| Lung cancer has developed into the world’s highest incidence and mortality rates of cancer. Most of lung cancer patients already advanced at the time of diagnosis. Invasion and metastasis are the main factors for lung cancer treatment failure and relapse. So research on the material basis and mechanism of a selected compound that inhibits the invasion and metastasis of lung cancer helps to understand the scientific connotation. This topic is based on the overall concept of traditional Chinese medicine and the "structural components" theory. Astragalus was selected to be the model medicine through the anti-tumor activity screening. Coupled with cell immobilization chromatography, the active components were isolated and identified. The effect of components on A549 cell migration and invasion was evaluated and the mechanism was studied. In vivo metabolism profile was studied subsequently. A tumor targeting nanomicelles was prepared and evaluated with physical characterization, drug release, cellular uptake, in vivo image and in vivo anti-cancer assessment. This research provides a scientific basis for revealing the material base of Astragalus against cancer migration and invasion. The main contents include the following sectionsScreening of anti-cancer drugsThe anti-caner effect of Jinfukang oral liquid and its fellowship were evaluated with tumor beared mice. The results indicated that the oral liqid with good tumor inhibition activity. Astragalus among the fellowship showed the strongest anti-tumor effect. "When Astragalus was knocked out, the effect of oral liquid composed with orther medicine reduced significantly.Astragalus showed inhibitory rate more than 30% repeatedly among herbs screening.Cell immobilized chromatography researchWe established A549 cell membrane immobilized chromatography coupled with UPLC-q-TOF-MS2. The effective compounds immobilized on cell membrane was identifed. The effect of different times, different concentration on cell immobilization was compared and resulted with concertration:2x10-4 mg/mL for 60 min incubation. Six components of astragalus were immobilized and identified namely:calycosin-7-O-glucoside, thorn formononetin-7-O-glucoside, calycosin, formononetin, Astragaloside IV, Astragaloside II.Cell invasion and metastasis of lung cancer suppressed by Astragaloside IVAstragaloside Ⅳ inhibited the proliferation of A549 cells and cell adhesion. Astragaloside IVsuppressed cell invasion and metastasis in wound healing, transwell experiments. Astragaloside IV restrained inflammatory factors TGF-β1，TNF-a, IL-6 levels in cell supernatants, which may affect tumor cell microenvironment. Astragaloside Ⅳ inhibit MMP-2, MMP-9, Integrin β1 increased protein expression of E-cad, enhanced cell adhesion, reducing metastasis. Signaling pathway proteins showed Astragaloside IV could inhibit PKC-a (membrane), p-ERK1/2, NF-κB protein expression achieve inhibit migration and invasion of A549 cells. Astragaloside Winhibited translocation of PKC-a to cell membrane. Along with AEB071, Astragaloside Ⅳcan inhibit PKC-a expression of A549 more and furtherly inhibition on MMP-2, MMP-9 and Integrin β1 expression levels while increasing the level of E-cad expression, suppression A549 cell invasion and metastasis. Astragaloside IV inhibiting ERK1/2 phosphorylation, combination with U0126, Astragaloside IV can inhibit A549 of ERK1/2 phosphorylation, further inhibit MMP-2, MMP-9 and Integrin β1 expression levels while increasing levels of E-cad expression, suppressing A549 cell invasion and metastasis. Astragaloside combined with PDTC can inhibit the A549 of NF-κB (p65) expression, further inhibit MMP-2, MMP-9 and Integrin β1 while increasing E-cad expression levels, inhibit A549 cell invasion and metastasis. The results confirmed that Astragaloside IV inhibited inhibited A549 cell invasion and metastasis by PKC-α-ERK1/2-NF-κB signaling pathway.Suppression of cell invasion and metastasis of lung cancer by calycosinCalycosin inhibit the proliferation of A549 cells, increased apoptosis of A549 cells. Statistical results showed that apoptosis control group was 3.44%,20μM,30μM and 40μM calycosin enhance apoptosis A549 from 23.39%,33.61% and 43.77%. Calycosin inhibited on TPA-induced A549 cell adhesion, migration and invasion. Calycosin suppressed Integrin β1, LnR, MMP-2 and MMP-9 levels, increased levels of E-cadherin, inhibited metastasis of A549 cells. PKC-a protein expression of A549 cells after TPA-induced was significantly increased, Calycosin inhibited TPA-induced ERK1/2 phosphorylation activation. Calycosin combined with AEB071 can inhibit TPA-induced overexpression of PKC-a, and further inhibit MMP-2, MMP-9, and Integrin β1 LnR expression levels while increasing E-cad expression levels, inhibit A549 cell invasion and metastasis. Calycosin combined with PD98059 can inhibit TPA-induced p-ERK1/2 overexpression, further inhibit MMP-2, MMP-9, and Integrin β1 LnR expression levels while increasing E-cad expression levels, inhibit A549 cell invasion and metastasis. The results confirmed that calycosin inhibited TPA-induced A549 cell invasion and metastasis by suppressing PKC-a-ERK1/2 signaling pathway.Astragaloside Ⅳ metabolism in vivo studiesEstablished UPLC-Q-TOF-MS/MS method for detecting applied orally plasma, bile, urine and feces samples metabolite Astragalus rats. A total of 22 metabolite detection and identification, and of which 15 were reported for the first time. Astragalus major metabolic pathway in rat Astragaloside IV is deglycosylation, vulcanization and glucuronidation.Study on Carycosin nanomicelles preparationThe prepared calycosin micelles with average particle size 111.91±5.82nm. PDI 0.11± 0.03, encapsulation efficiency 89.54 ± 1.49%. TPGS contained micelles in PBS 0.1% Tween 80 (pH 7.4) solution of 120 h to release 44.88 ± 4.94% compared with 90.95 ± 3.78% within 24 hours of free calycosin.Cellular uptake experiments showed micelles containing TPGS model can deliver drugs into tumor cells, an effect. The results also confirmed that cytotoxicity of calycosin micelles was better in cell proliferation than free micelles. In vivo imaging results showed that DiR micelles showed good tumor targeting properties. The fluorescence of DiR remains strong in DiR micelles. after administration, free DiR showed ineffective targeting, showing the distribution of the whole body shape, and eliminated within 24h. The results show that TPGS micelles containing a high tumor targeting efficiency.Tumor-bearing mice administrated with calycosin micelles, had tumor volume significantly reduced compared with the control group, but also less than the free calycosin groups, more than cisplatin group. Free calycosin treatment group, the median survival (median survival,12 days) than the control group (median survival,10 days) of mullein isoflavone micelles survival was significantly higher higher than cisplatin group. Free calycosin, calycosin micelles, the inhibition rate of cisplatin group were 34.03%,67.39%, and 76.97%, indicating calycosin micelles can significantly improve calycosin anti-tumor effect. Mice body weight of cisplatin decreased significantly (p<0.05), weight calycosin group and calycosin micelles group did not change significantly. The results from the in vivo anti-tumor tests showed that the micelles calycosin effectively improve the anti-tumor efficacy and safety of treatment of tumor-bearing mice.In summary, the active compound of Astragalus was selected and identified along with drug screening and cell immobilization experiments. These chemicals showed activity oncancer cell invasion and metastasis inhibition. The pharmaceutical preparation design indicated that with nanotechnology the preparation showed more effects and targeting distribution. This study revealed antitumor mechanism of astragalus at least partly.. |
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