Methoxyflavone derivatives modulate the effect of TRAIL-induced apoptosis in human leukemic cell lines

5,7-Dimethoxyflavone (DMF), 5,7,4'-trimethoxyflavone (TMF) and 3,5,7,3',4'-pentamethoxyflavone (PMF), which were isolated and purified from rhizomes of K. parviflora as previously described [6]. 5-Methoxyflavone, 2'-methoxyflavone, histopaque, MTT (3-(4,5-dimethyl)-2,5-diphenyl tetrazolium bromide, propidium iodide (PI), 3,3'-dihexyloxacarbocyanine iodide (DiOC₆) and 2',7'-dichlorofluorescin diacetate (DCFH-DA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). TRAIL was obtained from R&D system, USA. RPMI-1640 medium, DEVD-AFC (Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin) and IETD-AFC (Ile-Glu-Thr-Asp-amino-4-trifluoromethylcoumarin) were obtained from Invitrogen, USA. Mouse monoclonal antibodies to Mcl-1, BAX and rabbit polyclonal antibody to Bid, cFLIP and horseradish peroxidase (HRP) conjugated secondary antibodies were purchased from Abcam, Cambridge, UK. Mouse monoclonal antibodies to beta-actin, DR4 and DR5 were obtained from Santa Cruz Biotechnology, USA. SuperSignal West Pico Chemiluminecent Substrate was obtained from Pierce, Rockford, IL, USA. Annexin V-Fluos staining kit and complete mini protease inhibitor cocktail was obtained from Roche, Basel, Switzerland.

Human lymphoblastic leukemic MOLT-4 and monocytic U937 cells were gifts from Professor Watchara Kasinroek (Faculty of Associated Medical Sciences, Chiang Mai University). Peripheral blood mononuclear cells (PBMCs) were donated from healthy volunteers. PBMCs were isolated from heparinized blood by density gradient centrifugation using histopaque according to standard protocols. The blood was obtained from adult volunteers with Institutional Review Board approval, at Faculty of Medicine, Chiang Mai University. The cells were cultured in RPMI-1640 medium with 25 mM NaHCO₃, 20 mM HEPES, 100 units/mL penicillin, 100 μ g/mL streptomycin and supplemented with 10% fetal bovine serum. The cell lines were grown at 37°C in a 5% CO₂ atmosphere. The PBMCs and human leukemic cells (1 × 10⁶) were treated with MF derivatives at indicated concentrations and durations. MF derivatives were dissolved in DMSO as a vehicle and the maximal volume used did not exceed 10 μ l/ml of media.

Following MF derivative treatment, cell viability was assessed by the MTT (3-(4,5-dimethyl)-2,5-diphenyl tetrazolium bromide) assay [10]. This method is based on the ability of viable cells to reduce MTT and form a blue formazan product. MTT solution (sterile stock solution of 5 mg/ml) was added to cell suspension at a final concentration of 100 μ g/ml and the solution incubated for 4 h at 37°C in a humidified 5% CO₂ atmosphere. The medium was then removed and cells were treated with DMSO for 30 min. The optical density of the cell lysate was measured at 540 nm, with a reference wavelength of 630 nm, using a microtiter plate reader (Biotek, USA). The number of viable cells was calculated from the number of untreated cells, and the data were expressed as percent cell viability.

After treatment with methoxyflavone derivatives at a concentration of IC₂₀ for 0, 3, 6, 12, 18 and 24 h, the cells were washed with PBS and centrifuged at 200 × g for 5 minutes and suspended in 100 μ l of binding buffer from a kit containing annexin V-FITC and PI, for 15 min. The samples were analyzed using a flow cytometer (Beckton Dickinson, USA).

To measure mitochondrial membrane potential and intracellular ROS, either 40 nM 3,3'-dihexyloxacarbocyanine iodide (for mitochondrial transmembrane potential determination) or 5 μM 2',7'-dichlorofluorescin diacetate (for ROS detection) were added for 15 min at 37°C and the cells were then subjected to flow cytometry.

Cleavage of the fluorogenic peptide substrates DEVD-AFC and IETD-AFC was used to assay caspase-3-like and caspase-8-like enzyme activity. Cell lysates (1×10⁶ cells) and substrate (50 μM) were combined in a standard reaction buffer and added to a 96-well plate. Enzyme-catalyzed release of AFC was measured by a fluorescence plate reader (Bio-tek, USA) using 355 nm excitation and 460 nm emission wavelengths.

To obtain a cytosolic-rich fraction, MF derivative-treated cells were harvested and washed once in ice cold PBS and incubated at 4°C for 10 min with ice-cold cell lysis buffer (137 mM NaCl, 15 mM EGTA, 0.1 mM Na₂VO₄, 15 mM MgCl₂, 1% Triton X-100, with complete mini protease inhibitor cocktail). The cell suspension was centrifuged at 20,000 × g for 20 min. The supernatant was collected as the cytosolic-rich fraction. Protein concentration of the cytosolic-rich fraction was determined by the Bradford method. Cytosolic proteins (50 μg) were separated by 17% SDS-PAGE and transferred onto nitrocellulose membranes. After treating with 5% non-fat milk in TBS containing 0.2% Tween-20 (blocking buffer), membranes were incubated with mouse monoclonal antibodies to DR4, DR5, BAX and Mcl-1 and rabbit polyclonal antibody to Bid and cFLIP. For detection, appropriate horseradish peroxidase (HRP) conjugated secondary antibodies were used at 1:20,000 dilution. Protein bands were visualized on X-ray film with SuperSignal West Pico Chemiluminecent Substrate.

