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Analogs of (-)-EGCG containing a para-amino group on the D ring in place of the hydroxyl groups have been synthesized and their proteasome inhibitory activities were studied. We found that, the O-acetylated (-)-EGCG analogs possessing a p-NH2 or p-NHBoc (Boc; tert-butoxycarbonyl) D ring (5 and 7) act as novel tumor cellular proteasome inhibitors and apoptosis inducers with potency similar to natural (-)-EGCG and similar to (-)-EGCG peracetate. These data suggest that the acetylated amino-GTP analogs have the potential to be developed into novel anticancer agents.
Green tea, produced from Camellia sinensis, is a highly consumed beverage around the world and regular drinking of green tea has been claimed to reduce incidence of a variety of cancers1-3. Although a number of green tea polyphenols (GTPs) have been identified in green tea4, (-)-epigallocatechin-3-gallate [1, (-)-EGCG] is the most abundant constituent and is considered to be the most biologically active among the GTPs. A number of epidemiological and biological studies involving (-)-EGCG have been reported in the last decade and have shown that (-)-EGCG can reduce or inhibit tumour growth in breast5-7, lung8, urinary9-11 and GI tracts.12
The eukaryotic proteasome is a large multi-catalytic, multi-subunit protease complex possessing at least three distinct activities, which are associated with three different β subunits, chymotrypsin-like (with β5 subunit), trypsin-like (with β2 subunit) and peptidyl-glutamyl peptide-hydrolyzing-like (PGPH- or caspase-like; with β1 subunit)13-15. Inhibition of the chymotrypsin-like, but not the trypsin-like, activity of the proteasome has been found to be associated with induction of tumour cell apoptosis.16-18 Inhibition of the proteasome prevents ubiquitin-targeted proteolysis which can affect multiple signaling cascades within the cell. Since this disruption of normal homeostatic mechanisms can lead to cell death, the discovery of new proteasome inhibitors with little or no toxicity is highly desirable in anticancer therapy.19,20
We have previously reported that (-)-EGCG inhibits the chymotrypsin-like activity of the proteasome in vitro (IC50 0.1-0.2 μM) and in intact tumour cells (1-10 μM).21,22 Enantiomerically synthesised (+)-EGCG and other synthetic analogs of green tea catechins with an ester bond have also been shown to inhibit the proteasomal chymotrypsin-like activity,22,23 leading to accumulation of proteasome target proteins (such as IκB-α, p27 and Bax) and apoptosis in human cancer cell lines, as measured by activation of caspases and cleavage of poly(ADP-ribose) polymerase (PARP).24 Furthermore, in silico docking studies have indicated that (-)-EGCG predictably binds to the N-terminal threonine (Thr) of the proteasomal chymotrypsin active site. The orientation of (-)-EGCG bound to the proteasome is suitable for nucleophilic attack by the hydroxyl group of Thr 1 to the carbonyl carbon of (-)-EGCG, thus inhibiting the proteasomal chymotrypsin-like activity (Fig. 1).25 Furthermore, the hydroxyl groups of the (-)-EGCG D-ring were found to form hydrogen-bonds with Gly47 or Ser131 of the proteasome, thus contributing to the binding stability of (-)-EGCG to the proteasome (Fig. 1). In support of this model, compound 2 (Fig. 2), which contains only one p-hydroxy group on the D-ring was found to be a much weaker proteasome inhibitor than (-)-EGCG (Table 1).26 Since an amino group is capable of forming hydrogen bonds both as a donor and an acceptor, we are therefore interested in studying the replacement of the hydroxyl group in the D-ring by an amino group in an attempt to increase the potency of compound 2.
