The Study Of Anti-lung Cancer Effect And The Mechanism Of Polyphylla ? And The Pre-formulation Study On It

Objective:Lung cancer is one of the most common malignant tumors,the rate of its morbidity and mortality rank the first among cancers around the world.With the development of NSCLC,it's easily for cancer cells to invade and migrate,which is the main reason that lung cancer is hard to cure.Currently,the therapy for lung cancer is deficient,and it is imperative to develop new effective anti-lung cancer drugs to improve the survival rate and life quality of the patients.Rhizoma Paridis is a well-known Traditional Chinese medicine?TCM?with heat-clearing and detoxicating function,which has been used for anti-cancer more than one thousand years.Modern pharmacological studies have showed that Rhizoma Paridis has a variety of pharmacological activities,including anti-tumor,anti-bacterial anti-inflammatory,tranquilizing and so on.Steroidal saponins is the main pharmacological active ingredients of it.Nowadays most researches are focused on polyphylla?,polyphylla?and polyphylla?,actually,there are many other steroidal saponins in Rhizoma Paridis also have strong anti-tumor activities.In addition,most of the relevant papers are about the apoptosis and autophagy of cancer cells induced by steroidal saponins;but the effect of steroidal saponins on the invasion and metastasis of cancer cells and the involved mechanism has not been widely reported.Based on the possessed literature and previous research,we focus on nine kinds of steroidal saponins which can be absorbed into blood and accumulated in lung tissue partially,and the purpose of this study was to investigate the inhibitory effect of these nine steroidal saponins on proliferation,invasion,migration of human lung cancer A549 cells;then discuss the involved mechanism of steroidal saponins with stronger inhibitory effect.Finally,we investigated the equilibrium solubility,stability and oil-water partition coefficient of polyphylla?,lay the foundation on new pharmaceutic and the prosibility for polyphylla?to be a new drug.Method:1.Human lung adenocarcinoma A549 cells were cultured.The growth inhibitory effect of nine kinds of steroidal saponins with different concentrations on A549 cells was detected by MTT assay,and the appropriate concentration and time were selected for the further experiments.2.Transwell invasion assay was performed to detect the invasion inhibitory effect of nine kinds of steroidal saponins with different concentrations on A549cells.3.Wound-healing assay was performed to detect the migration inhibitory effect of nine kinds of steroidal saponins on A549 cells.4.A549 cells were treated with or without Polyphyllin?for 24h,the expression level of MMP-1,MMP-2 and MMP-9 genes of A549 cells were detected by Real-time PCR.5.A549 cells were treated with or without Polyphyllin?for 48h,the expression level of MMP-1,MMP-2 and MMP-9 protein were analyzed by Western blot;the protein related to PI3K/AKT/mTOR signaling pathway were also analyzed by Western blot.6.The equilibrium solubility,stability and oil/water partition coefficient of Polyphyllin?were detected by ultraviolet spectrophotometry.Results:1.After treated with a series of concentration of different steroidal saponins for 24h and 48h,the obviously inhibitory effect was found in A549cells,and the results showed dose-dependent and time-dependent manner.The half inhibitory concentration(IC₅₀)of Polyphyllin?,Polyphyllin?,Polyphyllin?,Polyphyllin?,Polyphyllin?,Polyphyllin?,Polyphyllin H,Gracillin,17-hydroxy diosgenin in A549 cells were 0.65,0.87,1.51,2.13,5.87,2.44,3.05,2.58,8.14?mol/L at 24h respectively;the half inhibitory concentration(IC₅₀)of Polyphyllin?,Polyphyllin?,Polyphyllin?,Polyphyllin?,Polyphyllin?,Polyphyllin?,Polyphyllin H,Gracillin,17-hydroxy diosgenin in A549 cells were 0.52,0.56,1.04,1.57,3.90,1.85,2.60,1.89,6.82?mol/L at 48h respectively.2.All these nine kinds of steroidal saponins suppressed the invasion of A549 cells.Compared to the control group,after treated with Polyphyllin??0.25,0.5?mol/L?,Polyphyllin??0.3,0.6?mol/L?,Polyphyllin??0.5,1.0?mol/L?,Polyphyllin??0.75,1.5?mol/L?,Polyphyllin??2.5,5.0?mol/L?,Polyphyllin??0.75,1.5?mol/L?,Polyphyllin H?1.25,2.5?mol/L?,Gracillin?0.75,1.5?mol/L?,17-hydroxy diosgenin?3.0,6.0?mol/L?for 24h respectively,the number of A549 cells passed through the membrane was significantly decreased,and showed a dose-dependent manner.3.All these nine kinds of steroidal saponins inhibited the migration of A549 cells.Compared to the control group,after treated with Polyphyllin??0.5?mol/L?,Polyphyllin??0.6?mol/L?,Polyphyllin??1.0?mol/L?,Polyphyllin??1.5?mol/L?,Polyphyllin??5.0?mol/L?,Polyphyllin??1.5?mol/L?,Polyphyllin H?2.5?mol/L?,Gracillin?1.5?mol/L?,17-hydroxy diosgenin?6.0?mol/L?for 48h respectively,the wound-healing rate was decreased by 47.90%,55.15%,36.66%,32.43%,62.53%,47.23%,63.72%,50.46%,34.80%respectively.4.The mRNA levels of MMP-1,MMP-2,MMP-9 in A549 cells treated with Polyphyllin?for 24h were detected by real-time PCR,the concentration-dependent down-regulation of MMP-1,MMP-2,MMP-9 were detected in Polyphyllin?treated group.5.The protein levels of MMP-1,MMP-2,MMP-9 in A549 cells treated with Polyphyllin??0.5,1.0?mol/L?for 48h were analyzed by western blot,the concentration-dependent down-regulation of MMP-1,MMP-2,MMP-9 were detected in Polyphyllin?treated group.To further investigate the involved mechanism,we examined the upstream PI3K/AKT/mTOR signaling pathway by western blot,P-PI3K/PI3K,P-AKT/AKT and P-mTOR/mTOR level were significantly decreased by Polyphyllin?in A549 cells.6.The equilibrium solubility of Polyphyllin?were 33.680,21.487,23.371,29.626,32.146,69.001,81.286?g/mL in buffer solutions with pH values of 2.0,3.0,3.8,5.0,6.8,7.0,7.8 respectively.The oil-water partition coefficients were0.116,0.218,0.266,0.288,0.538,0.436,0.493 in buffer solutions with pH values of 2.0,3.0,3.8,5.0,6.8,7.0,7.8 respectively.And the stability test result showed that the Polyphyllin?was stable within 9 h.Conclusion:1.All these nine kinds of steroidal saponins have obviously inhibitory effect on A549 cells,it showed great anti-lung cancer effects in vitro;2.All these nine kinds of steroidal saponins can inhibited the invasion and metastasis of A549 cells;3.Polyphyllin?can effectively inhibiting invasion and metastasis via down-regulation the mRNA and protein level of MMP-1,MMP-2,and MMP-9in A549 cells.4.Polyphyllin?inactivated PI3K/AKT/mTOR signaling pathway in A549cells,which mediated the inhibition of cell invasion and metastasis induced by Polyphyllin?.5.Polyphyllin?is poor solubility and unstability for long time.The equilibrium solubility of Polyphyllin?was higher in weak alkaline condition,and was increased with the increasing of pH values;all the lgP value are less than 1,indicating that the Polyphyllin?can hardly pass the lipid membrance,the fat-solubility is poor,so it's not suitable for using as conventional oral formulation.