Apoptotic and anti-metastatic effects of the whole skin of Venenum bufonis in A549 human lung cancer cells

Abstract

In the present study, the effects of the whole skin of Venenum bufonis on apoptotic and anti-invasive activity in A549 human lung cancer cells were investigated. Treatment with extract of the whole skin of V. bufonis (SVB) resulted in a significant decrease in cell growth of A549 cells, depending on dosage, which was associated with apoptosis induction, as proved by chromatin condensation and accumulation of apoptotic fraction. SVB treatment induced expression of death receptor-related proteins, such as death receptor 4, which further triggered activation of caspase-8 and cleavage of Bid. In addition, the increase in apoptosis by SVB treatment was correlated with dysfunction of mitochondria, activation of caspase-9 and -3, downregulation of IAP family proteins, such as XIAP, cIAP-1 and cIAP-2, and concomitant degradation of activated caspase-3-specific target proteins, such as poly (ADP-ribose) polymerase and β-catenin proteins. However, z-DEVD-fmk, a caspase-3-specific inhibitor, blocked SVB-induced apoptosis and increased the survival rate of SVB-treated cells, indicating that activation of caspase-3 plays a key role in SVB-induced apoptosis. In addition, within concentrations that were not cytotoxic to A549 cells, SVB induced marked inhibition of cell motility and invasiveness. Activities of matrix metalloproteinase (MMP)-2 and MMP-9 in AGS cells were dose-dependently inhibited by treatment with SVB, and this was also correlated with a decrease in expression of their mRNA and proteins, and upregulation of tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 mRNA expression. Further studies are needed; however, the results indicated that SVB induces apoptosis of A549 cells through a signaling cascade of death receptor-mediated extrinsic as well as mitochondria-mediated intrinsic caspase pathways. Our data also demonstrated that MMPs are critical targets of SVB-induced anti-invasiveness in A549 cells.

Figure 1

Inhibition of cell growth and induction of apoptosis by SVB treatment in A549 human lung carcinoma cells. Cells were plated at 4×10⁴ cells per 60-mm plate, and incubated for 24 h. Cells were treated with varying concentrations of SVB for 24 h, and cell viability and proliferation were measured by the metabolic-dye-based MTT assay (A) and hemocytometer counts of trypan blue-excluding cells (B), respectively. Data are expressed as mean ± SD of three independent experiments. Values marked as ^* indicate significant differences from other treatments (^*p<0.05). (C) Following treatment of cells with the indicated concentrations of SVB, they were observed using an inverted microscope (magnification ×200). (B) Cells grown under the same conditions as (C) were stained with DAPI for 10 min, washed with PBS, and then photographed with a fluorescence microscope using a blue filter (magnification ×400). (E) To quantify the degree of apoptosis induced by SVB, cells grown under the same conditions were evaluated for sub-G1 DNA content, which represents fractions undergoing apoptotic DNA degradation, using a flow cytometer. Each point represents the average of two independent experiments.

Figure 2

Loss of MMP (ΔΨ_m) by SVB treatment in A549 cells. Cells grown under the same conditions as those in Fig. 1 were stained with JC-1 and then incubated at 37°C for 20 min, after which the mean JC-1 fluorescence intensity was detected using a flow cytometer. Data represent the mean ± SD of representative experiments performed at least three times. Significance was determined by a Student’s t-test (^*p<0.05 vs. untreated control).

Figure 3

Effects of SVB treatment on levels of apoptosis-related proteins in AGS cells. Cells were treated with the indicated concentrations of SVB for 24 h. Equal amounts of cell lysates were resolved on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were probed with the indicated antibodies, and proteins were visualized using the ECL detection system. Actin was used as an internal control.

Figure 4

Activation of caspases and degradation of PARP and β-catenin proteins by SVB treatment in AGS cells. (A) Cells treated with various concentrations of SVB for 24 were lysed and cellular proteins were separated by SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. Membranes were probed with anti-caspase-3, -8, and -9, anti-PARP, and anti-β-catenin antibodies. Proteins were visualized using the ECL detection system. Actin was used as an internal control. (B) Cells grown under the same conditions as (A) were collected and lysed. Aliquots were incubated individually with DEVD-pNA, IETD-pNA, and LEHD-pNA for caspase-3, -8, and -9 at 37°C for 1 h. Released fluorescent products were measured. Data represent the mean of three independent experiments. The statistical significance of results was analyzed by a Student’s t-test (^*p<0.05).

Figure 5

Inhibition of SVB-induced apoptosis by caspase-3 inhibitor in A549 cells. (A) A549 cells were treated with z-DEVD-fmk (50 μM) for 1 h before challenge with 5 μg/ml of SVB for 24 h. Cells were stained with DAPI for 10 min and photographed with a fluorescence microscope using a blue filter (magnification ×400). (B) Cell proliferation was determined using the MTT assay after 24 h in the presence of the caspase-3 inhibitor z-DEVD-fmk (50 μM) for 1 h before SVB (5 μg/ml) treatment. Data are expressed as mean ± SD of three independent experiments. (C) Cells grown under the same conditions as (A) were evaluated for sub-G1 DNA content using a flow cytometer. Data are reported as mean ± SD of three independent experiments. The significance was determined by Student’s t-test (^*p<0.05 vs. untreated control).

Figure 6

Inhibition of cell motility and invasion by SVB treatment in A549 cells. (A) Cells were grown to confluency on 30-mm cell culture dishes and then a scratch was made through the cell layer using a pipette tip. After washing with PBS, serum-free media (to prevent cell proliferation) containing either vehicle or 2 μg/ml of SVB was added for 48 h. Photographs of the wounded area were taken in order to evaluate cell movement into the wounded area. (B) Cells pretreated with the indicated concentrations of SVB for 6 h were plated onto the apical side of matrigel coated filters in serum-free medium containing either vehicle or SVB. Medium containing 20% FBS was placed in the basolateral chamber to act as a chemoattractant. After 48 h, cells on the apical side were wiped off using a Q-tip. Next, cells on the bottom of the filter were stained using hematoxylin and eosin Y, and were then counted. Data are shown as the mean of triplicate samples and represent invasive cell numbers, compared with those of control cells. The significance was determined using a Student’s t-test (^*p<0.05 versus untreated control).

Figure 7

Effect of SVB treatment on expression of TIMPs and MMPs, and activity of MMPs in A549 cells. (A) Cells were treated with the indicated concentrations of SVB for 24 h. Total RNAs were isolated and reverse-transcribed. The resulting cDNAs were then subjected to PCR with TIMP-1, TIMP-2, MMP-2, and MMP-9 primers and the reaction products were subjected to electrophoresis in a 1% agarose gel and visualized by EtBr staining. Representative results from two independent experiments are shown. GAPDH was used as an internal control. (B) Cells grown under the same conditions as (A) were lysed and cellular proteins were separated by electrophoresis on SDS-polyacrylamide gels. Western blotting was then performed using anti-MMP-2 and anti-MMP-9 antibodies, and an ECL detection system. Actin was used as an internal control. (C) Following incubation with the indicated concentrations of SVB for 24 h, medium was collected, and the activities of MMP-2 and MMP-9 were measured by zymo-graphy.