A critical issue concerning the potential application of (-)-EGCG as an anti-cancer agent is its known low bioavailability which is thought to be partly due to the poor stability of (-)-EGCG in alkaline or neutral solutions.27,28 Because the pH values of intestine and body fluids are neutral or slightly alkaline, (-)-EGCG is potentially unstable inside the human body.27 Additionally, in vivo metabolic transformations of (-)-EGCG by glucuronidation, sulfonation or methylation into various metabolites may also contribute to its reduced bioavailability.29
Recently, we suggested that (-)-EGCG peracetate (3, Pro-E), a synthetic derivative of (-)-EGCG, can act as a pro-drug. 27 Pro-E is also converted under cellular conditions by esterases to (-)-EGCG with enhanced bioavailability in vivo.30 Consistently, even though Pro-E has no inhibitory effect against a purified 20S proteasome, it nevertheless showed much higher potency than (-)-EGCG to inhibit proliferation and transforming activity and to induce apoptosis in human prostate, breast, leukemic and simian virus 40-transformed cells.31 Recently, in a related study, we showed that Pro-E can be converted to (-)-EGCG in human breast cancer MDA-MB-231 cell cultures and xenographs, leading to a higher intracellular concentration (>2.4 fold) of (-)-EGCG than those cells treated with same dose of (-)-EGCG.32 Pro-E proved to be more efficacious in inhibiting breast cancer tumor growth in mice than (-)-EGCG.32
With the knowledge that O-acetyl protected GTPs could be cytotoxic against tumour, but not normal cells,31,32 we have continued to search for more potent anticancer agents. We have previously studied the biological activities of some EGCG analogs with modifications of ABC-ring moiety and gallate (D-ring) moiety.25-27, 31-32 Here we report structure-activity relationship (SAR) analysis with newly designed analog compounds, which possess a para-amino substituent on the D ring (compound 4), as well as the O-acetyl derivatives 5, and their corresponding N-t-butoxycarbonyl (Boc) derivatives 6 and 7.
The syntheses of compounds 4-7 were achieved as outlined in Scheme 1. We have previously reported on the total synthesis of (-)-EGCG in which (-)-(2R, 3R)-5,7-bis(benzyloxy)-2-[3,4,5-tris(benzyloxy)phenyl]chroman-3-ol (8) was prepared as the key intermediate.33 Esterification of 8 with boc-4-aminobenzoic acid (9) afforded 10 with excellent yield. Catalytic hydrogenation of 10 removed the benzyl protecting group readily to give 6. Deprotection of Boc group with trifluoroacetic acid furnished 4. On the other hand, acetylation of 6 with Ac2O and pyridine gave 7 which on subsequent deprotection of Boc group gave 5.
We examined these synthetic amino-GTPs, both unprotected (4 and 6) and O-acetyl protected (5 and 7), for their SAR in comparison with natural (-)-EGCG (1), 2, and Pro-E (3) against the chymotrypsin-like activity of a purified 20S proteasome (Table 1). Compound 4 exhibited reasonably strong proteasome inhibition (IC50 value, 0.84 μM). Even though the activity of 4 is not as high as (-)-EGCG (IC50 value, 0.20 μM34, it is much more active than compound 2 of which a hydroxyl group is in place of the amino group in compound 4 (Fig. 2 vs. Scheme 1). This suggests that an amino substituent is more effective than a hydroxyl in enhancing proteasome inhibition.
On the other hand, compound 6, with a p-NHBoc on the D ring, is less active (IC50=3.85 μM) than 4 but still much more active than compound 2. We also examined the O-acetyl protected analogs 5 and 7 to evaluate their proteasome-inhibitory activities. As expected, the O-acetyl protected analogs were not potent in inhibiting the chymotrypsin-like activity of the purified 20S proteasome due to the lack of cellular esterases for their conversion into de-acetyl and activated forms30,32,34. These acetylated analogs displayed exceptionally weak inhibitory activity against the purified 20S proteasome [less than 20% inhibition at 50 μM concentration (Table 1)]. This finding again demonstrates that O-acetyl analogs are inactive in a purified enzyme system.
In order to determine whether the synthetic amino-GTP analogs exhibit proteasomal inhibition in whole cells, leukaemia Raji B cells were treated with 25 μM of each amino-GTP analogue for 4 and 24 hrs (Fig. 3). Inhibition of intact tumour cell proteasome activity was assayed in whole cell extracts using a chymotrypsin-like activity assay. Compound 6 was unable to inhibit the chymotrypsin-like activity in whole cells after 4 and 24 hr incubation and compound 4 exhibited minimal proteasome activity at the same time points, while (-)-EGCG induced only 4 and 10% inhibition at 4 and 24 hrs respectively (Fig. 3). These findings suggest that 4 and 6 are unstable within the cellular environment, similar to (-)-EGCG. However, the O-acetylated analogs 5 and 7 exhibited far greater potency against the proteasomal chymotrypsin-like activity, 39% and 65% inhibition, respectively, after 24 hrs.
