Butein induces apoptosis and inhibits prostate tumor growth in vitro and in vivo

Abstract

Aim: Prostate cancer (PCa) is one of the most common cancers in men in the United States with similar trends worldwide. For several reasons, it is an ideal candidate disease for intervention with dietary botanical antioxidants. Indeed, many botanical antioxidants are showing promise for chemoprevention of PCa. Here, we determined the effect of an antioxidant butein (3,4,2',4'-tetrahydroxychalone) on cell growth, apoptosis, and signaling pathways in human PCa cells in-vitro and on tumor growth in athymic nude mice.

Results: Treatment with butein (10-30 μM; 48 h) caused a decrease in viability of PCa cells but had only a minimal effect on normal prostate epithelial cells. In butein-treated cells, there was a marked decrease in the protein expression of cyclins D1, D2, and E and cdks 2, 4, and 6 with concomitant induction of WAF1/p21 and KIP1/p27. Treatment of cells with butein caused inhibition of (i) phosphatidylinositol 3-kinase (p85 and p110), (ii) phosphorylation of Akt at both Ser(473) and Thr(308), (iii) nuclear factor-kappa B (NF-κB) and IκB kinaseα, (iv) degradation and phosphorylation of IκBα, (v) NF-κB DNA-binding activity, (vi) induction of apoptosis, and (vii) Poly (ADP-ribose) polymerase cleavage with activation of caspases-3, -8, and -9. Pretreatment of cells with caspase inhibitor (Z-VAD-FMK) blocked butein-induced activation of caspases. In athymic nude mice implanted with human PCa cells, butein caused a significant inhibition of tumor growth with a decrease in the serum prostate-specific antigen levels.

Innovation: For the first time, we have shown that butein caused inhibition of prostate tumor growth in-vivo.

Conclusion: We suggest that butein could be developed as an agent against PCa. Antioxid. Redox Signal. 16, 1195-1204.

FIG. 1.

Structure of butein.

FIG. 2.

Effect of butein on cell viability. (A,B) Effect of butein on cell-growth. As detailed in “Materials and Methods,” LNCaP, CWR22Rν1, PC-3, and PrEC cells were treated with butein (10–30 μM) for 24 and 48 h, and the viability of cells was determined by the MTT assay. The data are expressed as the percentage of cell viability and represent the means±SE of three experiments in which each treatment was performed in multiple wells. *p<0.05, **p<0.01, and ***p<0.001 versus control group.

FIG. 3.

Effect of butein on cell-cycle regulatory molecules. (A) Effect of butein on protein expression of cyclin D1, cyclin D2, and cyclin E in LNCaP cells. (B) Effect of butein on protein expression of cdk2, cdk4, and cdk6 in LNCaP cells. (C) Effect of butein on protein expression of WAF1/p21 and KIP1/p27 in LNCaP cells. (D) Effect of butein on protein expression of cyclin D1, cdk2, p21, and p27 in CWR22Rν1 cells. As detailed in “Materials and Methods,” the cells were treated with butein (10–30 μM; 48 h) and then harvested. Total cell lysates were prepared, and 40 μg protein was subjected to SDS-PAGE followed by immunoblot analysis and chemiluminescence detection. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-actin. The immunoblots shown here are representative of three independent experiments with similar results. The values below the figures represent relative density of the bands normalized to β-actin.

FIG. 4.

Effect of butein on nuclear factor-kappa B, phosphatidylinositol 3-kinase and phosphorylation of Akt. (A) Effect of butein on phosphorylation of NF-κB, IKKα, and phosphorylation and degradation of IκBα. in LNCaP cells. (B) ELISA for the NFκB binding complex was demonstrated by anti-p65 antibody. (C) Effect of butein on protein expression of PI3K (p85 and p110) and phosphorylation of Akt at Ser⁴⁷³ and Thr⁴⁰⁸ in LNCaP cells. (D) Effect of butein on protein expression of PI3K (p85 and p110) and phosphorylation of Akt at Ser⁴⁷³ in CWR22Rν1 cells. As detailed in “Materials and Methods,” the cells were treated with butein (10–30 μM; 48 h) and then harvested. Total cell lysates were prepared, and 40 μg protein was subjected to SDS-PAGE followed by immunoblot analysis and chemiluminescence detection. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-actin. The immunoblots shown here are representative of 3 independent experiments with similar results. The values below the figures represent relative density of the bands normalized to β-actin. IKKα, IκB kinase; ELISA, enzyme-linked immunosorbent assay; PI3K, phosphatidylinositol 3-kinase; NF-κB, nuclear factor-kappa B.

