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Caffeic acid phenethyl ester induced cell cycle arrest and growth inhibition in androgen-independent prostate cancer cells via regulation of Skp2, p53, p21Cip1 and p27Kip1.

Lin HP, Lin CY, Huo C, Hsiao PH, Su LC, Jiang SS, Chan TM, Chang CH, Chen LT, Kung HJ, Wang HD, Chuu CP - Oncotarget (2015)

Bottom Line: CAPE treatment decreased Skp2 and Akt1 protein expression in LNCaP 104-R1 tumors as compared to control group.Overexpression of Skp2, or siRNA knockdown of p21Cip1, p27Kip1, or p53 blocked suppressive effect of CAPE treatment.Co-treatment of CAPE with PI3K inhibitor LY294002 or Bcl-2 inhibitor ABT737 showed synergistic suppressive effects.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Cancer Research, National Health Research Institutes, Miaoli, Taiwan, ROC.

ABSTRACT
Prostate cancer (PCa) patients receiving the androgen ablation therapy ultimately develop recurrent castration-resistant prostate cancer (CRPC) within 1-3 years. Treatment with caffeic acid phenethyl ester (CAPE) suppressed cell survival and proliferation via induction of G1 or G2/M cell cycle arrest in LNCaP 104-R1, DU-145, 22Rv1, and C4-2 CRPC cells. CAPE treatment also inhibited soft agar colony formation and retarded nude mice xenograft growth of LNCaP 104-R1 cells. We identified that CAPE treatment significantly reduced protein abundance of Skp2, Cdk2, Cdk4, Cdk7, Rb, phospho-Rb S807/811, cyclin A, cyclin D1, cyclin H, E2F1, c-Myc, SGK, phospho-p70S6kinase T421/S424, phospho-mTOR Ser2481, phospho-GSK3α Ser21, but induced p21Cip1, p27Kip1, ATF4, cyclin E, p53, TRIB3, phospho-p53 (Ser6, Ser33, Ser46, Ser392), phospho-p38 MAPK Thr180/Tyr182, Chk1, Chk2, phospho-ATM S1981, phospho-ATR S428, and phospho-p90RSK Ser380. CAPE treatment decreased Skp2 and Akt1 protein expression in LNCaP 104-R1 tumors as compared to control group. Overexpression of Skp2, or siRNA knockdown of p21Cip1, p27Kip1, or p53 blocked suppressive effect of CAPE treatment. Co-treatment of CAPE with PI3K inhibitor LY294002 or Bcl-2 inhibitor ABT737 showed synergistic suppressive effects. Our finding suggested that CAPE treatment induced cell cycle arrest and growth inhibition in CRPC cells via regulation of Skp2, p53, p21Cip1, and p27Kip1.

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CAPE treatment dose-dependently reduced cell survival, proliferation, and soft agar colony formation of CRPC cells(A) LNCaP 104-R1 cells were treated with increasing concentrations of CAPE for 96 h to determine suppressive effect of CAPE on cell proliferation. Relative cell number was determined by either Hoechst 33258 fluorescence based-96 well proliferation assay or by MTT assay. Relative cell number was normalized to cell number of control (no treatment). (B) LNCaP 104-R1 cells were treated with increasing concentrations of CAPE for 24, 48, 72, 96 h to investigate the suppressive effects of CAPE. Relative cell number was normalized to cell number of control (no treatment) at 24 h and was determined by Hoechst 33258 fluorescence based-96 well proliferation assay. (C) LNCaP 104-R1, PC-3, DU-145, LNCaP C4–2, and 22Rv1 cells were treated with increasing concentrations of CAPE for 96 h to investigate the suppressive effects of CAPE. Relative cell number determined by Hoechst 33258 fluorescence based-96 well proliferation assay and was normalized to cell number of control (no treatment) for individual cell line. Asterisks *, **, and *** represented statistical significance in cell number of p < 0.05, p < 0.01, and p < 0.001, respectively, as compared to that of control. (D) Anticancer effect of CAPE was confirmed by the colony formation assay of LNCaP 104-R1 cells treated with 0, 10, or 20 μM CAPE for 14 days. Image is representative of three biological replicates.
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Figure 2: CAPE treatment dose-dependently reduced cell survival, proliferation, and soft agar colony formation of CRPC cells(A) LNCaP 104-R1 cells were treated with increasing concentrations of CAPE for 96 h to determine suppressive effect of CAPE on cell proliferation. Relative cell number was determined by either Hoechst 33258 fluorescence based-96 well proliferation assay or by MTT assay. Relative cell number was normalized to cell number of control (no treatment). (B) LNCaP 104-R1 cells were treated with increasing concentrations of CAPE for 24, 48, 72, 96 h to investigate the suppressive effects of CAPE. Relative cell number was normalized to cell number of control (no treatment) at 24 h and was determined by Hoechst 33258 fluorescence based-96 well proliferation assay. (C) LNCaP 104-R1, PC-3, DU-145, LNCaP C4–2, and 22Rv1 cells were treated with increasing concentrations of CAPE for 96 h to investigate the suppressive effects of CAPE. Relative cell number determined by Hoechst 33258 fluorescence based-96 well proliferation assay and was normalized to cell number of control (no treatment) for individual cell line. Asterisks *, **, and *** represented statistical significance in cell number of p < 0.05, p < 0.01, and p < 0.001, respectively, as compared to that of control. (D) Anticancer effect of CAPE was confirmed by the colony formation assay of LNCaP 104-R1 cells treated with 0, 10, or 20 μM CAPE for 14 days. Image is representative of three biological replicates.