Inhibition of proteasomal activity should lead to accumulation of ubiquitinated proteins and proteasome target proteins, such as IκB-α, p27 and Bax16,17. Both 4 and 6 slightly increased levels of ubiquitinated proteins in a time-dependent manner, although they had little effect on levels of IκB-α, p27 and Bax proteins (Fig. 4). In contrast, 5 and 7 were more potent with respect to accumulation of ubiquitinated proteins and proteasome target proteins, after both 4 hr and 24 hr treatment (Fig. 4A, B). Consistent with the level of proteasome inhibition observed in whole cells (Fig 3), 7 induced higher levels of ubiquitinated proteins than 5 at both 4 and 24 hrs (Fig. 4A). After 4 hr treatment with 7, IκB-α, p27 and Bax were accumulated by 1.4-, 2.1- and 3.5-fold respectively. In contrast, a treatment with 5 did not accumulate IκB-α and accumulated only p27 and Bax by 2.0- and 3.3-fold, respectively, after 4 hrs (Fig. 4A).
It has been shown that inhibition of the proteasomal chymotrypsin-like activity is associated with induction of apoptosis in wide variety of cancer cells.16, 17 We therefore determined the cytotoxicity of the O-acetylated and un-acetylated forms of the amino-GTP analogues by trypan blue dye exclusion analysis. Leukaemia Raji B cells were treated with each pair of the acetylated and unacetylated analogs at 25 μM for 4 and 24 hrs, followed by trypan blue analysis (Fig. 5A). Blue cells and cells with apoptosis-associated morphological changes (shrunken, blebbing, etc.) were scored as dead cells. We found that the un-acetylated amino-GTP analogs (4 and 6) induced no more than 10% cell death at up to 24 hrs (Fig. 5A) supporting the idea that they are either unstable or inactive within the cell30,32,34. In contrast, the acetylated analogs (5 and 7) were much more potent in inducing the cell death (Fig. 5A). Cell death was increased by 3.5-, and 5-fold in cells treated with the acetyl protected amino-GTP analogs 5 and 7, respectively, after 24 hr incubation compared to cells treated with (-)-EGCG (Fig. 5A). To determine whether the observed cell death (Fig. 5A) was representative of apoptosis, aliquots of the cell extracts from the same experiment were utilized for measurement of caspase-3 activation (Fig. 5B) and poly(ADP-Ribose) polymerase (PARP) cleavage (Fig. 5C). After 24 hr treatment, 5 and 7 induced a 4.7- and 5.5-fold increase in caspase-3 activity, respectively (Fig. 5B). Additionally Western blot analysis revealed that apoptosis-specific PARP cleavage was observed in cells treated with analogs 5 and 7 after 24 hrs (Fig. 5C). In contrast, the PARP cleavage was not observed in cells treated with the unacetylated amino-GTP analogs 4 and 6 (Fig. 5C). These results indicate that the acetyl protected analogs are more potent than the un-acetylated analogs in inhibiting proteasome activity and inducing apoptotic cell death in a time-dependent manner. These results are consistent with the hypothesis that compounds 5 and 7 are behaving as pro-drugs of the un-acetylated analogs 4 and 6, in the same manner as Pro-E serving as the pro-drug for EGCG.27,32
In conclusion, we have demonstrated that a para-amino substituent on the D ring of green tea polyphenols exhibits proteasome-inhibitory activity with potency similar to that of (-)-EGCG and ProE. Furthermore, the O-acetylated amino-GTP analogs appear to behave as prodrugs and the mechanism of action involves targeting the proteasome in tumour cells thereby inducing cell death. Although the para-amino compounds are not inherently more potent than ProE, the presence of the para-amino substituent could increase the bioavailability of these compounds by limiting biotransformation reactions to the hydroxyls, such as glucuronidation, sulfonation and methylation.29 Since the p-aminobenzoic acid moiety is a normal constituent of folic acid and generally presumed to be innocuous,35 the study of these p-amino substituted green tea polyphenols may offer the possibility of discovering novel anticancer drugs.
