Apoptosis-inducing Effect of Hydroquinone 5-O-Cinnamoyl Ester Analog of Renieramycin M on Non-small Cell Lung Cancer Cells

Materials and Methods

Preparation of CIN-RM. A solution of RM (15.0 mg, 0.026 mmol) in EtOAc (10 ml) was added with 20% Pd(OH)₂ on carbon (7.5 mg, 50% w/w). A hydrogen balloon was attached to the reaction flask. The heterogeneous reaction was stirred vigorously at room temperature (25°C) and 1 atm for 5 h. The reaction was filtered through a pad of celite and washed with EtOAc (10 ml, 3 times) and CHCl₃ (10 ml, 3 times). The filtrates were combined and concentrated in vacuo to yield HQ-RM as a colorless solid. The product was employed in the next step without further purification. Next, the resulted HQ-RM was placed in an oven dried-rounded bottom flask. To a solution of HQ-RM (15.0 mg, 0.026 mmol) in dry CH₂Cl₂ (5 ml) was stirred at room temperature (25°C) under Ar atmosphere. The reaction was added 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI.HCl, 4.0 mg, 0.026 mmol) and 4-dimethylaminopyridine (DMAP, 3.2 mg, 0.026 mmol), followed by cinnamoyl chloride (4.3 mg, 0.026 mmol). The reaction mixture was stirred for 3 h. After completion, the reaction mixture was quenched by addition of water (5 ml). The organic layer was separated by separatory funnel and the aqueous layer was extracted with CHCl3 (10 ml, 3 times). The organic layers were combined, washed with brine (30 ml), dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. Purification by the silica gel flash chromatography eluting with hexanes:EtOAc (3:1) gave 7.9 mg (43%) of CIN-RM as a yellow amorphous solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.8 (1H, d, J=16.0 Hz, CH=CH_β), 7.6 (2H, m, H-3’and H-5’), 7.5 (3H, m, H-2’, H-6’, and H-4’), 6.7 (1H, d, J=16.0 Hz, CH_α=CH), 6.0 (1H, qq, J=7.4, 1.2 Hz, H-26), 5.8 (1H, s, OH-8), 4.5 (1H, d, J=11.2 Hz, H-22b), 4.3 (1H, br t, J=4.0 Hz, H-1), 4.1 (1H, d, J=2.0 Hz, H-21), 4.1 (1H, dd, J=11.2, 4.0 Hz, H-22a), 4.0 (1H, br d, J=3.6 Hz, H-11), 3.9 (3H, s, 17-OCH₃), 3.8 (3H, s, 7-OCH₃), 3.4 (1H, dt, J=7.2, 2.0 Hz, H-13), 3.2 (1H, d, J=12.0 Hz, H-3), 2.8 (1H, dd, J=20.8, 7.2 Hz, H-14α), 2.6 (1H, dd, J=14.0, 2.0 Hz, H-4α), 2.3 (1H, d, J=20.8 Hz, H-14β), 2.3 (3H, s, 12-NCH₃), 2.1 (3H, s, 6-CH₃), 1.9 (3H, dq, J=7.2, 1.4 Hz, H₃-27), 1.9 (3H, s, 16-CH₃), 1.7 (3H, dq, J=1.4, 1.2 Hz, H₃-28), 1.7 (1H, overlapped, H-4β); ¹³C NMR (CDCl₃, 100 MHz) δ 186.0 (C-15), 182.6 (C-18), 167.1 (C-24), 164.7 (5-OC=O), 155.5 (C-17), 146.8 (CH=CH_β), 143.8 (C-7), 143.1 (C-8), 141.5 (C-20), 140.0 (C-26), 139.1 (C-5), 135.7 (C-19), 134.1 (C-1’), 131.0 (C-4’), 129.1 (C-2’ and C-6’), 128.8 (C-16), 128.4 (C-3’ and C-5’), 126.8 (C-25), 124.5 (C-10), 122.6 (C-6), 117.6 (21-CN), 117.2 (C, C-9), 116.4 (CH_α=CH), 64.5 (C-22), 61.2 (7-OCH₃), 60.6 (17-OCH₃), 59.5 (C-21), 56.5 (C-1), 55.3 (C-3), 54.9 (C-13), 54.6 (C-11), 41.5 (12-NCH₃), 27.8 (C-4), 21.1 (C-14), 20.6 (C-28), 15.9 (C-27), 10.1 (6-CH₃), 8.6 (16-CH₃). Chemicals were purchased from Tokyo Chemical Industry. NMR spectroscopy was performed on JEOL-JNM AL 400 FT-NMR spectrometer (19).