Mentions: Treatment of CAPE (dissolved in ethanol) at 10–40 μM for 96 h significantly reduced the cell number of AR-rich androgen-independent LNCaP 104-R1 cells dose-dependently as determined by light microscopy (Supplementary Figure 1). The ethanol control did not affect cell number of LNCaP 104-R1 cells as compared to no treatment (data not shown). Examination using fluorescent microscopy with Hoechst dye staining and DAPI staining indicated that cell survival and proliferation of commonly used CRPC cell lines, including LNCaP 104-R1 (Figure 1), AR-positive 22Rv1 (Supplementary Figure 2), AR-negative DU-145 (Supplementary Figure 3), and AR-positive LNCaP C4–2 cells (Supplementary Figure 4) were all significantly suppressed by CAPE treatment dose-dependently. The suppressive effects of CAPE on survival of CRPC cells were further confirmed by MTT assay and Hoechst 33258 96-well proliferation assay. MTT assay and Hoechst 33258 proliferation assay indicated an IC50 of 16.5 μM and 18.9 μM, respectively, for CAPE to cause growth inhibition on LNCaP 104-R1 cells (Figure 2A). The growth inhibitory effect of CAPE was evident within 24 hours of treatment but the suppressive effect accumulated over time (Figure 2B). The IC50 of 24, 48, 72, and 96 h CAPE treatment on LNCaP 104-R1 cells was 64.0, 30.5, 20.5, and 18.0 μM, respectively. We compared the sensitivity of LNCaP 104-R1 cells to CAPE treatment with the four other CRPC cell lines. CAPE treatment dosage-dependently suppressed the proliferation of LNCaP 104-R1, LNCaP C4–2, 22Rv1, PC-3, and DU-145 cells (Figure 2C) with an IC50 of 18.9, 10.9, 19.1, 23.2, and 22.6 μM, respectively. CAPE treatment caused the CRPC cells to proliferate slower. The doubling time of LNCaP 104-R1, LNCaP C4–2, 22Rv1, and DU-145 is 30.7, 37.4, 37.4, and 36.0 h, respectively. Under the treatment of 10 μM CAPE, the doubling time of these cells increased to 47.5, 75.8, 106.8, and 40.5 h, respectively. When being treated with 20 μM CAPE, the doubling time of LNCaP 104-R1 and DU-145 further extended to 68.6 and 44.2 h, respectively. We did not examine the doubling time of LNCaP C4–2 and 22Rv1 under treatment of 20 μM CAPE, as they proliferated too slow under this condition. Colony formation assay revealed that treatment with 10 μM CAPE reduced colony formation of LNCaP 104-R1 cells by 90% while treatment with 20 μM CAPE completely blocked the formation of LNCaP 104-R1 colonies (Figure 2D). These results confirmed the anti-cancer effect of CAPE against CRPC cells.