Fetal Bovine Serum was purchase from Tissue Culture Biologicals (Tulare, CA). RPMI 1640, penicillin, and streptomycin were purchased from Invitrogen (Carlsbad, CA). Dimethyl sulfoxide (DMSO) and (-)-EGCG were purchased from Sigma (St. Louis, MO). Suc-Leu-Leu-Val-Tyr-AMC (a proteasomal chymotrypsin-like substrate) and Ac-DEVD-AMC (a caspase-3 substrate) were obtained from Biomol (Plymouth Meeting, PA). Purified 20S proteasome from rabbit was acquired from Boston Biochem (Cambridge, MA). Monoclonal antibodies to Bax (H280) and Ubiquitin (P4D1), polyclonal antibodies to IκB-α (C15) and Actin (C11), and anti-goat, anti-rabbit, and anti-mouse IgG-horseradish peroxidase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibody to p27 (554069) was from BD Biosciences (San Diego, CA). The polyclonal antibody to PARP was purchased from Biosource (Camarillo, CA).
The synthesis was accomplished according to Scheme 1. All reactions were performed under an atmosphere of N2, and glassware was dried completely in an oven at 110 °C prior to use. Tetrahydrofuran (THF) was dried by distillation over sodium benzophenone, and dry dichloromethane (CH2Cl2), dimethylformamide (DMF) and toluene were obtained by distillation from CaH2. Unless otherwise stated, solvents or reagents were used as received without further purification. (-)-(2R, 3R)-5,7-Bis(benzyloxy)-2-[3,4,5-tris(benzyloxy)benzyl]-3-chroman-3-ol (8) was prepared by following reported procedure.33
To a solution of 4-N-Boc-aminobenzoic acid (9, 166 mg, 698 μmol) in CH2Cl2 (1.00 mL) was added N, N’-dicyclohexylcarbodiimide (217 mg, 1.05 mmol). The mixture was stirred at room temperature for 10 min, cooled to 0 °C. 4-Dimethylaminopyridine (21.4 mg, 175 μmol) was added to the solution and the mixture was stirred for 5 min. A solution of (-)-(2R, 3R)-5,7-bis(benzyloxy)-2-[3,4,5-tris(benzyloxy)phenyl]-chroman-3-ol (8, 264 mg, 349 μmol) in CH2Cl2 (2.50 mL) was added dropwise at 0 °C and the mixture was stirred at room temperature overnight. The solvent was evaporated in vacuo and the resulting oil was purified by flash SiO2 column chromatography (hexane/EtOAc, 4:1) to give 308 mg (90%) of the title compound as a pale yellow amorphous solid: [α]D20 = –60° (c 1.05, CHCl3); 1H NMR (CDCl3) δ 7.91 (d, J = 8.6 Hz, 2H), 7.48-7.17 (m, 27H), 6.78 (s, 2H), 6.61 (s, 1H), 6.34 (br s, 1H), 6.29 (br s, 1H), 5.66 (br s, 1H), 5.08-4.91 (m, 8H), 4.76 (d, J = 11.9, 2H), 3.14-3.04 (m, 2H), 1.51 (s, 9H); 13C NMR (CDCl3) δ 165.95, 159.72, 158.92, 156.51, 153.74, 152.87, 143.95, 139.14, 138.73, 137.94, 137.82, 137.76, 134.28, 132.10, 129.57, 129.49, 129.33, 129.02, 128.99, 128.86, 128.70, 128.66, 128.57, 128.42, 128.16, 125.02, 118.17, 107.53, 101.91, 95.67, 94.88, 82.14, 78.82, 76.04, 72.10, 71.11, 70.92, 69.04, 29.21, 27.07; HRMS m/z calculated for C62H57O10Na (M + Na) 998.3880, found 998.3845.
