Effects of matrine against the growth of human lung cancer and hepatoma cells as well as lung cancer cell migration

Abstract

The purpose of this study is to investigate in vitro and ex vivo effects of matrine on the growth of human lung cancer and hepatoma cells and the cancer cell migration as well as the expressions of related proteins in the cancer cells. Matrine significantly inhibited the in vitro and ex vivo growth of human non-small cell lung cancer A549 and hepatoma SMMC-7721 cells. Matrine induced the apoptosis in A549 and SMMC-7721 cells. Western blot analysis indicated that matrine dose-dependently down-regulated the expression of anti-apoptotic protein Bcl-2 and up-regulated the level of pro-apoptotic protein bax, eventually leading the reduction of ratios of Bcl-2/Bax proteins in A549 and SMMC-7721 cells. Furthermore, matrine significantly suppressed the A549 cell migration without reducing the cell viability. In addition, matrine dramatically reduced the secretion of vascular endothelial growth factor A in A549 cells. More importantly, matrine markedly enhanced the anticancer activity of anticancer agent trichostatin A (the histone deacetylase inhibitor) by strongly reducing the viability and/or the ratio of Bcl-2/Bax protein in A549 cells. Our findings suggest that matrine may have the broad therapeutic and/or adjuvant therapeutic application in the treatment of human non-small cell lung cancer and hepatoma.

Fig. 1

In vitro (A, C) and ex vivo (B) effects of matrine and sera from matrine-treated rabbits on the growth of human lung cancer A549 cells and the synergistic effects of matrine with anticancer agent trichostatin A against A549 cell growth. The cells were treated for 24, 48, and 72 h with matrine at the concentrations indicated (A) or the sera taken from rabbits (n = 6 for each group at different time points) at 0 (as the control group), 0.5, 1 and 2 h after oral administration of matrine in rabbits (B), respectively. C Synergistic effects of matrine with anticancer agent trichostatin A against A549 cell growth. The cells were treated for 48 h with matrine at 50–500 μg/mL (MA50, MA100, MA250, and MA500) in the absence or presence of the anticancer agent trichostatin A at 1 and 5 μg/L (TSA1 and TSA5, the histone deacetylase inhibitor) and Bay at 2.5 μM (BAY2.5, the inhibitor of NF-κB). The in vitro and ex vivo effects on A549 cell growth were examined by the MTT assay as described in the section of “Materials and methods”. The data are presented as the mean ± SD (Bar) for each group (n = 6). The figures (A, B and C) are the representative of 3 similar experiments. Comparison was made by two-way ANOVA followed by Bonferroni post hoc test to establish whether significant differences existed between the groups. Values with different letters (a–d) differ significantly (P < 0.05). c/s represents the significant synergistic effects (P < 0.05) compared with the treatment with its individual compound alone. Statistically significant synergistic effects on the growth were observed in A549 cells treated with MA50 + TSA1 or MA50 + TSA5 compared with the individual MA50, TSA1, and/or TSA5 treatment alone (MA50 + TSA1, P < 0.001, two-way ANOVA; MA50 + TSA5, P < 0.001, two-way ANOVA)

Fig. 2

In vitro (A) and ex vivo (B) effects of matrine and sera from matrine-treated rabbits on the growth of human hepatoma SMMC-7721 cells. The cells were treated for 24, 48, and 72 h with matrine at the concentrations indicated (A) or the sera taken from rabbits (n = 6 for each group at different time points) at 0 (as the control group), 0.5, 1 and 2 h after oral administration of matrine in rabbits (B), respectively. The in vitro and ex vivo effects on the SMMC-7721 cell growth were examined by the MTT assay as described in the section of “Materials and methods”. The data are presented as the mean ± SD (Bar) for each group (n = 6). The figures (A, B) are the representative of 3 similar experiments performed. Statistical analysis was carried out using the one-way ANOVA followed by Bonferroni post hoc test. * P < 0.05

