The Inhibitory Effect And Mechanism Of Aqueous Extract Of Radix Astragali And Calycosin On Lung Cancer

ObjectiveLung cancer is one of the malignant tumors with the highest morbidity and mortality rate in the world.And it owns the greatest threat to human society among all cancers.More and more studies have found that Traditional Chinese Medicine has a non-negligible role in the prevention and treatment of lung cancer.Chinese medical science generally recognizes Traditional Chinese Medicine as an adjuvant treatment of lung cancer.Rudix Astragali is used as medicine for thounds of years.It is reported has the function of enhancing human body's immune system,protecting liver,diuresis,anti-aging and anti-tumor effect.Radix Astragali is always alone or in combination with other Traditional Chinese Medicines used im various types of advanced cancer patients to improve the life quality of patients.In recent years,studies have reported that Rudix Astragali has anti-tumor effect in vivo and in vitro.But the anti-lung cancer effect and mechanism of Radix Astragali on lung cancer cells A549 and H1299 in vitro is little reported.Based on the above information,this paper aimed to study the anti-lung cancer cffect and mechanism of aqueous extract of Radix Astragali and its active ingredient calycosin on lung cancer.This study might provide a theoretical basis for the further development and utilization of Radix Astragali against lung cancer.Methods1.Effect of Radix Astragali aqueous extract and calycosin on cell viabilityMTT assay was used to evaluale cell viability.A549 and H1299 cell lines were selected as in vitro models.We used MTT assay to detect the effect of different concentrations of Rudix Astragali aqueous extract(0?1000 mg/mL)and calycosin(0?200?M)on A549 and 111299 cells for 48 h.We calculated IC50 using GraphPad Prism 5.0 software according to the results.Then we selected the appropriate drug concentrations for our next study.2.Effect of Radix Astragali aqueous extract and calycosin on cell proliferation and migrationClony formation assay was used to evaluate the clone formation ability of A549 and H1299 cells.EdU assay was used to further confirme the proliferation of A549 and H1299 cells.Flow cytometry was performed to investigate the cell cycle effect of Radix Astragali aqueous extract and calycosin on A549 and H1299 cells.Wound healing assay was used to assess the effect of Radix Astragali aqueous extract and calycosin on the migration of A549 and H1299 cells.3.Effect of Radix Astragali aqueous extract and calycosin on the key proteins of PI3K/Akt signaling pathwayWe used Western blot assay to evaluate the protein expression levels of the key proteins of PI3K/Akt signaling pathway.After treated with Radix Astragali aqueous extract and calycosin for 48 h,total proteins were extracted and the protein expressions of PI3K,AKT,p-AKT,p-PDK1,GSK3? and p-GSK3? in A549 and H1299 cells were detected.4.Effect of Radix Astragali aqueous extract on lung cancer in miceA benzopyrene-induced lung cancer model of A/J mice was established.Then low-dose and high-dose Radix Astragali aqueous extract were administered to mice.After 30 weeks,lung tissue was taken and the number and size of lung nodules in mice were recorded.5.Statistical analysisData were analyzed using SPSS 19.0 statistical software.One-way ANOVA was used to compare statistical significance of the data and p<0.05 was considered statistically significant.Graphs were presented using GraphPad Prism 5.0 software.The statistical results are expressed as Mean ±SD.Results1.Radix Astragali aqueous extract and calycosin could inhibit the cell viability of A549 and H1299 cellsAfter 48 h administration of Radix Astragali aqueous extract and calycosin,the viability of A549 and H1299 cells was inhibied.According to the results of MTT experiments,the concentrations of Radix Astragali aqueous extract and calycosin were determined.Radix Astragali aqueous extract were 0,6.25,12.5 and 25 mg/mL,and calycosin were 0,25,50 and 100 ?M.2.Radix Astragali aqueous extract and calycosin could inhibit the proliferation of A549 and H1299 cellsThe results of clony formation assay showed that the the clone formation ability of A549 and H 1299 cells was significantly inhibited by drugs.And the proliferation ability of A549 and H 1299 cells was significantly inhibited by Rudix Astragali aqueous extract and calycosin according to EdU assay.From the result of flow cytometry,the cell cycle of'A549 and H1299 cells was arrested in S phase by Radix Astragali aqueous extract,and was arrested in G0/G1 phase by calycosin treatment.3.Radix Astragali aqueous extract and calycosin could inhibit the migration of A549 and H1299 cellsThe migration ability of A549 and H1299 cells was significantly inhibited by Radix Astragali aqueous extract and calycosin according to wound healing assay.4.The effect of Radix Astragali aqueous extract and calycosin on PI3K/Akt signaling pathwayAccording to Western blot assay,we found that Radix Astragali aqueous extract could down-regulate the protein levels of PI3K,p-PDK1?p-Akt and p-Akt/Akt ratio level,as well as up-regulate the expression level of p-GK3?,while the level of PI3K,p-PDKI,Akt,p-Akt,GSK3? and p-GSK3? proteins were not signif icantly influenced by calycosin.5.Radix Astragali aqueous extract could inhibit lung tumor growth in miceThe in vivo experiment of benzopyrene-induced lung cancer model result showed that,compared with the model group,both low-dose and high-dose Radix Astragali aqueous extract could significantly inhibit the number and size of lung tumors in mice.Which to some extent reflected the inhibitory effect of Radix Astragali aqueous extract on lung cancer in vivo.ConclusionsThis study aimed to explore the inhibitory effect and mechanism of aqueous extract of Radix Astragali and calycosin on lung cancer.The results of in vitro experiments showed that Radix Astragali aqueous extract could inhibit the proliferation and migration of A549 and H1299 cells and arrest the cell cycle in S phase by regulating PI3K/Akt pathway.Invivo experiment showed that Radix Astragali aqueous extract could inhibit the growth of lung tumor in mice.Therefore,this study could provide a theoretical basis for the further development and utilization of Radix Astragali against lung cancer.Furthermore,calycosin could inhibit the proliferation and migration of lung cancer cells,but calycosin could not regulate PI3K/Akt signaling pathway,so the mechanism remained to be further studied.