Figure 1.
Effect of physalin F on viability in human renal cancer cells.
Renal cancer cell lines (A498, ACHN, and UO-31) were treated with various concentrations (0.3, 1, 3, and 10 µg/mL) for 24 hr for MTT assay (A) and for 48 hr for SRB assay (B) in A498 cells. A498 cells were treated with vehicle or physalin F at the indicated concentration (1, 3, and 10 µg/mL) for 24 hr and viewed by fluorescence microscope followed by TUNEL (C) and DAPI (D) staining (magnification × 400). (E) To quantitatively evaluate cell death after treatment of physalin F for 24 hr, we used cell death ELISAPLUS kit to detect. Data are expressed as the mean percentage of control ± S.D. of three independent experiments. **, ##, && P<0.01 and ***,###, &&& P<0.001 compared with the control group.
Figure 2.
Effect of physalin F on cell cycle distribution in A498 cells.
(A) Cells were incubated with vehicle or various concentrations (1, 3, and 10 µg/mL) of physalin F for 24 hr. (B) Cells were incubated with vehicle or 10 µg/mL of physalin F for indicated time period. The cell cycle analysis and cell apoptosis were determined using FACS analysis as described in Methods and Materials. * P<0.05, ** P<0.01, and *** P<0.001 compared with the 0-time point group in sub-G1 phase. # P<0.05 compared with the 0-time point group in G0/G1 phase.
Figure 3.
Effect of physalin F on reduction of Δψm and release of cytochrome c.
A498 cells were incubated in the absence or presence of physalin F (10 µg/mL) for indicated time, and cells were harvested and prepared for detection (A) mitochondria membrane potential by using FACScan analysis, (B) release of cytochrome c in cytosol (The fractions were collected by using the protocol of preparation of cytosolic and mitochondrial fractions as mentioned in Materials and Methods.), and (C) Bcl-2, Bcl-xL and Bax expression by using Western blotting analysis. * P<0.05 compared with the 12 hr-time point control group. # P<0.05 compared with the 18 hr-time point control group. && P<0.01 compared with the 24 hr-time point control group.
Figure 4.
Effects of physalin F on the expression of apoptosis-related proteins in A498 cells.
A498 cells were incubated in the absence or presence of physalin F for indicated time, and cells were harvested and prepared for detection of (A) pro-caspase-8, 9, caspase-3, PARP, and (B) p53, p21 by using Western blotting.
Figure 5.
Effect of physalin F on cellular ROS accumulation in A498 cells.
A498 cells were incubated in the absence or presence of physalin F (10 µg/mL) for indicated time. The fluorescent intensity of DCFH-DA was detected by flow cytometric analysis. ** P<0.01 compared with the 0.5 hr-time point control group. # P<0.05 compared with the 1 hr-time point control group. && P<0.01 compared with the 3 hr-time point control group. (B) Different ROS scavengers were preincubated for 30 min and cell viability was determined by MTT assay. *** P<0.001 compared with the control group. ### P<0.001 compared with the physalin F-treated group. Treatment of NAC and GSH inhibited physalin F-induced ROS generation (C, * P<0.05 and ** P<0.01 compared with the physalin F-treated group.) and the loss of Δψm (D, *** P<0.001 compared with the control group. # P<0.05 compared with the physalin F-treated group.) in A498 cells. (E) NAC (10 mM) and GSH (3 mM) reduced the apoptosis-related results in physalin F-treated cells for 24 hr.
Figure 6.
Effect of physalin F on NF-κB.
A498 cells were incubated in the absence or presence of physalin F (10 µg/mL) for indicated time and detection the expression of phospho-IκBα and IκBα (A). (B) The nuclear and cytosol extract were prepared as indicated in Methods and Materials and were analyzed by Western blotting for the detection of specific protein, as indicated (p-p65, p65, p50, GAPDH, and nucleolin). (C) cells were exposed to physalin F (10 µg/mL) for 6 hr and nuclear extracts were incubated with a hot NF-κB probe (lane 2–4) or cold probe (lane 1, indicate “cold”) and demonstrate the specificity of the bands obtained on EMSA. The data are representative of three independent experiments. P indicates positive nuclear extract. ** P<0.01 compared with the control group. (D) Effect of ROS scavengers on physalin F-inhibited NF-κB nuclear translocation in A498 cells. Cells were pretreated with NAC (10 mM) and GSH (3 mM) for 0.5 hr and then treated with 10 µg/mL of physalin F for 6 hr. The nuclear extract were prepared as indicated in Methods and Materials. The levels of p65 and p50 were detected by Western blotting.