Caffeic acid phenethyl ester induced cell cycle arrest and growth inhibition in androgen-independent prostate cancer cells via regulation of Skp2, p53, p21Cip1 and p27Kip1.

Lin HP, Lin CY, Huo C, Hsiao PH, Su LC, Jiang SS, Chan TM, Chang CH, Chen LT, Kung HJ, Wang HD, Chuu CP - Oncotarget (2015)

CAPE treatment dose-dependently reduced cell survival, proliferation, and soft agar colony formation of CRPC cells(A) LNCaP 104-R1 cells were treated with increasing concentrations of CAPE for 96 h to determine suppressive effect of CAPE on cell proliferation. Relative cell number was determined by either Hoechst 33258 fluorescence based-96 well proliferation assay or by MTT assay. Relative cell number was normalized to cell number of control (no treatment). (B) LNCaP 104-R1 cells were treated with increasing concentrations of CAPE for 24, 48, 72, 96 h to investigate the suppressive effects of CAPE. Relative cell number was normalized to cell number of control (no treatment) at 24 h and was determined by Hoechst 33258 fluorescence based-96 well proliferation assay. (C) LNCaP 104-R1, PC-3, DU-145, LNCaP C4–2, and 22Rv1 cells were treated with increasing concentrations of CAPE for 96 h to investigate the suppressive effects of CAPE. Relative cell number determined by Hoechst 33258 fluorescence based-96 well proliferation assay and was normalized to cell number of control (no treatment) for individual cell line. Asterisks *, **, and *** represented statistical significance in cell number of p < 0.05, p < 0.01, and p < 0.001, respectively, as compared to that of control. (D) Anticancer effect of CAPE was confirmed by the colony formation assay of LNCaP 104-R1 cells treated with 0, 10, or 20 μM CAPE for 14 days. Image is representative of three biological replicates.
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Figure 2: CAPE treatment dose-dependently reduced cell survival, proliferation, and soft agar colony formation of CRPC cells(A) LNCaP 104-R1 cells were treated with increasing concentrations of CAPE for 96 h to determine suppressive effect of CAPE on cell proliferation. Relative cell number was determined by either Hoechst 33258 fluorescence based-96 well proliferation assay or by MTT assay. Relative cell number was normalized to cell number of control (no treatment). (B) LNCaP 104-R1 cells were treated with increasing concentrations of CAPE for 24, 48, 72, 96 h to investigate the suppressive effects of CAPE. Relative cell number was normalized to cell number of control (no treatment) at 24 h and was determined by Hoechst 33258 fluorescence based-96 well proliferation assay. (C) LNCaP 104-R1, PC-3, DU-145, LNCaP C4–2, and 22Rv1 cells were treated with increasing concentrations of CAPE for 96 h to investigate the suppressive effects of CAPE. Relative cell number determined by Hoechst 33258 fluorescence based-96 well proliferation assay and was normalized to cell number of control (no treatment) for individual cell line. Asterisks *, **, and *** represented statistical significance in cell number of p < 0.05, p < 0.01, and p < 0.001, respectively, as compared to that of control. (D) Anticancer effect of CAPE was confirmed by the colony formation assay of LNCaP 104-R1 cells treated with 0, 10, or 20 μM CAPE for 14 days. Image is representative of three biological replicates.
Mentions: Treatment of CAPE (dissolved in ethanol) at 10–40 μM for 96 h significantly reduced the cell number of AR-rich androgen-independent LNCaP 104-R1 cells dose-dependently as determined by light microscopy (Supplementary Figure 1). The ethanol control did not affect cell number of LNCaP 104-R1 cells as compared to no treatment (data not shown). Examination using fluorescent microscopy with Hoechst dye staining and DAPI staining indicated that cell survival and proliferation of commonly used CRPC cell lines, including LNCaP 104-R1 (Figure 1), AR-positive 22Rv1 (Supplementary Figure 2), AR-negative DU-145 (Supplementary Figure 3), and AR-positive LNCaP C4–2 cells (Supplementary Figure 4) were all significantly suppressed by CAPE treatment dose-dependently. The suppressive effects of CAPE on survival of CRPC cells were further confirmed by MTT assay and Hoechst 33258 96-well proliferation assay. MTT assay and Hoechst 33258 proliferation assay indicated an IC50 of 16.5 μM and 18.9 μM, respectively, for CAPE to cause growth inhibition on LNCaP 104-R1 cells (Figure 2A). The growth inhibitory effect of CAPE was evident within 24 hours of treatment but the suppressive effect accumulated over time (Figure 2B). The IC50 of 24, 48, 72, and 96 h CAPE treatment on LNCaP 104-R1 cells was 64.0, 30.5, 20.5, and 18.0 μM, respectively. We compared the sensitivity of LNCaP 104-R1 cells to CAPE treatment with the four other CRPC cell lines. CAPE treatment dosage-dependently suppressed the proliferation of LNCaP 104-R1, LNCaP C4–2, 22Rv1, PC-3, and DU-145 cells (Figure 2C) with an IC50 of 18.9, 10.9, 19.1, 23.2, and 22.6 μM, respectively. CAPE treatment caused the CRPC cells to proliferate slower. The doubling time of LNCaP 104-R1, LNCaP C4–2, 22Rv1, and DU-145 is 30.7, 37.4, 37.4, and 36.0 h, respectively. Under the treatment of 10 μM CAPE, the doubling time of these cells increased to 47.5, 75.8, 106.8, and 40.5 h, respectively. When being treated with 20 μM CAPE, the doubling time of LNCaP 104-R1 and DU-145 further extended to 68.6 and 44.2 h, respectively. We did not examine the doubling time of LNCaP C4–2 and 22Rv1 under treatment of 20 μM CAPE, as they proliferated too slow under this condition. Colony formation assay revealed that treatment with 10 μM CAPE reduced colony formation of LNCaP 104-R1 cells by 90% while treatment with 20 μM CAPE completely blocked the formation of LNCaP 104-R1 colonies (Figure 2D). These results confirmed the anti-cancer effect of CAPE against CRPC cells.

