Role of p53, PUMA, and Bax in wogonin-induced apoptosis in human cancer cells

Abstract

We observed that treatment of prostate cancer cells for 24 h with wogonin, a naturally occurring monoflavonoid, induced cell death in a dose- and time-dependent manner. Exposure of wogonin to LNCaP cells was associated with increased intracellular levels of p21(Cip-1), p27(Kip-1), p53, and PUMA, oligomerization of Bax, release of cytochrome c from the mitochondria, and activation of caspases. We also confirmed the role of p53 by noting that knock-in in p53 expression by transfecting p53 DNA increased wogonin-induced apoptosis in p53-null PC-3 cells. To study the mechanism of PUMA up-regulation, we determined the activities of PUMA promoter in the wogonin treated and untreated cells. Increase of the intracellular levels of PUMA protein was due to increase in transcriptional activity. Data from chromatin immunoprecipitation (ChIP) analyses revealed that wogonin activated the transcription factor p53 binding activity to the PUMA promoter region. We observed that the up-regulation of PUMA mediated wogonin cytotoxicity. Further characterization of the transcriptional response to wogonin in HCT116 human colon cancer cells demonstrated that PUMA induction was p53-dependent; deficiency in either p53 or PUMA significantly protected HCT116 cells against wogonin-induced apoptosis. Also, wogonin promoted mitochondrial translocation and multimerization of Bax. Interestingly, wogonin (100 microM) treatment did not affect the viability of normal human prostate epithelial cells (PrEC). Taken together, these results indicate that p53-dependent transcriptional induction of PUMA and oligomerization of Bax play important roles in the sensitivity of cancer cells to apoptosis induced by caspase activation through wogonin.

Fig. 1. Chemical structures of baicalein, baicalin, and wogonin

Fig. 2. Effects of baicalein, baicalin, and wogonin on the cell viability of human prostate carcinoma LNCaP cells

Cells (1×10⁵) were plated, allowed to attach overnight, and then treated with DMSO (control) or 10-100 μM of baicalein, baicalin, or wogonin for 24 h at 37°C. (A, B, C) Cell viability was determined by trypan blue dye exclusion assay. Error bars represent the mean ± S.E from three separate experiments. (D, E, F) Cells were harvested and samples were prepared for analysis of the cleavage of PARP-1 using Western blot analysis. Actin was shown as an internal standard.

Fig. 3. Wogonin induces apoptosis in LNCaP cells

Cells were treated with various concentrations (10-100 μM) of wogonin or 0.1% DMSO for 24 h. (A), Cell morphology was examined under a light microscope. Magnification, × 200. Wog: wogonin. (B), DNA isolated from cells that were untreated (C, lane 2), or treated with DMSO (D, lane 3) or wogonin (lane 4-7) was fractionated by electrophoresis. Oligonucleosomal length DNA fragments were visualized by staining gels with ethidium bromide. Lane 1, DNA ladder. Similar results were obtained in two separate experiments.

Fig. 4. Wogonin activates caspases in LNCaP cells

In vitro treatment of LNCaP cells with wogonin caused the cleavage of caspase-8 (A), caspase-9 (B), and caspase-3 (C). Cells were treated with various concentrations of wogonin (5-50 μM) for 24 h, and then cells were harvested. Lysates containing equal amounts of protein (20 μg) were separated by SDS-PAGE and immunoblotted. Actin was used to confirm the equal amount of proteins loaded in each lane.

Fig. 5. Wogonin treatment causes release of cytochrome c from mitochondria to the cytosol in LNCaP cells

Cytochrome c release from mitochondria to cytosol was examined in LNCaP cells which were incubated overnight with wogognin (5, 10, 25, and 50 μM) or without (C; control). Lysates containing equal amounts of protein (20 μg) were separated by SDS-PAGE and immunoblotted with anti-cytochrome c antibody. We used actin as a cytosolic marker and COX IV as a mitochondrial marker. The top and third panels represent mitochondrial and cytosolic fractions, respectively.

Fig. 6. Wogonin induces cytotoxicity in human prostate cancer LNCaP cells, but not in prostate epithelial cells (PrEC)

LNCaP and PrEC cells were treated with various concentrations (10-100 μM) of wogonin for 24 h. (A) The cytotoxic effect of wogonin on LNCaP and PrEC cells was determined using the trypan blue dye exclusion assay. Error bars represent standard error of the mean (SEM) from three separate experiments. (B, C) Equal amounts of protein (20 μg) from cell lysates of PrEC or LNCaP cells were separated by SDS-PAGE and immunoblotted with anti-phospho-Akt, anti-Akt, anti-PARP-1, or anti-p53 antibody. Actin was shown as an internal standard.

