Anti-proliferative And Anti-metastasis Effect Of Cardamonin On Lewis Lung Cancer In Vivo And In Vitro Via The MTOR Pathway

1 Background and AimsMammalian target of rapamycin is involved in tumor metastasis. The migration ability of cell increases when mTOR is activited. Therefore, activated mTOR promotes tumor cell to migrate to the surrounding tissue and distant organs. The reduction of cell adhesion is the beginning process of tumor metastasis. E-cadherin is an important member of the adhesion molecules, which maintains the epithelial cell morphology, and reduces the separation of cancer cells. Snail is a zinc-finger protein transcription factor, which combines with E-cadherin promoter and reduces the expression of intracellular E-cadherin. Activated mTOR promotes the phosphorylation of downstream target p70s6K kinase 1 (p70ribosomal s6 kinase, p70s6K1) and transcription of eukaryotic initiation factor 4E binding protein 1 (4E-binding protein-1,4E-BP1), which play an important role in translation of protein and metastasis of tumor. The expression of E-cadherin is inhibited by mTOR/p70S6K through increasing the expression of Snail. In this condition, the adhesion of cancer cells decreases. Many studies have demonstrated that mTOR inhibitor (rapamycin and its derivatives) could inhibite tumor metastasis; however, rapamycin has many adverse reactions, for example, immune suppression, high cholesterol, liver damage and non-infectious pneumonia. It is important to develope a new inhibitor that has fewer side effects.Our previous study had confirmed that cardamonin (CAR) could effectively inhibit the proliferation of human umbilical arterial smooth muscle cells (HUASMCs) which induced by phosphatidic acid (PA). Another experiment confirmed that CAR inhibited the proliferation of human non-small cell lung cancer (A549). Our further explore had found that the mechanism of CAR might realate to the mTOR signaling pathway. The transduction of mTOR signaling pathway and the activity of S6K1 were inhibited by CAR.In this study, we investigate the effect and mechainsm of CAR on the proliferation and metastasis of Lewis lung cancercells in vivo/vitro.2 Methods2.1 Cell cultureLewis lung cancer cells were cultured in DMEM high glucose medium with 10% FBS,100 U·mL-1 penicillin and 100μg·mL-1 streptomycin at 37℃with 5% CO2. Cells were passaged when the monolayer adherent growth to 70%-80%.2.2 ProtocolIn vitroAfter passaged, cells were set up the control group, solvent control group (0.1% DMSO), Rap group, CAR different dose groups; PA stimulated, were given different doses of Rap and CAR. The final concentrations of drug were PA(10-4mol·L-1), Rap(10-7 mol·L-1), CAR(10-4,3×10-5,10-5 mol·L-1,10-6 mol·L-1,10-7 mol·L-1 ).In vivoWe collected the Lewis lung cancer cell with trypsin and resuspended in serum free medium into a 1×107/ml, vaccinated in 48 C57BL/6 mice forelimb armpit with each of 0.2 ml, randomly divided into 6 groups(n=8),,①model group:added normal saline;②positive control group:added rapamycin 1.12 mg·kg-1;③solvent control group(VdH2O: V吐温80: V无水乙醇=72:10:18),④high-dose cardamonin group: 10.5mg·kg-1;⑤middle-dose cardamonin group:7mg·kg-1;⑥low-dose cardamonin group:3.5mg·kg-1, intraperitoneal injections with drug were performed once a day and repeated continuously for 40 days. And mice were sacrificed on 41 days, weighed the isolated tumor, and calculated inhibitory rate. Removed the complete lungs of mice, weighed and observed the metastatic foci of lung surface, calculated the metastasis suppressed ratio of lung surface nodules.2.3 Measurement2.3.1 Proliferation ability in vitro:After treated with CAR for 48 h, the proliferation of Lewis lung cancer cells was measured with MTT method (λ=490 nm).2.3.2 Metastatic ability in vitro:Cell adhesion:drug added for 30,60 and 90 min, MTT method (λ=490 nm) was used for measurement of matrix adhesion of Lewis cell; invasion assay:used Matrigel to simulated extracellular matrix components, drug added for 40 h, fixed and stained, observed the cell number of across the small room under the surface by using the microscope; Cell migration:drug added for 24,48 h, observed the situation of healing scratches of each groups under the microscope.2.3.3 Tumor morphology:body weight and the tumor volume were measured per day, used by caliper, measured the tumor length、width, V=1/2×L×D2, inhibition rate(%)=(1-the average tumor weight of the treated group/the average tumor weight of control group)×100%. Inhibition rate of metastasis(%)=(1-the average metastatic foci number of treated group/the average metastatic foci number of control group)×100%.2.3.4 Protein expression:extracted the total protein in tumor tissue by using the cell lystae, determined the expression of mTOR, p70s6k, P-mTOR, P-p70s6k, E-cadherin and Snail using by Western blotting technique,β-actin antibody dy as a control sample.3 ResultsIn vitro3.1 Effects of CAR on proliferation of Lewis cells①The proliferation of Lewis lung cancer cell was inhibited by CAR in a dose-dependent manner (0.423±0.0089 vs.0.406±0.0049,0.361±0.0039, 0.277±0.0041,0.208±0.0062,0.127±0.0059; P<0.01);②The proliferation of Lewis lung cancer cell which induced by PA was inhibited by CAR in a dose-dependent manner (0.549±0.0112 vs 0.390±0.0078,0.318±0.0068,0.242±0.005,0.152±0.0053; P<0.01).3.2 Effects of CAR on Lung metastasis of Lewis cells Treated with CAR for 30,60,90 minutes, the adhesion of Lewis lung cells was reduced, especially at 90 minutes. In addition, the invasion and migration were inhibited by CAR.In vivo3.3 Effects of CAR on growth and metastasis of tumor①Compared with the normal group, the growth of tumor in mice was inhibited by CAR in a dose-dependent manner(P<0.01), and the inhibition rate were 24.6%,41.2% and 68.6%;②Compared with the normal group, the lung metastasis of cancer was decreased by CAR in a dose-dependent manner, the inhibition rate were 37.8 %,51.6% and 65.7%(P<0.01).③The phosphorylation of mTOR, p70s6k1 was decreased by RAP and RAP in vivo, (mTOR:0.0893±0.0083; 0.3758±0.0079,0.1003±0.0073,0.0102±0.0058, P70s6k1:0.1874±0.0048; 0.5226±0.0064,0.1985±0.0032,0.0213±0.0076). The expression of Snail was inhibited but the express of E-cadherin was increased by CAR, (Snail:0.1726±0.0053; 0.4273±0.0048, 0.1837±0.0073,0.0534±0.0042; E-cadherin:0.4802±0.0072; 0.1924±0.0048,0.4521±0.0062,0.6724±0.0073)(P<0.01);④The total protein of mTOR and p70s6kl was not affected by RAP and CAR.4 Conclusions1.The proliferation, adhesion, invasion and migration of Lewis lung cancer cell was inhibited by CAR in a dose-dependent manner;2.The tumor growth and its lung metastasis were inhibited by CAR in a dose-dependent manner in vivo.3.The antitumor effect of CAR related to the mTOR pathways. The expression of Snail was inhibited but the express of E-cadherin was increased by CAR through inhibiting the activity of mTOR and p70s6k1.