Matrine suppresses the migration and invasion of NSCLC cells by inhibiting PAX2-induced epithelial-mesenchymal transition

Abstract

Non-small cell lung cancer (NSCLC) is the major cause of deaths among all the cancer types worldwide. Most of the NSCLC is diagnosed at an advanced stage and the 5-year overall survival rate is low. The reason for the low survival rate of patients with NSCLC is mainly due to distant metastasis. Matrine, a traditional Chinese medicine, has been shown a significant anti-proliferation and anti-invasive effect in tumors. However, little is known on the anti-invasive mechanism of matrine in lung cancer. Therefore, we tried to investigate the molecular mechanism of matrine on the invasive ability of NSCLC cells in vitro. Cell Counting Kit-8 assay was used to evaluate the cell viability. Transwell assay was used to detect the migration and invasion abilities. Microarray assay was used to analyze the differentiated expression genes with or without matrine treatment. Western blotting and real-time polymerase chain reaction were applied to detect the expressions of PAX2, E-cadherin and N-cadherin. Our study showed that matrine could suppress the proliferative activity of NSCLC cells in a dose- and time-dependent manner. Further investigation discovered that the migration and invasion of NSCLC cells were significantly inhibited by treatment with different concentrations of matrine. Microarray assay, real-time polymerase chain reaction and western blotting showed that matrine could significantly decrease the expression of PAX2. In addition, epithelial-mesenchymal transition and related proteins were decreased. In conclusion, matrine may block PAX2 expression to interfere with epithelial-mesenchymal transition signaling pathway that ultimately inhibit the migration and invasion of NSCLC cells in vitro. Matrine might serve as a potential agent for NSCLC treatment.

Keywords: PAX2; epithelial-mesenchymal transition; invasion; matrine; migration.

Figure 1

Matrine inhibited the proliferation of A549 and H1299 cells. Notes: After being treated by matrine with different concentrations (0, 0.25, 0.5, 1, 2, 4 and 8 mg/mL) cultured for 24, 48 and 72 hours, the viability of A549 (A) and H1299 (B) cells was detected by CCK-8 assay. All data are presented as the mean values ± SD from three independent experiments. *p<0.05 and **p<0.01 vs control group. Abbreviation: CCK-8, cell counting kit-8.

Figure 2

The migration and invasion ability of NSCLC cells were attenuated with the treatment of matrine in a dose-dependent manner in A549 (A) and H1299 (B) cells. Notes: For migration assay, cells were treated with different concentrations (0, 0.5, 1 and 2 mg/mL) of matrine following which the cell was transferred to a Transwell chambers without matrigel, and the numbers of cells migration were counted 24 hours later. For invasion assay, cells were pretreated with different concentrations of matrine for 24 hours cultured in matrigel invasion chambers, and the numbers of cells invasion were counted 24 hours later. The numbers of migration and invasion cells were counted and shown as the mean values ± SD from three independent experiments. *p<0.05 and **p<0.01 vs control group. Magnification: 200×. Abbreviation: NSCLC, non-small cell lung cancer.

Figure 3

Heat map analysis of microarray data in A549 cell treated with or without matrine. Notes: (A) About 2,419 differentially expressed genes with a cut-off threshold of 2-fold were analyzed and shown. (B) Approximately 36 genes associated with epithelial-mesenchymal transition or migration and invasion were selected from the differential express genes for mapping heat map. The expression levels of genes were expressed in different colors, red representing increase and blue representing decrease.

Figure 4

Matrine inhibited the expression of PAX2 in NSCLC cells. Notes: (A) The PAX2 mRNA levels of PAX2 in A549 and H1299 cells were detected by real-time polymerase chain reaction after treatment with different concentrations (0, 0.5, 1 and 2 mg/mL) of matrine for 24 hours. (B) The PAX2 protein levels in A549 and H1299 cells were detected by western blot analysis after treatment with different concentrations of matrine for 24 hours. Data are presented as the mean ± SD of 3 independent experiments. *p<0.05 and **p<0.01 vs control group. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NSCLC, non-small cell lung cancer.

Figure 5

The effects of matrine on the expression of EMT and migration associated molecules. Notes: The mRNA expression levels of N-cadherin, E-cadherin, MMP2, MMP9 in A549 (A) and H1299 (B) cells were detected by real-time polymerase chain reaction after treatment with different concentrations of matrine as indicated (0, 0.5, 1, 2 mg/mL). The protein expression levels of N-cadherin, E-cadherin, MMP2, MMP9 in A549 (C) and H1299 (D) cells were detected by western blotting after treatment with different concentrations of matrine as indicated (0, 0.5, 1, 2 mg/mL). Data are presented as the mean ± SD for mRNA expression levels of A549 and H1299 cells of three independent experiments. *p<0.05 and **p<0.01 vs control group. Abbreviations: EMT, epithelial-mesenchymal transition; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MMP, matrix metalloproteinase.