Fig. 7. Wogonin increases the intracellular level of p53 and its downstream cell cycle-related proteins as well as apoptosis-related protein in human prostate carcinoma LNCaP cells, but not in p53-null PC-3 cells

LNCaP PC-3 cells were treated with various concentrations (0-50 μM) of wogonin for 24 h or LNCaP cells were treated with 50 μM wogonin for various times (4-24 h), and Western blot analysis was done for PARP-1, p53, p27^Kip-1, p21^Cip-1, or PUMA. Lysates containing equal amounts of protein (20 μg) were separated by SDS-PAGE and immunoblotted. Actin was used to confirm the equal amount of proteins loaded in each lane.

Fig. 8. Involvement of p53 and PUMA in wogonin-induced apoptosis

(A), HCT116 p53^+/- and HCT116 p53^-/- cells were treated with various concentrations (10-100 μM) of wogonin for 24 h. Cell viability was determined using the trypan blue dye exclusion assay (Upper panel). Error bars represent the mean ± SE from three separate experiments. Comparisons of IC50 were made by dose response curve fitting. Equal amounts of protein (20 μg) from cell lysates cells were separated by SDS-PAGE and immunoblotted with anti-PARP-1, anti-p53, or anti-PUMA antibody (Lower panel). Actin was shown as an internal standard. (B) PC-3 cells were transiently transfected with pcDNA3 vector containing empty (pcDNA3) or wild-type p53 cDNA (PcDNA-p53). After 48 h incubation, cells were treated with or without wogonin (50 μM) for 24 h. Equal amounts of protein (20 μg) from cell lysates cells were separated by SDS-PAGE and immunoblotted with anti-PARP-1, anti-p53, anti-Mdm2, or anti-PUMA antibody. Actin was shown as an internal standard.

Fig. 9. Transcription factor binding activity in PUMA promoter during treatment with wogonin in LNCaP cells

Cells were treated with 50 μM wogonin for 24 h. (A) Cells were sonicated and chromatin fragments were immunoprecipitated with anti-p53 antibody. PUMA promoter contains p53 binding sites (-1409 ∼ -76, 8 putative binding sites). The binding of p53 on PUMA promoter was analyzed by PCR. (B), chromatin fragments were immunoprecipitated with anti-p53 antibody. Interaction between p53 and histone H3 was performed with anti-histone H3 antibody.

Fig. 10. Role of PUMA in wogonin-induced apoptosis

HCT116 PUMA^+/+ and HCT116 PUMA^-/- cells were treated various concentrations (10-100 μM) of wogonin for 24 h. (A), Cell viability was determined using the trypan blue dye exclusion assay. Error bars represent the mean ± SE from three separate experiments. Comparisons of IC50 were made by dose response curve fitting. (B) Equal amounts of protein (20 μg) from cell lysates were separated by SDS-PAGE and immunoblotted with anti-phospho-PARP-1, anti-p53, anti-PUMA, or anti-Bax antibody. Actin was shown as an internal standard.

Fig. 11. Oligomerization of Bax during wogonin treatment

LNCaP, HCT116 PUMA^+/+, or HCT116 PUMA^-/- cells were treated with 50 μM wogonin for 24 h. (A), Cell lysates for LNCaP cells were immunoprecipitated with anti-Bax antibody or mock antibody (IgG) and immunoblotted with anti-Bcl-xL antibody (upper panels). The presence of Bax in the lysates was verified by immunoblotting (lower panel). (B, C), Mitochondrial and cytosolic fractions were isolated and cross-linked and then subjected to immunoblotting with an antibody to Bax. Bax monomers (1×) and multimers (2× – 4×) are indicated. We used actin for a cytosolic marker and COX IV for a mitochondrial marker as fractional markers and loading controls.

Fig. 11. Oligomerization of Bax during wogonin treatment

LNCaP, HCT116 PUMA^+/+, or HCT116 PUMA^-/- cells were treated with 50 μM wogonin for 24 h. (A), Cell lysates for LNCaP cells were immunoprecipitated with anti-Bax antibody or mock antibody (IgG) and immunoblotted with anti-Bcl-xL antibody (upper panels). The presence of Bax in the lysates was verified by immunoblotting (lower panel). (B, C), Mitochondrial and cytosolic fractions were isolated and cross-linked and then subjected to immunoblotting with an antibody to Bax. Bax monomers (1×) and multimers (2× – 4×) are indicated. We used actin for a cytosolic marker and COX IV for a mitochondrial marker as fractional markers and loading controls.

Fig. 12. Role of Bax in wogonin-induced apoptosis

HCT116 Bax^+/- and HCT116 Bax^-/- cells were treated with various concentrations (10-100 μM) of wogonin for 24 h. (A), Cell viability was determined using the trypan blue dye exclusion assay. Error bars represent the mean ± SE from three separate experiments. Comparisons of IC50 were made by dose response curve fitting. (B) Equal amounts of protein (20 μg) from cell lysates were separated by SDS-PAGE and immunoblotted with anti-phospho-PARP-1, anti-p53, anti-PUMA, or anti-Bax antibody. Actin was shown as an internal standard.