Curcumin Induces a p53-Dependent Apoptosis in Human Basal Cell Carcinoma Cells  Shiou-Hwa Jee  Journal of Investigative Dermatology  Volume 111, Issue 4, Pages 656-661 (October 1998) DOI: 10.1046/j.1523-1747.1998.00352.x Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Apoptosis occurring in human basal cell carcinoma cells after treatment with curcumin. BCC cells were treated with solvent control (0.1% dimethylsulfoxide) (A) or curcumin (50 μM) for 12 h (B). After removal of the curcumin, the cells were fixed and examined as described in Materials and Methods. Original magnification, 600×;scale bar: 6 μm. Journal of Investigative Dermatology 1998 111, 656-661DOI: (10.1046/j.1523-1747.1998.00352.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Internucleosomal DNA fragmentation in BCC cells treated with curcumin. (A) BCC cells were exposed to 50 μM curcumin for 0–48 h. DNA from cells exposed to different time points was extracted, electrophoresed through 2% agarose gels, and visualized by staining with ethidium bromide. (B) The apoptotic cells in the sub-G1 region were quantitated by flow cytometry. The data presented here are the mean value of three independent experiments. Bars indicate standard errors. Journal of Investigative Dermatology 1998 111, 656-661DOI: (10.1046/j.1523-1747.1998.00352.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Increase of p53 protein was closely associated with curcumin-induced apoptosis. (A) BCC cells were treated with 50 μM curcumin, staurosporine (50 ng per ml; STP), or pretreated with cycloheximide (10 μM; CHX), actinomycin D (2 ng per ml; Act D) for 3 h followed by 50 μM curcumin treatment. The expression of p53 protein was detected by western blotting. (B) The percentage of hypodiploid cell in BCC cells under different treatment as described in (A) was measured by flow cytometric analysis. (C) Induction of p53 binding activity by curcumin was detected by gel retardation assay in BCC cells. wt, 25-fold molar excess of the nonradioactive p53CON oligonucleotide was added prior to the addition of [32P] p53CON; mut, 25-fold molar excess of the mutated p53CON was added prior to the addition of [32P] p53CON. Journal of Investigative Dermatology 1998 111, 656-661DOI: (10.1046/j.1523-1747.1998.00352.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Identification of the state of p53 proteins in BCC cells. Cell extracts from BCC cells were immunoprecipitated by pAb1801 (specific to both wt p53 and mut p53 protein), pAb 240 (specific to mut p53), or pAb 246 (specific to wt p53), respectively, and followed by western blotting to detect p53 protein using pAb 1801 as prob. mut, control of mutated p53 protein from SW620 cell extracts, which were immunoprecipitated by pAb 240 antibody and detected by western blotting described in Materials and Methods. Journal of Investigative Dermatology 1998 111, 656-661DOI: (10.1046/j.1523-1747.1998.00352.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Flow cytometric analysis of DNA content in BCC cells treated with curcumin. One million BCC cells were treated with 50 μM curcumin for the indicated time and then permeabilized and subjected to flow cytometric analysis as described in Materials and Methods. Journal of Investigative Dermatology 1998 111, 656-661DOI: (10.1046/j.1523-1747.1998.00352.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Induction of CIP1 and Gadd45 proteins by curcumin in BCC cells. (A) Immunoblot analysis of CIP1 and Gadd45 proteins in BCC cells. Cell extracts were prepared and immunoblotting performed as described in Materials and Methods. (B) Immunostaining of intracellular p53, Gadd45, CIP 1 protein by specific antibody. Cells were treated with curcumin (50 μM) for 12 h and fixed in methanol at –20°C followed by staining with anti-p53 (Oncogene Science), anti-CIP1 (Transduction), or anti-Gadd45 (Santa Cruz Technology) antibody, and observed with fluorescence microscope. Control, cells cultured with 0.1% dimethylsulfoxide for 12 h; 12 h, cells treated with 50 μM curcumin for 12 h. Journal of Investigative Dermatology 1998 111, 656-661DOI: (10.1046/j.1523-1747.1998.00352.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Attenuation of curcumin induced the increase of p53 protein and apoptosis by anti-sense p53 oligonucleotides. (A) BCC cells were pretreated with 20 μM sense (S) or anti-sense (AS) oligonucleotides for 16 h followed by 50 μM curcumin (Cur) treatment for 24 h. The percentage of apoptotic cell was measured by flow cytometric analysis. C, control group. (B) Flow cytometric analysis of p53 protein levels in BCC cells in the presence of anti-sense or sense p53 S-oligos. Cell samples were trypsinized and fixed in 75% ethanol. Then 0.5% Triton X-100-premeabilized samples were stained with fluorescent isothiocyanate-conjugated anti-p53 monoclonal antibody to determine the p53 protein. Journal of Investigative Dermatology 1998 111, 656-661DOI: (10.1046/j.1523-1747.1998.00352.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions