Synthesis and Pro-Apoptotic Activity of Novel Glycyrrhetinic Acid Derivatives

Triterpenoids are used for medicinal purposes in many countries. Some, such as oleanolic and glycyrrhetinic acids, are known to be anti-inflammatory and anticarcinogenic. However, the biological activities of these naturally occurring molecules against their particular targets are weak, so the synthesis of new synthetic analogues with enhanced potency is needed. By combining modifications to both the A and C rings of 18βH-glycyrrhetinic acid, the novel synthetic derivative methyl 2-cyano-3,12-dioxo-18βH-olean-9(11),1(2)-dien-30-oate was obtained. This derivative displays high antiproliferative activity in cancer cells, including a cell line with a multidrug-resistance phenotype. It causes cell death by inducing the intrinsic caspase-dependent apoptotic pathway.


Introduction
Organic molecules synthesized by plants constitute a rich reservoir of biologically active compounds. For centuries extracts from various plants have been extensively used in traditional medicines for the treatment of a wide variety of human ailments; even today, many cultures still employ them directly for medicinal proposes. [1][2][3][4] Among the classes of recognized therapeutically useful products, pentacyclic triterpeniods have been studied intensively for their diverse biological, pharmacological, and medicinal activities, which are similar to those of retinoids and steroids. [5,6] However, these triterpeniods exhibit only weak effects on the biological activity of their molecular targets; therefore these compounds have been used as building blocks for the synthesis of more active analogues. [6] Oleanolic acid, an abundantly occurring triterpene, has been converted into 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and other structurally related analogues (CDDO-Me, CDDO-Im, CDDO-CN; Scheme 1 A). [7][8][9] All of these synthetic derivatives were reported to display various bioactivities: cytoprotection, cancer cell growth inhibition, apoptosis induction, and inhibition of the production of NO induced by INF-g in mouse macrophages. [7][8][9][10][11][12] CDDO and CDDO-Me are currently in clinical trials for cancer treatment, and have been shown to effectively suppress the growth of a broad spectrum of solid and hematologic cancer cell types, both in vitro and in mouse models bearing xenografted human tumors. [9][10][11][12] During the development of CDDO, it was found that the 2-cyano-1-en-3-one in ring A, and the 9(11)-en-12-one in ring C are essential for the biological activity of CDDO and its analogues. [13][14][15] 18bH-Glycyrrhetinic acid (Scheme 1 B), the aglycon of glycyrrhizin, is abundant in licorice root (Glycyrrhiza glabra and Glycyrrhiza uralensis Fischer). The glycyrrhizin content in triterpene extracts from licorice root amounts to 90 %. Recent reviews have described the wide spectrum of glycyrrhetinic acid bioactivity, such as anti-inflammatory, antiviral, hepatoprotective, antitumor, and immunomodulatory activities. [16,17] Several studies have reported that glycyrrhizin and glycyrrhetinic acid have moderate cytotoxic and apoptotic effects on cancer cells, although most reported only moderate or low potency.
Triterpenoids are used for medicinal purposes in many countries. Some, such as oleanolic and glycyrrhetinic acids, are known to be anti-inflammatory and anticarcinogenic. However, the biological activities of these naturally occurring molecules against their particular targets are weak, so the synthesis of new synthetic analogues with enhanced potency is needed. By combining modifications to both the A and C rings of 18bH-glycyrrhetinic acid, the novel synthetic derivative methyl 2cyano-3,12-dioxo-18bH-olean-9(11),1(2)-dien-30-oate was obtained. This derivative displays high antiproliferative activity in cancer cells, including a cell line with a multidrug-resistance phenotype. It causes cell death by inducing the intrinsic caspase-dependent apoptotic pathway.
In attempts to prepare more-potent analogues of glycyrrhetinic acid, we synthesized compounds 2-12, similar to CDDO-Me (1), by introducing modification at both rings A and C (Scheme 1 A). We investigated the effects of the novel derivatives on the growth of human cancer cells, and we identified methyl 2-cyano-3,12-dioxo-18bH-olean-9(11),1(2)-dien-30-oate 12 as a compound displaying significant antiproliferative activity toward cancer cells; the other glycyrrhetinic-acid derivatives did not display this activity. We compared 12 and CDDO-Me on several cell lines under the same conditions, and we showed that IC 50 was lower for 12 than CDDO-Me for all cell lines. Compound 12 induced cell-cycle arrest, the translocation of phosphatidylserine to the cell surface, and fragmentation of the nucleus. It also caused a dramatic dissipation of the mitochondrial potential, and induced activation of the caspase cascade; these effects were more pronounced for 12 than for CDDO-Me. The data indicate that 12 induces the death of cancer cells by the intrinsic caspase-dependent apoptosis pathway.

Chemical synthesis
The reaction sequence to introduce the 2-cyano-1-en-3-one and 9(11)-en-12-one in A and C rings of glycyrrhetinic acid is shown in Scheme 2.
18bH-Glycyrrhetinic acid acetate 2, obtained from a licorice extract, was used as the starting material. Compound 2 was esterified at 0 8C with ethereal diazomethane to give methyl glycyrrhetinate acetate 3, which was reduced by Zn/HCl in dioxane at 5-10 8C. The resulting methyl ester of 11-deoxoglycyrrhetinic acid acetate 4 was converted into 12-oxo derivative 5 by treating with hydrogen peroxide in acetic acid at 80 8C. The formation of the 9,11-double bond was achieved by bromination-dehydrobromination of ketone 5 with bromine in acetic acid at 80 8C. Finally the 9(11)en-12-one moiety in the C ring was obtained. Deprotection of acetate group by KOH in methanol (reflux) freed the 3-hydroxy group, then Jones oxidation gave the ketone 8. Subsequent formylation at C 2 was performed by condensation with HCO 2 Et/ NaOMe in benzene, and the re-sulting hydroxymethylene derivative 9 was cyclized into isoxazole 10 by reacting with hydroxylamine hydrochloride in aqueous ethanol (reflux). Opening of the isoxazole ring at the NÀO bond was promoted by NaOMe routinely to deliver the 2cyano group in 11. The new 1,2 double bond was formed by dehydrogenation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in benzene (reflux) to complete the synthesis of the 2-cyano-1-en-3-one moiety in A ring in 12.
It should be noted that the synthesis scheme for our endproduct 12 has been described by Chadalapaka et al. [18] Our investigations were conducted independently and in parallel. In addition to the synthesis scheme, a detailed description of the synthesis and physicochemical properties of the end product (and of the intermediates) is presented in this work (see the Experimental Section).
Glycyrrhetinic acid, 7, 8, 9, 10 and 12 were tested; the other derivatives were found to be insoluble in dimethylsulfoxide (DMSO). Cells were exposed to the compounds for 24 h and then assayed for growth by the MTT method. The cells were also incubated in the presence of CDDO-Me 1, whose ability to inhibit cancer-cell growth was established earlier (reviewed by Liby et al.). [19] Figure 1 shows the dose-response curves for 1 and 12 with KB-3-1 cells, and the IC 50 values for all the tested compounds (for the inhibition of KB-3-1 cell growth) are presented in Table 1. Compound 12 displayed the highest activity. The in vitro IC 50 values (the concentrations required for 50 % growth inhibition) were 0.3 and 1.2 mm for 12 and 1, respectively (Table 1). IC 50 values for the other glycyrrhetinic acid derivatives were greater than 10 mm.
We compared the effects of 12 and 1 on the growth of different human cancer cell-lines: KB-3-1 epidermoid carcinoma cells, KB-8-5 multidrug-resistant cancer cells (a derivative of KB-3-1), HeLa cervical epithelioid carcinoma cells, MCF-7 breast adenocarcinoma cells, and SKNMC neuroblastoma cells. The dose-response curves for 12 with the different cell lines are displayed in Figure 2. Compound 12 induces concentration-dependent cell death in all cell lines tested. IC 50 values for 12 and 1 are displayed in Table 2. The IC 50 values for compounds were similar for all cell lines, with the exception of MCF-7, for which IC 50 was more that ten times higher than for KB-3-1 (5 mm vs 0.3 mm for 12). IC 50 values for 12 were lower than for 1 for all tested cell lines.
One of the reasons for the failure of chemotherapy-based treatment is multidrug resistance (MDR). We tested the ability of 12 to suppress the growth of multidrug-resistant KB-8-5 cells. This cell line is characterized by overexpression of the MDR1 gene, which encodes P-glycoprotein, an ATP-dependent membrane pump that efficiently decreases the intracellular concentrations of various compounds. Treatment of KB-8-5 cells with 12 significantly decreased the number of living cells ( Figure 2); the IC 50 value for this cell line was only four times higher than the IC 50 for the drug-sensitive KB-3-1 (0.3 mm for KB-3-1 vs 1.2 mm for KB-8-5). Thus, this glycyrrhetinic acid derivative is not targeted at P-glycoprotein, and might be efficient against tumors exhibiting the P-glycoprotein-dependent MDR phenotype.    The effect of antioxidants on the cytotoxicity of 12: Similarly to CDDO and many other synthetic triterpenoids, 12 has potential electrophilic Michael acceptor sites at positions 1 and 9 of the triterpenoid nucleus (Figure 3 A). It is known that the presence of Michael acceptor groups at specific positions is essential for inhibition of proliferation, promotion of differentiation, and induction of apoptosis in various cell lines. This arises from the ability of Michael electrophiles to target specific nucleophiles, and to affect selective biological functions. [20][21][22] The involvement of the Michael electrophiles in a particular biological process can be proved by inhibition of their activity with antioxidants, for example glutathione (GSH).
We investigated whether reducing the nucleophilic agents would abrogate the cytotoxicity of 12. Cells were incubated in the presence of GSH (1, 5, 15 or 45 mm), either alone or in combination with 12 (1 mm; Figure 3 B). Incubation of cells in the presence of 12 with of GSH (5 or 15 mm) decreased the cytotoxicity of 12. The IC 50 value in the presence of GSH was 3 mm, but only 0.3 mm for 12 alone. Higher concentrations of GSH were toxic: incubation in the presence of 45 mm glutathione led to 95 % cell death (data not shown). It should be noted that incubation with ascorbic acid did not decrease the cytotoxicity of compound 12 (not shown). Thus, we demonstrated that 12 displays biologically active Michael acceptors.
Cell-cycle arrest: Flow cytometry was employed to determine whether 12 caused stage-specific inhibition of the cell cycle (Table 3). After 18 h incubation in the absence of 12, the number of cells with sub-G 1 (apoptotic) peak was insignificant. An increase in the concentration of 12 (0.3 to 1 mm ) yielded a corresponding increase in the population of cells in sub-G 1 (19.2 to 51.8 % ; values relative to the control) in a concentration-dependent manner (n = 3; p < 0.05). The increase in the population of cells in the sub-G 1 phase was accompanied by a decrease of cells in the G 1 and S phases ( Table 3). It has been reported that cells with these features are those dying of apoptosis. [23] The number of cells in the G2-M phase remained constant.
Morphological observation of nuclear change: There are several morphological characteristics for apoptotic cells, such as cell shrinkage, nuclear fragmentation and chromatin condensation. To examine cell death due to exposure to 12, we investigated the nuclear morphological changes in KB-3-1 cells treat-  [a] KB-3-1 human epidermoid cells were seeded into six-well plates to ensure that they had not reached confluency. After 24 h they were incubated either in the absence (control) or presence of 0.3 or 1 mm 12. After 18 h the percentage of cells in each phase of the cell cycle was determined by flow cytometry as described in the Experimental Section. Data were obtained from at least three separate experiments in duplicate. Quantification of apoptosis by annexin V binding and flow cytometry: Increases in morphologically changed cells, and in the number of cells in the sub-G0/G1 phase, are usually associated with apoptosis. We examined whether cell death was apoptotic when induced by the glycyrrhetinic acid derivatives by using annexin V and propidium iodide analysis ( Figure 5). KB-3-1 cells were exposed to 12, then subjected to flow cytometric analysis. Annexin V binds phosphatidylserine residues, which are asymmetrically distributed toward the inner plasma membrane, and migrate to the outer plasma membrane during apoptosis. [24] The data show that 12 induced apoptotic cell death in 50 % of KB-3-1 cells at concentrations equal to the IC 50 values. The number of apoptotic cells increased with the time of incubation, and with increasing compound concentration. 89.2 % of KB-3-1 cells were detected as apoptotic following 24 h of incubation in the presence of 1 mm 12, so 12 induces dose-and time-depended apoptotic cell death. Taken together, these data indicate that the decrease in viability of cancer cells exposed to the novel glycyrrhetinic acid derivatives occurred by apoptosis, and that 12 had the greatest potency.
Dissipation of the mitochondrial transmembrane potential: We investigated whether 12 utilizes the mitochondrial "intrinsic" pathway in the apoptotic death of KB-3-1 cells, as the pivotal role of mitochondria in the triggering of apoptosis is well established. We evaluated the mitochondrial transmembrane potential (Dy m ) in KB-3-1 cells exposed to 12, and compared this to that for 1, whose ability to decrease Dy m has been documented. [10,19,[25][26][27][28][29] Changes in Dy m were evaluated by cytofluorometric analysis. Cells were stained with the mitochondria-specific cationic dye JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazole carbocyanine iodide), which accumulates in the transmembrane region of polarized mitochondria where it forms "J-aggregates". These emit orange fluorescence that can be recorded on channel 2 of a cytofluorometer, or visualized via a red filter on a fluorescence microscope. A decrease in Dy m results in a decrease in J-aggregates and increase in JC-1 monomers, which emit a greenish-yellow fluorescence. The cytometric analysis of KB-3-1 cells stained with JC-1 is shown in Figure 6 B. In the control cells (incubated in the presence of 0.1 % DMSO) the majority of cells showed a high emission of fluorescence in both channels, because of the equilibrium between J-aggregates and monomers. The exposure of KB-3-1 cells to 12 leads to a significant decrease in fluorescence compared to the control (0.1 % DMSO). In fluorescent   (Figure 6 A), one can see that most of the cells turn green. After incubation in the presence of 1, cells can be seen to be somewhere between the control and 12-treated cells, both in the fluorescent micrograph and in the flow-cytometry histogram. One can conclude that 12 causes a dramatic dissipation of mitochondrial potential, and that this effect, consistent with the results of the MTT assay, is more pronounced than that for 1.
Activation of the caspase cascade in apoptosis induced by glycyrrhetinic acid derivatives: To determine whether activation of the caspase cascade is involved in 12-induced apoptosis, we used the fluorescein isothiocyanate (FITC)-labeled pan-caspase inhibitor FITC-VAD-FMK (FITC-valyl-alanyl-aspartyl-[O-methyl]fluoromethylketone). The conjugated compound is cell-permeable and binds irreversibly to activated caspase molecules, and thus serves as an in situ marker for apoptosis. [30] We compared the abilities of 12 and 1 to activate caspase (Figure 7). In control cells (18 h incubation in the presence of 0.1 % DMSO) only a faint green signal was seen: this equates to 9 % of cells with activated caspase (Figure 7 A and C). With the addition of 12 (0.3 and 1 mm), the number of cells with activated caspase increased (51 and 85 %, respectively; Figure 7 C), and green fluorescence was observed in the fluorescence microscope. Similar assays with 1 yielded data that lay between those for the control and 12, as had been the case for mitochondrial transmembrane potential dissipation.
The results provide evidence that the most-active glycyrrhetinic-acid derivative 12 induces caspase-dependent apoptosis in cancer cells. Caspase involvement in cell death is suggested also by the higher IC 50 for MCF-7 cells ( Table 2)-cells that are known to be caspase-3-deficient. [31] Conclusions In this report we describe the synthesis of the new glycyrrhetinic acid derivative methyl 2-cyano-3,12-dioxo-18bH-olean-  9(11),1(2)-dien-30-oate (12), obtained by the direct modification of the A and C rings of glycyrrhetinic acid. We provide a detailed description of synthesis and physicochemical characteristics of the end product, 12, and of the intermediate compounds. The modifications converted the well-known triterpenoid (exhibiting weak antitumor activity) to derivative 12, which displays high antiproliferative activity toward cancer cells. The intermediate products 7-10 did not display this activity.
We have shown that human epidermoid cancer cells are sensitive to 12, as are other tumor cell types, including cells exhibiting the multidrug-resistant phenotype. Compound 12 displays potent single-agent activity, at micromolar concentrations, against different human cancer cells in culture. The mechanism of action (MOA) of triterpenoids on cancer cells is not fully understood. Different mechanisms have been proposed for the cytotoxic activity of synthetic triterpenoids in various types of cancer and leukemia cells; this suggests that cellular context is important. Several studies point to an MOA dependent on the extrinsic apoptotic pathway (DR4/DR5/caspase-8 activation), [20,32] whereas other studies point to involvement of the intrinsic apoptotic pathway. [10,33,34] Our studies imply that the apoptotic MOA of 12 includes components of intrinsic pathways in epidermoid cancer cells.
We have compared the ability of 12 to cause cancer-cell death with that of CDDO-Me, a well known compound that is currently in late-stage clinical trials for the treatment of chronic kidney disease in type 2 diabetes mellitus patients. The antiproliferative activity of 12 exceeds that of CDDO-Me: the IC 50 value was lower for all tested cell lines, and 12 caused a dramatic dissipation of mitochondrial potential and caspase-cascade activation; these effects were more pronounced for 12 than for CDDO-Me. Moreover, we can suppose that commercial synthesis of 12 will be more amenable than that of CDDO-Me because 18bH-glycyrrhetinic acid, the starting material for the synthesis of 12, is more readily available than oleanolic acid. Indeed, the level of oleanolic acid in olive leaves ranges from 0.71 to 3.5 %, [35][36][37] and from 0.8 to 4.3 % in Ligustrum fruit; [38] the level of glycyrrhizin can reach 25-30 % in Glycyrrhiza glabra root, and 90 % in triterpene extracts of licorice root. [39][40][41] Experimental Section Reagents: CDDO-Me 1 was synthesized from oleanolic acid according to a previously described method [5] with 10 % yield (NMR 1 H and 13 C data).
18bH-Glycyrrhetinic acid acetate 2, obtained from a licorice extract, was used as starting material (purity~94 %). [39] General experimental procedures: Melting points were determined on a Hoover melting point apparatus and were uncorrected. The elemental composition of the products was determined from high-resolution mass spectra recorded on a DFS (Double Focusing Sector) Thermo Electron Corporation instrument. 1 H and 13 C NMR spectra were measured from CDCl 3 solutions on Bruker spectrometers: AM-400 (400.13 MHz for 1 H, 100.61 MHz for 13 C) and DRX-500 (500.13 MHz for 1 H, 125.76 MHz for 13 C). Chloroform was used as the internal standard (d H 7.24 ppm, d C 76.90 ppm). The structure of the compounds was determined by NMR from proton spin-spin coupling constants in 1 H, 1 H double-resonance spectra, and by analyzing 13 C NMR proton-selective and off-resonance saturation spectra, 2D 13 C, 1 H correlated spectroscopy on CH constants (COSY, 1 J C,H = 135 Hz; and COLOC, 2, 3 J C,H = 10 Hz, correspondingly), and 1D 13 C, 1 H long-range J modulation difference (LRJMD, J C,H = 10 Hz). Flash column chromatography was performed with silica gel (Merck, 60-200 mesh) and neutral alumina (Chemapol, 40-250 mesh).
