Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis in Prostate Cancer Cells after Treatment with Xanthohumol-A Natural Compound Present in Humulus lupulus L

Abstract

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is an endogenous ligand, which plays role in immune surveillance and anti-tumor immunity. It has ability to selectively kill tumor cells showing no toxicity to normal cells. We tested the apoptotic and cytotoxic activities of xanthohumol, a prenylated chalcone found in Humulus lupulus on androgen-sensitive human prostate adenocarcinoma cells (LNCaP) in combination with TRAIL. Cytotoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium reduction assay (MTT) and lactate dehydrogenase assay (LDH). The expression of death receptors (DR4/TRAIL-R1 and DR5/TRAIL-R2) and apoptosis were detected using flow cytometry. We examined mitochondrial membrane potential (ΔΨm) by DePsipher reagent using fluorescence microscopy. The intracellular expression of proteins was evaluated by Western blotting. Our study showed that xanthohumol enhanced cytotoxic and apoptotic effects of TRAIL. The tested compounds activated caspases-3, -8, -9, Bid, and increased the expression of Bax. They also decreased expression of Bcl-xL and decreased mitochondrial membrane potential, while the expression of death receptors was not changed. The findings suggest that xanthohumol is a compound of potential use in chemoprevention of prostate cancer due to its sensitization of cancer cells to TRAIL-mediated apoptosis.

Keywords: TRAIL; apoptosis; prostate cancer cells; xanthohumol.

Figure 1

(A) Female strobiles [38]; (B) section through the mature strobili [39]; and (C) structure of xanthohumol.

Figure 2

Cytotoxic and apoptotic effects of TRAIL in combination with xanthohumol in LNCaP cancer cells. The values represent mean ± SD of three independent experiments performed in quadruplicate (n = 12), *** p < 0.001 compared with control, ^### p < 0.001 compared with xanthohumol, ⁺⁺⁺ p < 0.001 compared with TRAIL. (A) Cytotoxic effect of TRAIL in combination with xanthohumol in LNCaP cells; (B) apoptotic effect of TRAIL in combination with xanthohumol in LNCaP cells; (C) representative histograms: (1) control cells; (2) cells incubated with 100 ng/mL TRAIL; (3) cells incubated with 50 µM xanthohumol; (4) cells incubated with 100 ng/mL TRAIL + 50 µM xanthohumol. Red dots show apoptotic cells and gray dots live cells.

Figure 2

Cytotoxic and apoptotic effects of TRAIL in combination with xanthohumol in LNCaP cancer cells. The values represent mean ± SD of three independent experiments performed in quadruplicate (n = 12), *** p < 0.001 compared with control, ^### p < 0.001 compared with xanthohumol, ⁺⁺⁺ p < 0.001 compared with TRAIL. (A) Cytotoxic effect of TRAIL in combination with xanthohumol in LNCaP cells; (B) apoptotic effect of TRAIL in combination with xanthohumol in LNCaP cells; (C) representative histograms: (1) control cells; (2) cells incubated with 100 ng/mL TRAIL; (3) cells incubated with 50 µM xanthohumol; (4) cells incubated with 100 ng/mL TRAIL + 50 µM xanthohumol. Red dots show apoptotic cells and gray dots live cells.

Figure 3

Necrotic effect of TRAIL and/or xanthohumol in LNCaP cancer cells. The percentage of necrotic cells was measured by lactate dehydrogenase assay (LDH) cytotoxicity assay. The values represent mean ± standard deviation (SD) of three independent experiments performed in quadruplicate (n = 12). No statistically significant differences were shown.

Figure 4

Effect of xanthohumol on death receptors expression on surface of LNCaP cancer cells. The expression of (A) DR4/TRAIL-R1 and (B) DR5/TRAIL-R2 on cancer cells together with representative histograms determined by flow cytometry. The values represent mean ± SD of three independent experiments performed in quadruplicate (n = 12). Statistical significance has not been shown for the results compared to the control.

Figure 5

Mitochondrial membrane potential (ΔΨm) in LNCaP cancer cells. (A) TRAIL in combination with xanthohumol caused loss of ΔΨm in LNCaP cells. The values represent the mean ± SD of three independent experiments performed in duplicate n = 6 (*** p < 0.001 compared with control, ^### p < 0.001 compared with xanthohumol and ⁺⁺⁺ p < 0.001 compared with TRAIL); (B) The loss of ΔΨm in cancer cells was evaluated by fluorescent microscopic analysis of DePsipher staining: (1) control cells; (2) cells incubated with 100 ng/mL TRAIL; (3) cells incubated with 50 µM xanthohumol; and (4) cells incubated with 100 ng/mL TRAIL + 50 µM xanthohumol. In cells with intact cell membrane aggregates of DePsipher emitting red fluorescence are formed in mitochondria. Green fluorescence is evidence of the monomeric form of the DePsipher molecule that is present in the cytosol after mitochondrial membrane depolarization (indicated by arrows); magnification 200×.

Figure 6

Effect of TRAIL and/or xanthohumol on the protein expression in LNCaP cells. Cancer cells were treated with 50 µM xanthohumol and/or 100 ng/mL TRAIL for 2 or 8 h. The expressions of (A–C) caspases (-3, -8, -9) and (D–F) Bcl-2 family members (Bid, Bax, Bcl-xL) were measured by the Western blot technique. The results are expressed as means ± SD obtained from three independent experiments. The β-actin was used as a control to show equal loading of proteins. The data were normalized to the control and β-actin level. A statistical significance of the differences between the treatment and control results is marked with * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

Effect of TRAIL and/or xanthohumol on the protein expression in LNCaP cells. Cancer cells were treated with 50 µM xanthohumol and/or 100 ng/mL TRAIL for 2 or 8 h. The expressions of (A–C) caspases (-3, -8, -9) and (D–F) Bcl-2 family members (Bid, Bax, Bcl-xL) were measured by the Western blot technique. The results are expressed as means ± SD obtained from three independent experiments. The β-actin was used as a control to show equal loading of proteins. The data were normalized to the control and β-actin level. A statistical significance of the differences between the treatment and control results is marked with * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 7

Inhibition of the apoptotic effect of TRAIL in combination with xanthohumol on LNCaP cells after application of caspase inhibitors: -3 (iC-3), -8 (iC-8), -9 (iC-9), and inhibitor of all caspases (iVAD). LNCaP cells were incubated with 100 ng/mL TRAIL and/or 50 µM xantohumol and/or 20 µM caspase inhibitors for 24 h. Apoptotic cells were detected using flow cytometer with Apoptotest-FITC (*** p < 0.001 compared with TRAIL in combination with xanthohumol).