To a solution of (-)-(2R, 3R)-5,7-bis(benzyloxy)-2-[3,4,5-tris(benzyloxy)phenyl]chroman-3-yl 4’-N-(tert-butoxycarbonyl)aminobenzoate (10, 101 mg, 104 μmol) in THF (10.0 mL) and MeOH (10.0 mL) was added palladium hydroxide on carbon powder [20% Pd (100 mg)]. The mixture was stirred under a H2 atmosphere at room temperature for 1 h, filtered and eluted with MeOH and the eluate was evaporated in vacuo. The obtained colourless oil (66.8 mg) was purified by flash SiO2 column chromatography (AcOEt/hexane, 2:1) to give 45.3 mg (83%) of the title compound as a pale yellow amorphous solid: [α]D20 = –88° (c 0.14, MeOH); 1H NMR (CDCl3) (complexity due to rotamers from the amide function) δ 7.81 (d, J = 9.5 Hz, 1/6×2H), 7.78 (d, J = 9.5 Hz, 5/6×2H), 7.45 (d, J = 9.5 Hz, 1/6×2H), 7.43 (d, J = 9.5 Hz, 5/6×2H), 6.54 (s, 1/6×2H), 6.52 (s, 5/6×2H), 6.05 (d, J = 2.4 Hz, 1/6×1H), 5.99-5.97 (m, 1H), 5.96 (d, J = 2.4 Hz, 5/6×1H), 5.54 (br s, 1/6×1H), 5.52 (br s, 5/6×1H), 5.10-4.68 (m, 5H), 5.02 (s, 1/6×1H), 5.00 (s, 5/6×1H), 3.03 (dd, J = 17.0, 4.4 Hz, 1/6×1H), 3.00 (dd, J = 17.0, 4.4 Hz, 5/6×1H), 2.91 (dd, J = 17.0, 2.0 Hz, 1/6×1H), 2.89 (dd, J = 17.0, 2.0 Hz, 5/6×1H), 1.51 (s, 1/6×9H), 1.49 (s, 5/6×9H); 13C NMR (CDCl3) δ 168.06, 158.74, 158.65, 158.01, 147.55, 146.23, 134.54, 132.60, 131.61, 125.71, 119.36, 107.56, 107.51, 100.13, 97.40, 97.32, 96.66, 96.58), 82.20, 79.37, 79.28, 71.31, 19.43, 29.40, 27.50; HRMS m/z calculated for C27H27NO10Na (M + Na) 548.1533, found 548.1537.
To a solution of (-)-(2R, 3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl 4-( tert-butoxycarbonyl)-aminobenzoate (6, 20.6 mg, 39.2 μmol) in CHCl3 (800 μL) was added trifluoroacetic acid (160 μL) and the mixture was stirred at room temperature for 30 min. The reaction mixture was directly evaporated in vacuo and 28.5 mg (>99%) of the title compound was obtained as a pale brown solid: [α]D20 = –72° (c 0.19, MeOH); 1H NMR (CDCl3) δ 7.67 (d, J = 9.8 Hz, 2H), 6.63 (d, J = 9.8 Hz, 2H), 6.54 (s, 2H), 6.00 (br s, 1H), 5.99 (br s, 1H), 5.51 (br s, 1H), 5.01 (s, 1H), 4.96-4.82 (m, 5H), 3.01 (dd, J = 16.8, 4.9 Hz, 1H), 2.89 (dd, J = 16.8, 3.3 Hz, 1H); 13C NMR (CDCl3) δ 168.97, 158.74, 158.68, 158.08, 151.05, 147.55, 134.57, 133.60, 133.51, 131.75, 119.52, 115.21, 115.09, 107.72, 107.61, 100.33, 97.35, 96.60, 79.48, 70.61, 27.58; HRMS m/z calculated for C32H29NO13 426.1189, found 426.1205.
To a solution of (-)-(2R, 3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl 4-( tert-butoxycarbonyl)-aminobenzoate (6, 94.0 mg, 179 μmol) in pyridine (1.00 mL) was added acetic anhydride (130 μL, 1.38 mmol) and the mixture was stirred at room temperature for 30 min. The solvent was evaporated in vacuo and the resulting yellow oil (113 mg) was purified by flash SiO2 column chromatography (hexane/EtOAc, 1:1) to give 58.1 mg (59%) of the title compound as a colourless amorphous solid: [α]D20 = –48° (c 1.11, CHCl3); 1H NMR (CDCl3) δ 7.78 (d, J = 8.5 Hz, 2H), 7.34 (d, J = 8.5 Hz, 2H), 7.27 (s, 2 H), 6.74 (d, J = 1.9 Hz, 1H), 6.68 (s, 1H), 6.59 (d, J = 1.9 Hz, 1H), 5.62-5.58 (m, 1H), 5.20 (s, 1H), 3.06 (br s, 2H), 2.30-2.27 (m, 6H), 2.26-2.23 (m, 9H), 1.50 (s, 9H); 13C NMR (CDCl3) δ 169.93, 169.42, 168.55, 167.72, 166.40, 155.77, 153.00, 150.66, 144.31, 143.95, 136.58, 135.19, 132.15, 124.43, 119.71, 118.21, 110.73, 109.84, 108.96, 82.09, 77.54, 68.14, 29.20, 26.94, 22.06, 21.75, 21.58, 21.10; HRMS m/z calculated for C37H37NO15Na (M + Na) 758.2061, found 758.2068.
