Cardamonin Inhibits Metastasis of Lewis Lung Carcinoma Cells by Decreasing mTOR Activity

Abstract

The mammalian target of rapamycin (mTOR) regulates the motility and invasion of cancer cells. Cardamonin is a chalcone that exhibits anti-tumor activity. The previous study had proved that the anti-tumor effect of cardamonin was associated with mTOR inhibition. In the present study, the anti-metastatic effect of cardamonin and its underlying molecule mechanisms were investigated on the highly metastatic Lewis lung carcinoma (LLC) cells. The proliferation, invasion and migration of LLC cells were measured by MTT, transwell and wound healing assays, respectively. The expression and activation of mTOR- and adhesion-related proteins were assessed by Western blotting. The in vivo effect of cardamonin on the metastasis of the LLC cells was investigated by a mouse model. Treated with cardamonin, the proliferation, invasion and migration of LLC cells were significantly inhibited. The expression of Snail was decreased by cardamonin, while that of E-cadherin was increased. In addition, cardamonin inhibited the activation of mTOR and its downstream target ribosomal S6 kinase 1 (S6K1). Furthermore, the tumor growth and its lung metastasis were inhibited by cardamonin in C57BL/6 mice. It indicated that cardamonin inhibited the invasion and metastasis of LLC cells through inhibiting mTOR. The metastasis inhibitory effect of cardamonin was correlated with down-regulation of Snail and up-regulation of E-cadherin.

Fig 1. Chemical structure of cardamonin.

Fig 2. Cardamonin inhibited the proliferation of LLC cells.

LLC cells were treated with rapamycin (0.1 μM) and cardamonin (0.1, 1 and 10 μM) for 48 h. The proliferation was analyzed at by MTT method. The cell viability for each group was presented by comparing to the control group. The data were presented as the mean ± SD (n = 6). **p < 0.01, compared to the control group.

Fig 3. Cardamonin inhibited the invasion and migration of LLC cells.

After incubation with different drugs for 24 h, the cells in the top on the chamber were removed. (A) The cells that had migrated onto the bottom of the chamber were stained with crystal violet and photographed at 200 × magnification. (B) The invaded cells were counted, and the invasive ratio for each group was presented by comparing to the control group. The percentage of the penetrated cells for each group was showed as the bar graph. In the migration assay, a wound was inflicted in the cell layer, and then treated with different drugs for 24 h. (C) The denuded zone was photographed at 100 × magnification. (D) The traveled distance of the different treatment group was expressed as a ratio to the original distance. The migration ratio of each group was showed as the bar graph. The data were presented as the mean ± SD (n = 5). **p < 0.01, compared to the control group.

Fig 4. Cardamonin inhibited the tumor growth and lung metastasis of LLC cells in vivo.

After 20 days of treatment, the mice were sacrificed and the tumors were isolated. The number of lung metastasis was determined by counting the number of metastatic nodules on the lung surface. (A) Tumors in different groups. (B) The initiation and growth of tumors were determined by measuring the average tumor volume. (C) Representative lung tissue sections were stained with H&E and photographed at 100 × magnification. (D) The number of lung surface metastases formed by LLC cells in each group. The data were presented as the mean ± SD (n = 6). The control group represents normal C57BL/6 mice without treatment. The model control group represents C57BL/6 mice that injected s.c. with Lewis lung carcinoma cells and treated with physiological saline.**p < 0.01, compared to the control group.

Fig 5. Effects of cardamonin on the phosphorylation of mTOR, S6K1 and the expression of E-cadherin, Snail.

LLC cells were treated with different drugs for 24 h, and then the total protein was extracted. Analysis of protein expression of mTOR, S6K1, p-mTOR p-S6K1, E-cadherin and Snail was performed by Western Blot method. Expressions of mTOR, p-mTOR, S6K1, p-S6K1, E-cadherin, Snail and actin were showed as the immunoblot band (n = 3).