Broad Spectrum Anti-tumor Activity Screening Of Traditional Chinese Medicine And Preliminary Mechanism Study Of The Hit Components

Objectives:To screen out traditional Chinese medicine extraction with broad spectrum of anti-tumor activityTo demonstrate the possible preliminary mechanism of anti-tumor activityMethods:1.To screen the traditional Chinese medicine with broad spectrum of anti-tumor activity:MTT assay was used to evaluate the in vitro inhibition effect of traditional Chinese medicine extracts on different kinds of tumor cells.1)Firstly,two lung cancer cell lines NCI-H1975 and PC-9/ZD were explored as biological evaluation platform.According to inhibition?>50%?,30 out of 102 traditional Chinese medicine were selected.2)The selected 30 extractions were further examined the anti-proliferation via a range of concentrations.And 5 of them were chosen for the next evaluation.3)Finally,one or two hit extracts were identified via evaluating on 9 tumor cell lines,including lung cancer cells NCI-H1975,PC-9/ZD,HCC827,A549,skin cancer cells A431,colon cancer cells SW620,liver cancer cells HepG2,prostate cancer cells PC-3,blood cancer cells Karpas299.2.Moreover,to study the anti-tumor mechanism of the hit extracts,a vary of experiment was perfomed.4)To study the effect of hit extracts on cell cycle and apoptosis through Propidium iodide?PI?staining with flow cytometry.5)To study the cell apoptosis effect of hit extracts by Annexin V-FITC/Propidium iodide?PI?staining with flow cytometry.6)To explore the effect of hit extracts on cell migration by scratch repair and transwell migration models.7)To explore the expression of apoptosis related protein Bcl-2 in hit extracts by Western blotting assay.8)To investigate the effect of hit extracts on the phosphorylation of EGFR,c-MET and downstream signal transduction pathways key molecular Akt and Erk1/2 by Western blotting assay.Results:1.To screen the traditional Chinese medicine extracts with broad spectrum of anti-tumor activity:1)102 traditional Chinese medicine extract were selected to evaluate the anti-proliferation on NCI-H1975 and PC-9/ZD at 250?g/mL or 500?g/mL.After incubation for 48 hours,in vitro,32 of 102 extracts were selected based on the inhibition?above 50%?.2)To further evaluate the anti-proliferative activities(IC₅₀)of the 32 extract,an array of concentrations were set.And 5 of them were chosen with an excellent IC₅₀ value.IC₅₀ of NP-23,NP-39,NP-49,NP-50 and NP-103 extracts on NCI-H1975 respectively was 0.03,30,15,27 and 17?g/mL;IC₅₀ of NP-23,NP-39,NP-50 and NP-103 extracts on PC-9/ZD respectively was 0.012,22,29 and 23?g/mL.3)To explore the anti-tumor activities on different cancer cell lines,in vitro,the selected5 extracts were tested on 9 tumor cell lines,such as lung cancer cell lines NCI-H1975,PC-9/ZD,HCC827,A549,skin cancer cell line A431,colon cancer cell line SW620,liver cell line HepG2,prostate cancer cell line PC-3,blood tumor cell line Karpas299.The Results showed that the five extracts exhibited good anti-proliferation inhibition.Among them,NP-23 displayed excellent proliferation inhibition and broad spectrum anti-tumor activity in vitro.Next,we will further study the possible mechanism of NP-23 and its antitumor activity.2.Preliminary mechanism study of the hit extract NP-23:4)NP-23 displayed its anti-tumor activity through blocking the NCI-H1975 cell cycle:when separately incubated with 1,2,5 and 10?g/mL of NP-23 for 72 hours.The cell cycle changes of NCI-H1975 were investigated by PI staining with flow cytometry.Cells rate in G1phase was inhibited by NP-23 with 40.84%,44.09%,55.55%and 56.78%,respectively.These findings suggested that NP-23 displayed its anti-tumor activity by blocking cellular DNA synthesis of NCI-H1975 cell.5)Inducing cell apoptosis displayed an important role in anti-tumor activity mechanisms of NP-23.?1?Negative control?DMSO?group and NP-23 20?g/mL group on NCI-H1975 cell were performed for 72hours,and then the cell apoptosis of NCI-H1975 was studied by PI staining with flow cytometry.Percentage of apoptotic peak increased to 83.9%in NP-23 group from 4.07%in the negative control group.Results indicated that high concentrations of NP-23displayed anti-tumor activity through inducing the apoptosis of NCI-H1975 cells.?2?Negative control?DMSO?group,positive control afatinib?0.5,1,and 5?g/mL?group and the NP-23?0.5,1,and 5?g/mL?group on NCI-H1975 cell were performed for 72 hours,and the cell apoptosis of NCI-H1975 was studied by Annexin V-FITC/Propidium iodide?PI?staining with flow cytometry.Cell apoptosis rate of Negative control group was 10.63%;while those of NP-23?0.5,1,and 5?g/mL?group were increased to 16.13%,56.90%and 98.07%;and cell apoptosis rates of afatinib?0.5,1,and 5?g/mL?group were increased to 23.63%,63.76%and 96.93%,respectively.Results suggested that NP-23 can induce cell apoptosis and positively correlated with the concentration trends of NP-23;and the trend in inducing apoptosis showed no significantly difference between the NP-23 groups and positive control group?afatinib?.6)NP-23 can inhibit cell migration.After dosing for 48 hours,the cell-covered rates?%?were 53.53 and-4.23 on NP-23 groups?0.25 and 0.5?g?,respectively.In transwell migration studies,migrating cell number decreased with the increasing concentration of NP-23;and migrating cell number increased as times went on.Results suggested that NP-23 at the concentration of 0.25?g/mL and aboves could significantly inhibit NCI-H1975 cell migration;and the cell number of migration showed dose-related decline.7)NP-23 significantly inhibited the phosphorylation of EGFR and c-Met.At the same time,the expression of Akt and Erk1/2 of the downstream signal transduction pathway was also decreased.The negative control?DMSO?group and NP-23?1,2 and10?g/mL?groups were designed.After NCI-H1975 cells starved for 1 hour,collected protein samples dosing for 6 hours.Western blotting was used to detect the expression of target proteins.Expression of phosphorylation of EGFR,c-Met and Erk1/2 in NP-23?1,2 and 10?g/mL?groups were significantly lower than that in the negative control group;while the expression of p-Akt in high concentration of NP-23?10 g/mL?group was down regulated significantly.8)NP-23 may display an important role in apoptosis by inhibiting the expression of anti apoptotic protein Bcl-2 and Bcl-xl.7).The expression of anti apoptotic protein Bcl-2 in high concentration of NP-23?10 g/mL?group was significantly lower than that of negative control group.The effect of the expression of anti apoptotic protein Bcl-xl was not obvious.Conclusion:NP-23 has broad spectrum anti-tumor activity in vitro.It has been proved that cell migration can be inhibited obviously.Moreover,NP-23 inhibits the expression of apoptosis protein Bcl-2 and induces apoptosis arrest of tumor cells.In addition,NP-23inhibits the phosphorylation of EGFR and c-Met,which affects the phosphorylation of key molecules Akt and Erk1/2 in downstream signaling pathways,and displays a role in cell proliferation inhibition.In conclusion,NP-23 has obvious anti-tumor activity and displays an important role in cell proliferation,apoptosis,cycle and migration.Through optimized separation and structural modification,it is expected a candidate with excellent antitumor activity could be discovered and make a basis for the further drug discovery.