Results were expressed as mean ± S.D. (standard deviation). Statistical difference between control and treated group was determined by a nonparametric one-way ANOVA (Kruskal Wallis test) with a limit of p < 0.05 in three independent experiments. For comparison between two groups, data were analyzed using Student's t-test.

All five MF derivatives were toxic to MOLT-4 and U937 cells with IC₅₀ values as shown in Figure 1 and Table 1. DMF, TMF and PMF were not cytotoxic to PBMCs whereas 5-MF and 2'-MF were also toxic to PBMCs (Figure 1). TRAIL was not cytotoxic to U937 and MOLT-4 cells, viz. both cell lines were resistant to TRAIL (Figure 2). The IC₂₀ level of MF derivatives was chosen for further experiments. In TRAIL combination with MF, MOLT-4 cells were induced to die more in a dose response manner (Figure 2).

5-MF induced MOLT-4 cell apoptosis mostly at 3 h when combined with TRAIL for 24 h (Figure 3a) and 2'-Methoxyflavone induced apoptosis (the time dependence is shown in Figure 3b). Percent apoptotic cells increased when combined with TRAIL compared to the absence of TRAIL. Meanwhile DMF, TMF and PMF also induced MOLT-4 cell apoptosis synergistically to TRAIL (Figure 3c). Notably, all five MF derivatives and TRAIL synergistically induced MOLT-4 cell apoptosis, but this phenomenon did not occur in U937 cell line (data not shown).

The percentage of MOLT-4 cells (treated with 5-MF and/or TRAIL) with reduction of MTP was increased more than those treated with 5-MF alone at 3, 6, 12, 18 and 24 h (Figure 4A). The combined treatment also increased the cells with mitochondrial transmembrane reduction compared to TRAIL alone.

For the 2'-MF treatment for 24 h without TRAIL, percent cells with reduced mitochondrial transmembrane potential was increased compared to other incubation time points. The percentage of cells with loss of mitochondrial transmembrane potential significantly increased when treated with TRAIL for 24 h compared to TRAIL alone (control) as shown in Figure 4B.

For DMF, TMF and PMF treatment for 24 h in combination with TRAIL for another 24 h, the percentage of cells with reduction of mitochondrial transmembrane potential significantly increased when compared with control or TRAIL alone (Figure 4C).

Curcumin [11] and zerumbone [12] could induce cancer cells to undergo apoptosis via the excessive production of ROS. This led to the investigation of the ROS production in MF derivative (5-MF and 2'-MF) treatment in the MOLT-4 cells. When MOLT-4 cells were treated with 5-MF and measured for ROS production, the fluorescence intensity of DCF increased, with the maximum effect at 30 min treatment. For incubation with 2'-MF, ROS was greatest at 60 min treatment (Figure 5).

To determine how 5-MF and 2'-MF could facilitate TRAIL-induced apoptosis, the effects of both MF derivatives on TRAIL receptor (DR4 and DR5) expression were determined. MOLT-4 cells were treated with 5-MF or 2'-MF for various incubation times. The whole cell extracts were prepared and examined for the expression of DR4 and DR5. 5-MF induced both DR4 and DR5 (Figure 6A and 6B) expression in a time dependent manner, whereas the expression of DR4 and DR5 induced by 2'-MF was unaltered (Figure 6C and 6D). These results indicated that 5-MF up-regulated the expression of both DR4 and DR5.

The treatment with 5-MF at various times (3, 6, 12, 18 and 24 h) resulted in a time dependent reduction in the levels of antiapoptotic proteins cFLIP, Mcl-1 and an increase in the proapoptotic protein BAX (Figure 7). BH3 domain-only protein Bid (proform, 22 kDa) was reduced due to the cleavage to obtain the truncated Bid (tBid, 15 kDa). The 2'-MF treated cells had a lesser effect on the reduction of Bid, antiapoptotic protein cFLIP and Mcl-1 and an increase of BAX compared to 5-MF (Figure 7).

Caspase-8 is activated by association with death ligand and receptor activation via the extrinsic pathway. In MOLT-4 cells treated with 5-MF and TRAIL, caspase-8 activity increased at 3 and 6 h of 5-MF treatment (Figure 8A). For the 2'-MF and TRAIL treatment, the caspase-8 activity increased between 18 and 24 h (Figure 8B). When MOLT-4 cells were treated with MF derivatives alone, caspase-8 activity did not change. In the presence of TRAIL, 5-MF and 2'-MF induced MOLT-4 cell apoptosis via the extrinsic pathway by activation of caspase-8 activity.

Caspase-3 plays a central role in the apoptotic cascade. When MOLT-4 cells were treated with 5-MF combined with TRAIL, caspase-3 activity increased at 3, 6, 12, 18 and 24 h of treatment (Figure 8C). MOLT-4 cells treated with 2'-MF and TRAIL, had increased caspase-3 activity at 3, 6, 12, 18 and 24 h of treatment (Figure 8D). However, when the cells were treated with MF derivatives alone, caspase-3 activity was unaltered. In the combined treatment, MOLT-4 cells were activated to undergo apoptosis via caspase-3 activation.