Chalcones and dihydrochalcones augment TRAIL-mediated apoptosis in prostate cancer cells

Abstract

Chalcones and dihydrochalcones exhibit chemopreventive and antitumor activity. TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a natural endogenous anticancer agent. We examined the cytotoxic and apoptotic effect of chalcones and dihydrochalcones on TRAIL-mediated apoptosis in LNCaP prostate cancer cells. The cytotoxicity was evaluated by the MTT and LDH assays. The apoptosis was detected using annexin V-FITC by flow cytometry and fluorescence microscopy. The DeltaPsim was evaluated using DePsipher staining by fluorescence microscopy. Our study showed that two tested chalcones (chalcone and 2',6'dihydroxy-4'-methoxychalcone) and three dihydrochalcones (2',6'-dihydroxy-4'4-dimethoxydihydrochalcone, 2',6'-dihydroxy-4'-methoxydihydro- chalcone, and 2',4',6'-trihydroxydihydrochalcone, called phloretin) markedly augmented TRAIL-induced apoptosis and cytotoxicity in LNCaP cells and confirmed the significant role of chalcones in chemoprevention of prostate cancer.

Figure 1

Chemical structures of the studied chalcones and dihydrochalcones.

Figure 2

Cytotoxic and apoptotic effects of chalcones and dihydrochalcones on LNCaP prostate cancer cells. The cancer cells were incubated for 48 hours with compounds C1–C5 at the concentrations of 100 μM. The values represent mean ±SD of three independent experiments performed in quadruplicate (n = 12) for cytototoxicity, or in duplicate (n = 6) for apoptosis (p < 0.05). (A) Cytotoxic activity of chalcones and dihydrochalcones in LNCaP cells. The percentage of cell death was measured by MTT cytotoxicity assay. (B) Apoptotic activity of chalcones and dihydrochalcones in LNCaP cells. Detection of apoptotic cell death by annexin V-FITC staining using flow cytometry.

Figure 3

Cytotoxic and apoptotic effect of TRAIL on LNCaP prostate cancer cells. The cancer cells were incubated for 48 hours with TRAIL at the concentrations of 20–200 ng/mL. The values represent mean ±SD of three independent experiments performed in quadruplicate (n = 12) for cytototoxicity, or in duplicate (n = 6) for apoptosis (p < 0.05). (A) Cytotoxic activity of TRAIL in LNCaP cells. The percentage of cell death was measured by MTT cytotoxicity assay. (B) TRAIL-induced apoptosis in LNCaP cells. Detection of apoptotic cell death by annexin V-FITC staining using flow cytometry.

Figure 4

Cytotoxic activity of TRAIL in combination with chalcones and dihydrochalcones in LNCaP prostate cancer cells. The cancer cells were incubated for 48 hours with TRAIL at the concentrations of 20–100 ng/mL and the compounds C1–C5 at the concentrations of 50–100 μM. The percentage of cell death was measured by MTT cytotoxicity assay. The values represent mean ±SD of three independent experiments performed in quadruplicate (n = 12). ^*** = significantly different from control or TRAIL (p < 0.0001).

Figure 5

Statistical analysis of cytotoxic activity of TRAIL in combination with chalcones and dihydrochalcones in LNCaP prostate cancer cells. The data are expressed as mean ±SD (n = 12) (p < 0.05).

Figure 6

TRAIL induced apoptosis in combination with chalcones and dihydrochalcones in LNCaP prostate cancer cells. The cancer cells were incubated for 48 hours with TRAIL at the concentrations of 50–100 ng/mL and the compounds C1–C5 at the concentrations of 50–100 μM. Detection of apoptotic cell death by annexin V-FITC staining using flow cytometry. The values represent mean ±SD of three independent experiments performed in duplicate (n = 6). ^*** = significantly different from control or TRAIL (p < 0.0001).

Figure 7

Statistical analysis of apoptotic activity of TRAIL in combination with chalcones and dihydrochalcones in LNCaP prostate cancer cells. The data are expressed as mean ±SD (n = 6) (p < 0.05).

Figure 8

TRAIL in combination with chalcone induced apoptosis in LNCaP prostate cancer cells. The cancer cells were incubated for 48 hours with TRAIL at the concentration of 100 ng/mL and chalcone at the concentration of 100 μM. Detection of apoptotic cell death by annexin V-FITC staining using fluorescent microscopy. The healthy cells (stained with Hoechst 33342) emitted blue fluorescence and apoptotic cells. Apoptotic cells (stained with Annexin V-FITC and Hoechst 33342) emitted green and blue fluorescence (indicated by arrows). (A) Control cells, (B) cells incubated with TRAIL (100 ng/mL), (C) cells incubated with chalcone (100 μM), (D) cells incubated with TRAIL (100 ng/mL) and chalcone (100 μM).

Figure 9

TRAIL in combination with chalcone altered the mitochondrial transmembrane potential (ΔΨm) and induced apoptosis in LNCaP prostate cancer cells. The cancer cells were incubated for 48 hours with TRAIL at the concentration of 100 ng/mL and chalcone at the concentration of 100 μM. DePsipher has the property of aggregation upon membrane polarization forming a red fluorescent compound. If the potential is disturbed, the dye cannot access the transmembrane space and remains or reverts to its green monomeric form. The fluorescence is observed and analyzed by microscope. Apoptotic cells with loss ΔΨm are indicated by arrows. (A) Control cells, (B) cells incubated with TRAIL (100 ng/mL), (C) cells incubated with chalcone (100 μM), (D) cells incubated with TRAIL (100 ng/mL) and chalcone (100 μM).