Methyl 18 bH-Glycyrrhetinate acetate (3): [42] A solution of diazomethane in ether was added dropwise at 0 8C to a stirred suspension of 2 (10 g, 19.0 mmol) in methanol (200 mL) until the originally colorless mixture turned yellow. The resulting mixture was allowed to stand at room temperature overnight. The solvent was removed and the product was purified by crystallization (chloroform/methanol; yield = 9.1 g, 89 %  Methyl 3 b-Acetoxy-18 bH-olean-12-en-30-oate (4): [43] A solution of conc. hydrochloric acid (50 mL) was added dropwise at 10 8C to a stirred suspension of 3 (9.1 g, 17.3 mmol) and zinc powder (18.2 g, 280 mmol) in dioxane (300 mL) over 2 h. The reaction mixture was stirred for a further 3 h at 5-10 8C, concentrated in a vacuum, diluted with water (1 L), and filtered. The solid was dried and subjected to flash column chromatography (silica gel; benzene followed by chloroform) to give crude 4 (yield = 6.8 g, 77 %). This material was used for the next reaction without further purification. An analytically pure sample was obtained by recrystallization from a mixture chloroform/methanol. M.p. 265-267 8C; 1  Methyl 3b-acetoxy-12-oxo-18 bH-olean-30-oate (5): A mixture of hydrogen peroxide (~30 %, 25 mL) and acetic acid (25 mL) was added dropwise at 80 8C to a stirred suspension of 4 (3.0 g, 5.7 mmol) in acetic acid (100 mL) over 1 h. The reaction mixture was stirred for a further 30 min at 80 8C, cooled to room temperature, and diluted with water (500 mL). The solid was filtered, washed with water, and dried to give crude 5 (yield = 6.8 g , 96 %). This material was used for the next reaction without further purification. An analytically pure sample was obtained by recrystallization from a mixture chloroform/methanol. M.p. 296-299 8C;
Glycyrrhetinic acids derivatives were dissolved in DMSO (10 mmol L À1 ), and stock solution were stored at À20 8C.
After treatments, both floating and adherent scraped cells were collected by centrifugation, and used for further analysis.
Cell viability analysis by MTT assay: Cancer cells, growing in log phase, were seeded in triplicate 96-well plates at a density of 5 10 3 cells per well for HeLa cells, 7 10 3 for KB-3-1, KB-8-5 and MCF-7 cells, and 30 10 3 for SKNMC cells. The plates were incubated at 37 8C in humidified 5 % CO 2 atmosphere. Cells were allowed to adhere to the surface for 24 h, then treated with varying doses of the compounds for 24 h. Aliquots of [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) solution (10 mL, 5 mg mL À1 ) were added to each well, and the incubation was continued for an additional 3 h. The dark blue formazan crystals (formed within healthy cells) were solubilized with DMSO, and the absorbance was measured at 570 nm in a Multiscan RC plate reader (Thermo LabSystems , Finland). The IC 50 was determined as the compound concentration required to decrease the A 570 to 50 % of the control (no compound, DMSO), and was determined by interpolation from dose-response curves.
Analysis of antioxidant effect on the cytotoxicity of compound 12: KB-3-1 cells growing in the log phase were seeded in triplicate in 96-well plates (7 10 3 cells per well). The plates were incubated at 37 8C in a humidified 5 % CO 2 atmosphere. Cells were allowed to adhere to the surface for 24 h, then treated with GSH (1, 5, 15 or 45 mm) or with ascorbic acid (1, 3 or 5 mm), both alone and in combination with 12 (1 mm). Cells were incubated with the compounds for 24 h and cell viability was analyzed by the MTT assay as described above.
Morphological observation of nuclear change: KB-3-1 cells were seeded into 24-well plates (10 5 cells per well) containing glass cover slips. Cells were allowed to adhere to the surface for 24 h. Cells were treated with 12 (1 mm) or with DMSO (0.1 % (v/v)) for 6, 18 or 24 h at 37 8C in a humidified 5 % CO 2 atmosphere. After incubation, cells were fixed with 4 % formaldehyde for 15 min, and then stained for 30 min with Hoechst 33258 (200 ng mL À1 ). Cells were analyzed for the presence of fragmented nuclei and condensed chromatin by fluorescent microscopy.