Fig. 3

In vitro effects of matrine on induction of apoptosis (A) and reduction of Bcl-2/Bax protein ratio (B) in A549 cells by matrine with its synergistic anticancer agent trichostatin A. A Induction of apoptosis in A549 cells by treatment for 48 h with matrine at concentrations of 0 (0.1% DMSO vehicle as the control), 100 and 250 μg/mL. The treated cells were stained with Hoechst 33258 and the apoptotic morphological changes in the nuclear chromatin were observed under a fluorescent microscope as described in the “Materials and methods” section. B Reduction of Bcl-2/Bax protein ratio in A549 cells by treatment for 48 h with matrine at the concentrations of 50–500 μg/mL (MA50, MA100, MA250, and MA500) in the absence or presence of Bay at 2.5 μM (BAY2.5, the inhibitor of NF-κB), celecoxib at 10 μM (S10, the inhibitor of cyclooxygenase-2), and trichostatin A at 5 μg/L (TSA5). The protein expressions of Bcl-2 and Bax were analyzed by Western blotting as described in the “Materials and methods” section. Anti-β-Actin antibody was used as a sample loading control. The ratio of Bcl-2 and Bax, (the ratio of relative density of each band normalized to β-actin), shown as mean ± SD (Bar) is relative to that of 0 (0.1% DMSO vehicle) as the control (designated as 100%). For one experiment, 3 assays were carried out and only one set of gels is shown. Comparison was made by two-way ANOVA followed by Bonferroni post hoc test to establish whether significant differences existed between the groups. Values with different letters (a–f) differ significantly (P < 0.05); f/s represents the significant synergistic effect (P < 0.05) compared with the treatment with its individual compound alone. Statistically significant synergistic effect on the Bcl-2/Bax ratio was observed in A549 cells treated with MA50 + TSA5 compared with the individual MA50 or TSA5 treatment alone (P < 0.001, two-way ANOVA)

Fig. 4

In vitro effects of matrine on induction of apoptosis (A) and reduction of Bcl-2/Bax protein ratios (B) in SMMC-7721 cells by matrine. A Induction of apoptosis in SMMC-7721 cells by treatment for 48 h with matrine at concentrations of 0 (0.1% DMSO vehicle as the control), 0.5 and 1 mg/mL. The treated cells were stained with Hoechst 33258 and the apoptotic morphological changes in the nuclear chromatin were observed under a fluorescent microscope as described in the “Materials and methods” section. B Reduction of Bcl-2/Bax protein ratios in SMMC-7721 cells by treatment for 48 h with matrine at concentrations of 0 (0.1% DMSO vehicle as the control), 0.5 and 1 mg/mL. The protein expressions of Bcl-2 and Bax were analyzed by Western blotting as described in the “Materials and methods” section. Anti-β-Actin antibody was used as a sample loading control. The ratio of Bcl-2 and Bax, (the ratio of relative density of each band normalized to β-actin), shown as mean ± SD (Bar) is relative to that of 0 (0.1% DMSO vehicle) as the control (designated as 1.00). For one experiment, 3 assays were carried out and only one set of gels is shown. Statistical analysis was carried out using the one-way ANOVA followed by Bonferroni post hoc test. * P < 0.05

Fig. 5

In vitro effects of matrine on the migration of human lung cancer A549 cells. The A549 cell migration was examined by the migration assay as described in the section of “Materials and methods”. A The photos show the propidium iodide-stained A549 cells that migrated through the fibronectin-coated transwell chamber. The cells were treated for 6 h with either the vehicle (0.1% DMSO as the control) or matrine at 25 μg/mL. B Suppression of A549 cell migration by matrine. The cells were treated for 6 h with matrine at the concentrations indicated in figure. Relative migration (%) ± SD (n = 6) are shown for the indicated matrine concentrations and the control received DMSO vehicle (0.1%, final concentration). The figure is the representative of 3 similar experiments. Statistical analysis was carried out using the one-way ANOVA followed by Bonferroni post hoc test. * P < 0.05

Fig. 6

In vitro effects of matrine (MA) on the excretion of VEGF-A in A549 cells. The cells were treated for 24 h with matrine at concentrations of 50–500 μg/mL. Then each supernatant of the cell culture was respectively collected and analyzed by using an ELISA kit (VEGF-A) as described in the section of “Materials and methods”. Values are shown as mean ± SD (bar) for the indicated concentration (n = 3). The figure is the representative of 3 similar experiments. Statistical analysis was carried out using the one-way ANOVA followed by Bonferroni post hoc test. * P < 0.05.; ** P < 0.01