saponin Steroidal Compounds On NSCLC Cell Lines And Its Mechanism

Lung cancer is divided into two types:small cell lung cancer and non-small cell lung cancer (NSCLC). And NSCLC accounts about75%of lung cancer. In NSCLC, adenocarcinoma and squamous carcinoma are the most common types, accounting for more than80%of NSCLC.Chinese medicine has unique advantages on stabilizing the tumor, reducing the clinical symptoms, preventing relapse, and improving the quality of life during the treatment of cancer. Chinese medicine has became an essential component in comprehensive cancer treatment. But its mechanism remains to be further clarified, and scientific application remains to be further improved.Mai men dong and Qian jin wei jing decoction which comes from Golden Chamber (Jin) and "Ke Jinyan Prescription" which comes from State Medical Master Zhou Zhongying are both verified to have significant inhibition effect on Lewis lung cancer or suppressed the growth of NCI-H157or A549transplanted lung tumor in nude mice. Radix Ophiopogonis is an important component in these two prescriptions. After screening at the cellular level, we found that ophiopogonins were the main components inhibiting the NSCLC cell proliferation. However, further investigation still needed to be performed to find out the main cytotoxic components and its inhibition mechanisms on different subtypes of NSCLC cell lines.In this paper, the commonly over-activated signaling pathway PI3K/Akt/mTOR in NSCLC was chosen as a targeted pathway to study the different pharmacological effects and mechanisms of different Ophiopogonins on NSCLC cell lines, and to provide foundation for clinical research and develop molecular targeted therapies. The details of work were as follows:1Anti-NSCLC screening of Ruscogenin、ophiopogonin B (OP-B)、 ophiopogonin D (OP-D) Through investigating the effect of the three Ophiopogon saponin steroidal compounds on cell cycle or the cell survival pathway PI3K/Akt/mTOR in NCI-H157cell line, we found that OP-D and Ruscogenin had much less effects on cell cycle or cell survival pathway. But OP-B significantly inhibited the pathway and induced cell cycle arrested in G0/G1phase. The results showed that OP-B was the main cytotoxicity compound among the three Ophiopogon saponin steroidal compounds.2Investigation of the pharmacological activity of OP-B on NCI-H157and H460cells Further investigation of OP-B in NCI-H157and H460cell lines demonstrated that the cells treated with OP-B grew more slowly and accumulated vacuoles in their cytoplasm compared to untreated cells. However, expression of caspase-3and Bcl-2, detected by western blot, nuclear morphology (stained by Hoechst33258), and fluorescence intensity (labeled by AnnexinV/PI/Hoechst33258) detected by high content screening (HCS) Kinetic Scan Reader all showed that OP-B did not induce apoptosis or necrosis in either cell line. Instead, both the results of transmission electron microscopy (TEM) and the expression of microtubule-associated protein1light chain3-II (LC3-II) determined that OP-B treatment induced autophagy in both cell lines. Next, the PI3K/Akt/mTOR signaling pathway were examined and the results showed that OP-B inhibited phosphorylation of Akt and p70S6K in a time and dose-dependent manner in both cell lines. And the inhibition on them was more specific than LY294002, the inhibitor of PI3K, or Rapmycin, the inhibitor of mTORCl. Additionally, insulin-mediated activation of the PI3K/Akt/mTOR pathway provided evidence that inhibition of this pathway may correlate with induction of autophagy in the cells.3Apoptosis and autophagy induction of OP-B on A549cells Adenocarcinoma has the highest incidence in NSCLC and the highest mortality in clinic. Herein, we found that in lung adenocarcinoma cell line A549, OP-B induced autophagy following apoptosis in it through inhibiting phosphorylation of Akt and p70S6K in PI3K/AKT/mTOR signaling pathway. Beclinl SiRNA interference experiments showed that autophagy was the key reason of the induction of apoptosis.4Investigation of the difference of the regulation on cell cycle in H460and A549cells Further investigation of the different pharmacological effect of OP-B on these cell lines showed that OP-B had different regulation on cell cycles in them. Otherthan in H460cell line, OP-B induced cell cycle arrested in S and G2/M phase in A549cell line mainly through increasing p27and p21, the inhibitor of cell cycle progression.5The effects of OP-B on cell invasion and migration on A549in vitro Moreover, transwell and cell scratch experiments showed that OP-B also inhibited the migration and invasion of A549cells in vitro. The regulation on cell invasion was correlated with the inhibition on p-Akt and EphA2.Conclusion:OP-B is the main cytotoxity components among the three Ophiopogon saponin steroidal compounds. It has different pharmacological effects on different NSCLC cell lines. In NCI-H157and H460cell lines, it mainly induced autophagy, while in A549cells it induced both autophagy and apoptosis. Inhibition of OP-B on PI3K/AKT/mTOR signaling pathway seemed to have no significant difference among three cell lines, but the effects on cell cycle were significantly different among them. In NCI-H157and H460cells, it mainly induced cell cycle arrestted in G0/G1phase, while, in A549cells, it induced cell cycle arrested in S and G2/M phase. Furthermore, OP-B also inhibited cell invasion and migration in A549cells, which mean OP-B was more sensitive to A549cell line.