Bottom Line: CAPE treatment decreased Skp2 and Akt1 protein expression in LNCaP 104-R1 tumors as compared to control group.Overexpression of Skp2, or siRNA knockdown of p21Cip1, p27Kip1, or p53 blocked suppressive effect of CAPE treatment.Co-treatment of CAPE with PI3K inhibitor LY294002 or Bcl-2 inhibitor ABT737 showed synergistic suppressive effects.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Cancer Research, National Health Research Institutes, Miaoli, Taiwan, ROC.

ABSTRACT
Prostate cancer (PCa) patients receiving the androgen ablation therapy ultimately develop recurrent castration-resistant prostate cancer (CRPC) within 1-3 years. Treatment with caffeic acid phenethyl ester (CAPE) suppressed cell survival and proliferation via induction of G1 or G2/M cell cycle arrest in LNCaP 104-R1, DU-145, 22Rv1, and C4-2 CRPC cells. CAPE treatment also inhibited soft agar colony formation and retarded nude mice xenograft growth of LNCaP 104-R1 cells. We identified that CAPE treatment significantly reduced protein abundance of Skp2, Cdk2, Cdk4, Cdk7, Rb, phospho-Rb S807/811, cyclin A, cyclin D1, cyclin H, E2F1, c-Myc, SGK, phospho-p70S6kinase T421/S424, phospho-mTOR Ser2481, phospho-GSK3α Ser21, but induced p21Cip1, p27Kip1, ATF4, cyclin E, p53, TRIB3, phospho-p53 (Ser6, Ser33, Ser46, Ser392), phospho-p38 MAPK Thr180/Tyr182, Chk1, Chk2, phospho-ATM S1981, phospho-ATR S428, and phospho-p90RSK Ser380. CAPE treatment decreased Skp2 and Akt1 protein expression in LNCaP 104-R1 tumors as compared to control group. Overexpression of Skp2, or siRNA knockdown of p21Cip1, p27Kip1, or p53 blocked suppressive effect of CAPE treatment. Co-treatment of CAPE with PI3K inhibitor LY294002 or Bcl-2 inhibitor ABT737 showed synergistic suppressive effects. Our finding suggested that CAPE treatment induced cell cycle arrest and growth inhibition in CRPC cells via regulation of Skp2, p53, p21Cip1, and p27Kip1.

Show MeSH
Related in: MedlinePlus