To a solution of (-)-(2R, 3R)-5,7-diacetoxy-2-(3,4,5-triacetoxyphen)chroman-3-yl 4-(tert-butoxycarbonyl)-aminobenzoate (7, 60.0 mg, 81.6 μmol) in CHCl3 (1.60 mL) was added trifluoroacetic acid (320 μL) and the mixture was stirred at room temperature for 6.5 h. The reaction was quenched with saturated aqueous NaHCO3 at 0 °C and the mixture was extracted with CHCl3 (3 × 10.0 mL). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The obtained brown red amorphous solid (65.0 mg) was purified by flash SiO2 column chromatography (1% triethylamine, EtOAc/hexane, 2:1→EtOAc) to give 26.4 mg (51%) of the title compound as a yellow amorphous solid: [α]D20 = –54° (c 1.32, CHCl3); 1H NMR (CDCl3) δ 7.57 (d, J = 8.7 Hz, 2H), 7.20 (s, 2H), 6.65 (br s, 1H), 6.50 (br s, 1H), 6.46 (d, J = 8.7 Hz, 2H), 5.51 (br s, 1H), 5.10 (s, 1H), 3.00-2.90 (m, 2H), 2.20 (s, 3H), 2.19 (s, 3H), 2.17 (s, 3H), 2.15 (s, 6H); 13C NMR (CDCl3) δ169.32, 168.80, 167.95, 167.13, 166.07, 155.18, 151.01, 150.03, 149.93, 143.62, 136.08, 134.52, 132.18, 119.16, 114.31, 110.26, 109.11, 108.26, 76.97, 66.89, 26.35, 21.40, 21.09, 20.91, 20.45; HRMS m/z calculated for C32H29NO13 635.1638, found 635.1643.
Human leukaemia Raji B cells were cultured in RPMI supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cell cultures were maintained in a 5% CO2 atmosphere at 37 °C.
Whole cell extracts were prepared as described previously.36 Analysis of IκB-α, p27, Bax , PARP, and ubiquitinated proteins was performed using monoclonal or polyclonal antibodies, according to previously reported protocols.21 Densitometry was quantified using AlphaEase FC software (Alpha Innotech Corporation, San Leandro, CA).
Measurement of the chymotrypsin-like activity of the 20S proteasome was performed by incubating 35 ng of purified rabbit 20S proteasome with 40 μM of fluorogenic peptide substrate, Suc-Leu-Leu-Val-Tyr-AMC, with or without a natural or synthetic GT.37
Cells were treated with each compound at 25 μM for 4 or 24 hrs, harvested, and lysed as described previously.16 Whole cell extracts (10 μg) were incubated with Suc-Leu-Leu-Val-Tyr-AMC (40 μM) fluorogenic substrate at 37 °C in 100 μL of assay buffer (50 mM Tris-HCL, pH 8) for 2.5 hrs. After incubation, production of hydrolyzed 7-amino-4-methylcoumarin (AMC) groups was measured using a Victor 3 Multilabel Counter with an excitation filter of 380 nm and an emission filter of 460 nm (PerkinElmer, Boston, MA, USA).
Cells were treated with each compound at 25 μM for 4 or 24 h, harvested, and lysed as described previously.21 Ac-DEVD-AMC (40 μM) was then incubated with the prepared cell lysates for 2.5 h and the caspase-3 activity was measured as described previously.38
The trypan blue dye exclusion assay was used to ascertain cell death in Raji cells treated with either a natural or synthetic compound at 25 μM for 4 or 24 hrs. Cell morphology was assessed using phase-contrast microscopy as described previously.22,,31
We thank the Areas of Excellence Scheme established under the University Grants Committee of the Hong Kong Administrative Region, China (Project No. AoE/P- 10/01) for financial support. Funding support by National Cancer Institute (Grant Number: 1R01CA120009 and 5R03CA112625) is gratefully acknowledged.