Apoptosis detection by Annexin V staining: Log-phase KB-3-1 cells in six-well plates (5 10 5 cells per well) were treated with 12 (0.3 mm or 1 mm) or with DMSO (0.1 % (v/v)) for 4, 18 or 24 h. The cells were stained with Annexin V-FITC and propidium iodide by using the ApopNexin-FITC apoptosis detection kit (Chemicon Millipore) according to the manufacturer's instructions. Briefly, cells were collected by scraping, washed twice with cold PBS, and centrifuged (400 g, 5 min). Cells were resuspended in binding buffer (1 mL) at a concentration of 1 10 6 cells per mL, then a sample (200 mL) was transferred to a 5 mL culture tube, and Annexin V-FITC (3 mL) and 100 PI (2 mL) were added. Cells were incubated for 15 min at room temperature in the dark. Finally, binding buffer (300 mL) was added to each tube, and the quantity of apoptotic cells in samples was analyzed by flow cytometry (FC500, Beckman Coulter, USA). For each sample, 10 000 ungated events were acquired. Annexin V + /PI À cells represented early apoptotic populations. Annexin V + /PI + cells represented either late apoptotic or secondary necrotic populations.
Mitochondria depolarization analysis: Mitochondria involvement in apoptosis was measured by the mitochondrial depolarization that occurs early during the onset of apoptosis. KB-3-1 cells were treated with 1 (1 mm), 12 (1 mm) or DMSO (0.1 % (v/v)) for 6 h, and loss of mitochondrial potential was determined by using the mitochondrial potential sensor JC-1 (Molecular Probes, Invitrogen).
Flow cytometry assay: Cells were incubated for the appropriate time with the compounds, then collected, incubated in complete media in the dark with JC-1 (5 mg mL À1 ) at 378C for 15 min, and washed with PBS. At the end of the incubation period the cells were washed twice with cold PBS, and resuspended in PBS (400 mL). J-aggregate and J-monomer fluorescence were recorded in the channesl 2 (FL2) and 1 (FL1), respectively, of an FC500 flow cytometer. Necrotic fragments were electronically gated out, on the basis of morphological characteristics on the forward light scatter versus side light scatter dot plot.
Fluorescent microscopy assay: Cells were plated into 24-well plates (10 5 cells per well) containing glass cover slips, and allowed to adhere to the surface for 24 h. Cells were incubated for the appropriate time with the compounds. After incubation the cell culture media was removed and replaced with JC-1 reagent (5 mm) diluted in PBS. Cells were incubated at 37 8C in a 5% CO 2 incubator for 15 min, and analyzed by fluorescence microscopy.
Cytofluorimetric analysis of DNA content: Exponentially growing KB-3-1 cells in 6-well plates (5 10 5 cells per well) were treated with 12 (0.3 mm, or 1 mm) or DMSO (0.1 % (v/v)) for 18 h. After incubation, the cells were collected by centrifugation (400 g, 10 min), fixed with ice-cold 70 % ethanol for at least 1 h at 4 8C and treated with RNase A from bovine pancreas (1 mg mL À1 ; Sigma) for 30 min at 37 8C. PI (50 mg mL À1 ) was then added to the solution and the DNA content was quantitated by a flow cytometry. Cells in sub-G1 phase were considered apoptotic.
Flow cytometry assay: Cells were incubated for the appropriate time in the presence of the compounds, collected, suspended in PBS (0.5 mL), and FITC-VAD-FMK (1 mL, 5 mm) was added. The cells were gently mixed and incubated for 20 min at RT in the dark. Cells were washed twice with PBS, and the pellets resuspended in PBS (0.5 mL). Flow cytometry was conducted within 10 min.
Fluorescent microscopy assay: Cells were seeded (10 5 cells per well) into 24-well plates containing glass cover slips, and allowed to adhere to the surface for 24 h. After incubation for the appropriate time with the compounds, the cell culture medium was removed and replaced with JC-1 reagent (5 mm) diluted in PBS. Cells were incubated at 37 8C in a 5% CO 2 incubator for 15 min. The cells were