FIG. 5.

Effect of butein on poly (ADP-ribose) polymerase, Bax and Bcl2. (A) Effect of butein on cleavage of PARP in LNCaP cells. (B) Effect of butein on protein expression of Bax and Bcl2 in LNCaP cells. (C) Effect of butein on protein expression of Bax and Bcl2 in CWR22Rν1 cells. (D) Effect of butein on Bax/Bcl2 ratio in LNCaP cells. (E) Effect of butein on Bax/Bcl2 ratio in CWR22Rν1 cells. As detailed in “Materials and Methods,” the cells were treated with butein (10–30 μM; 48 h) and then harvested. Total cell lysates were prepared, and 40 μg protein was subjected to SDS-PAGE followed by immunoblot analysis and chemiluminescence detection. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-actin. The immunoblots shown here are representative of 3 independent experiments with similar results. The values below the figures represent relative density of the bands normalized to β-actin. PARP, poly (ADP-ribose) polymerase.

FIG. 6.

Effect of butein on induction of apoptosis and activation of caspases. (A) Effect of butein and Z-VAD-FMK on apoptosis. LNCaP cells were cells were incubated with 40 μM concentration of the general caspase inhibitor Z-VAD-FMK for 2 h, followed by addition of butein (20 μM; 48 h) and then harvested. The fluorescence was measured by a Nikon Eclipse Ti system (Nikon Instruments, Inc.). The details are described under “Materials and Methods,” and the data shown here are from one representative experiment repeated twice with similar results, magnification ×20 (To see this illustration in color, the reader is referred to the web version of this article at www.liebertonline.com/ars). (B) Effect of butein on protein expression of active caspases-3, -8, and -9 in LNCaP cells. (C) Effect of Z-VAD-FMK on butein-induced activation of caspases in LNCaP cells. As detailed in “Materials and Methods,” cells were incubated with 40 μM concentration of the general caspase inhibitor Z-VAD-FMK for 2 h, followed by addition of butein (20 μM; 48 h) and then harvested. Total cell lysates were prepared, and 40 μg protein was subjected to SDS-PAGE followed by immunoblot analysis and chemiluminescence detection. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-actin. The immunoblots shown here are representative of 3 independent experiments with similar results. The values below the figures represent relative density of the bands normalized to β-actin. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 7.

Effect of butein on human prostate cancer tumor growth, serum prostate-specific antigen levels, and on expression of Ki-67, vascular endothelial growth factor, and CD31. (A) Effect of butein on CWR22Rν1 tumor growth athymic nude mice. Twenty-four animals were then randomly divided into eight animals in group 1 and sixteen animals in group 2. Approximately one million CWR22Rν1 cells were s.c. injected in each flank of mouse to initiate tumor growth. Twenty-four hours after cell implantation, 8 animals of the first group of animals received an i.p. injection of DMSO (30 μl) and served as a control. The 16 animals of group 2 received an i.p. injection of butein (1 mg/animal) in 30 μl of DMSO twice weekly. Once tumors started to grow, their sizes were measured weekly, and the tumor volume was calculated. Average tumor volume of the control group and butein-treated mice plotted over days after tumor cell inoculation. Values represent mean±SD of 16 tumors in eight animals. *p<0.05, **p<0.01, and ***p<0.001 versus control group. (B) Number of mice remaining with tumor volumes of 1,200 mm³ after they received treatment with butein for the indicated days. (C) Serum PSA levels were analyzed by ELISA. Values represent mean±SE of eight animals. ***p<0.001 versus control group of mice. Details are described in “Materials and Methods.” PSA, prostate-specific antigen; DMSO, dimethyl sulfoxide. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 8.

Immunohistochemical staining showing the effect of butein on expression of Ki-67, VEGF, and CD31 in tumor sections of athymic nude mice. Tumor sections from control and butein-treated mice were stained using specific antibodies as detailed in “Materials and Methods.” Counterstaining was performed with hematoxylin. Sacle bar, 50 μm. Photomicrographs (magnification,×20) show representative pictures from two independent samples (To see this illustration in color, the reader is referred to the web version of this article at www.liebertonline.com/ars). VEGF, vascular endothelial growth factor.