Apoptosis Induction of Human Lung Carcinoma Cells by Chan Su (Venenum Bufonis) Through Activation of Caspases

Inhibition of the cell growth by SVB in A549 human lung carcinoma cells. The cells were plated at 4 × 10⁴ cells per 60 mm plate, and incubated for 24 hours. The cells were treated with varying concentrations of SVB for 24 hours and cell viability and growth inhibition were, respectively, measured by the metabolic-dye-based MTT assay (A) and hemocytometer counts of trypan blue-excluding cells (B). Data is expressed as mean ± SD of three independent experiments. Statistical analysis was carried out by t-test (*, p < 0.05).

Induction of apoptosis by SVB treatment in A549 cells. (A and B) After treating the cells with SVB for 24 hours, they were observed using an inverted microscope (A, magnification, ×200) or were stained with DAPI for 10 minutes, washed with PBS, and then photographed with a fluorescence microscope using a blue filter (B, magnification, ×400). (C) To quantify the degree of apoptosis induced by SVB, the cells were evaluated for sub-G1 DNA content using a flow cytometer. This represents the fractions undergoing apoptotic DNA degradation. Each point represents the average of two independent experiments.

Effects of SVB on the levels of apoptosis-related genes in A549 cells. After incubation with SVB for 24 hours, the cells were lysed and the cellular proteins were then separated in SDS-polyacrylamide gels, after which they were transferred onto nitrocellulose membranes. The membranes were then probed with the indicated antibodies and the proteins were visualized using an ECL detection system. Actin was used as the internal control.

Loss of MMP by SVB treatment of A549 cells. After being treated with SVB for 24 hours, the cells were stained with JC-1 and then incubated at 37°C for 20 minutes, 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. The significance was determined by a Student's t-test (*, p < 0.05 vs. untreated control).

Activation of caspases and the degradation of the PARP and β-catenin protein by SVB treatment in A549 cells. (A) After 24 hours incubation with SVB, the cells were lysed. The cellular proteins were separated by SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were probed with anti-caspase-3, anti-caspase-8, anti-caspase-9, anti-PARP and anti-β-catenin antibodies. The proteins were visualized using an ECL detection system. Actin was used as the internal control. (B) The cell lysates from the cells grown under the same conditions as (A) were assayed for in vitro caspase-3, caspase-8 and caspase-9 activity using the substrates DEVD-pNA, IETD-pNA and LEHD-pNA, respectively. The concentrations of the fluorescent products released were measured. Data are expressed as mean ± SD of three independent experiments. For statistical analysis, t-test was performed (*, p < 0.05).