Chemicals. Hoechst 33342, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethysulfoxide (DMSO), trypsin and phosphate-buffered saline (PBS) were obtained from Sigma Chemical, Inc. (St. Louis, MO, USA). Antibodies to apoptosis-inducing factor (AIF), phosphorylated AKT (p-AKT), total AKT, p53, BCL2, BAX, poly ADP ribose polymerase (PARP), caspase-3, caspase-9, and β-actin were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Cell culture and treatment. H292 Non-small cell lung cancer cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum, 2 mmol/l L-glutamine and 100 units/ml penicillin/streptomycin. Cells were maintained in a humidified incubator containing 5% CO₂ at 37°C. Compounds were dissolved in DMSO and achieve the working concentrations (0, 15, 30 and 60 μM). DMSO in the working solution was less than 0.1% and showed no toxicity towards H292 cells.

Cell viability assay. Cell viability was determined by the MTT assay, which measures the cellular capacity to reduce MTT (yellow) to formazan crystals (purple) by mitochondria dehydrogenase. After 24 h treatment, the medium was replaced by 100 μl/well of MTT solution (0.4 mg/ml) and the cells were incubated for 4 h at 37°C. Subsequently, the MTT solution was removed and a 100 μl/well of DMSO were added to dissolve the formazan crystals. The intensity was measured at 570 nm using a microplate reader (SpectraMax® M5, Molecular Devices, City, Ca, USA). All analyses were performed in at least three independent replicate cultures. The cell viability was calculated from the ratio of the optical density (OD) of treated to non-treated control cells and is presented as a percentage of the non-treated controls.

Nuclear staining assay. Cells were treated without or with different concentrations of CIN-RM in RPMI with serum for 24 h at 37°C. Apoptotic and necrotic cell death was determined by Hoechst 33342 staining: cells were incubated with 10 μM of Hoechst 33342 dye for 30 min at 37°C in the dark. Finally, cells were visualized under a fluorescence microscope (Olympus IX51 with DP70; Olympus, Center Valley, PA, USA). The blue fluorescent Hoechst dye was used for detection of nuclear condensation and DNA fragmentation in apoptosis cell. The data are presented as the percentage of apoptosis as follows: Apoptosis (%)= (number of apoptotic cells×100)/number of total cells.

Membrane integrity analysis. The cells collected after treatment with compounds were stained with trypan blue dye and subsequently morphologically visualized and quantified under an automated cell counter (TC10 automated cell counter; Bio-Rad, Hercules, CA, USA).

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Figure 1.

The synthesis of 5-O-cinnamoyl ester analog of renieramycin M (CIN-RM). CIN-RM was derived from hydrogenation of renieramycin M with 20% Pd(OH)2/C in ethyl acetate for 5 h to obtain the bishydroquinonerenieramycin M (HQ-RM). HQ-RM was subjected to esterification with cinnamoyl chloride in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI.HCl) and 4-dimethylaminopyridine (DMAP) yielding CIN-RM.

Western blot analysis. Treated and untreated cells were harvested and lysed on ice for 60 min. The protein content of cell lysate was determined using BSA protein assay kit (Pierce, Rockford, IL, USA). An equal amount of protein from each sample was separated by size using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred into nitrocellulose membranes. The membrane was blocked in 5% skim milk in 25 mmol/l Tris-HCl (pH 7.4), 125 mmol/l NaCl and 0.1% Tween 20 TBST) for 1 h at room temperature and then probed with appropriate primary antibodies at 4°C overnight. Subsequently, the membrane was washed three times with TBST for 8 min then incubated with horseradish peroxidase-conjugated secondary antibodies according to the primary antibodies for 2 h at room temperature. The signal of immunoreactive proteins was detected by enhanced chemiluminescence (Supersignal West Pico; Pierce, Rockford, IL, USA). Protein expression was investigated and β-actin was used as a loading of control in each treatment. Subsequently, the bands were then visualized using a film exposure with a chemiluminescence detection system and were quantified with analystPC densitometric software.

Statistical analysis. Mean data from at least three independent experiments were normalized to result for the non-treated control. All data are presented as the mean±standard error of the mean (S.E.M.). Statistical differences between means were determined using analysis of variance (ANOVA) and post hoc test at a significance level of p<0.05.