{gamma}-Tocopheryl quinone induces apoptosis in cancer cells via caspase-9 activation and cytochrome c release

Gabriella Calviello, Fiorella Di Nicuolo, Elisabetta Piccioni, M.Elena Marcocci, Simona Serini, Nicola Maggiano1, Kenneth H. Jones2, David. G. Cornwell3 and Paola Palozza

Institutes of General Pathology and
1 Pathology, Catholic University, L.go F.Vito, 1–00168 Rome, Italy,
2 Department of Anatomy and Medical Education and
3 Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recently, it was suggested the potential role of {gamma}-tocopheryl quinone ({gamma}-TQ), an oxidative metabolite of {gamma}-tocopherol, as a powerful chemotherapeutic agent, since it was shown that this molecule exerts powerful cytotoxic effects, induces apoptosis and escapes drug resistance in human acute lymphoblastic leukemia and promyelocytic leukemia cells. We have studied the apoptogenic potential of {gamma}-TQ in cultured human leukemia HL-60 and colon adenocarcinoma WiDr cells, and in murine thymoma cells growing in vivo in ascites form. The cells were treated with {gamma}-TQ and apoptosis was evaluated morphologically by acridine-orange staining and cytofluorimetrically by Annexin V binding assay. {gamma}-TQ-induced apoptosis in a dose- and time-dependent manner in all the cell types tested, although HL-60 and thymoma cells were much more sensitive than WiDr cells. In HL-60 cells apoptosis was mediated by the activation of the caspase-3 cascade. In particular, we observed a time- and dose-dependent increase in the activities of the upstream caspase-9 and caspase-8 and of the downstream caspase-3. The activation of caspase-9 preceded that of caspase-8 and its specific inhibition completely prevented apoptosis. These findings and data showing the precocious release of cytochrome c from mitochondria, a decrease in Bcl-2, and a change in mitochondrial transmembrane potential ({Delta}{psi}m), all suggest that the intrinsic mitochondrial pathway is primarily involved in the development of {gamma}-TQ-induced apoptosis. The late activation of caspase-8 and data showing the partial cleavage of pro-apoptotic protein BID suggest that the initial activation of caspase-9 may be potentiated by a feedback amplification loop involving the caspase-8/BID pathway.

Abbreviations: AMC, 7-amido-4-methylcoumarin; DiOC6(3), 3'-dihexyloxacarbocyanide iodide; DTT, dithiothreitol; {Delta}{Psi}m, mitochondrial transmembrane potential; MDR, multidrug resistance; {alpha}-T, {alpha}-tocopherol; {delta}-T, {delta}-tocopherol; {gamma}-T, {gamma}-tocopherol; {alpha}-TQ, {alpha}-tocopherylquinone; {delta}-TQ, {delta}-tocopherylquinone; {gamma}-TQ, {gamma}-tocopherylquinone.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In recent years, different components and derivatives of the vitamin E group of compounds including {alpha}-tocopherol ({alpha}-T) (1,2), {alpha}-tocopheryl succinate (3), {delta}-tocopherol ({delta}-T) (4), {gamma}-tocopherol ({gamma}-T) (5,6) and {alpha}-, {gamma}-, and {delta}-isoforms of tocotrienols (4,7) have received particular attention as chemotherapeutic agents. An anticancer therapeutic role for the oxidative derivatives {gamma}- and {delta}-tocopheryl quinone ({gamma}-TQ and {delta}-TQ) was put forward recently by Cornwell et al. (810). Quinones represent a clinically important category of chemotherapeutic agents with a wide range of applications in both antitumor and anti-microbial therapy (1113). Some peculiar features of tocopheryl quinones make them particularly interesting. In acute lymphoblastic and promyelocytic leukemia cell lines, {gamma}-TQ and {delta}-TQ were more cytotoxic than doxorubicin, a xenobiotic quinone currently used in chemotherapy (810). The cytotoxicity of {gamma}-TQ appeared related to its property as an arylating electrophile, as the non-arylating compounds, {alpha}-tocopherylquinone ({alpha}-TQ) and the glutathion-S-yl derivative of {gamma}-TQ, are not cytotoxic (810). {gamma}-TQ retained its cytotoxicity when tested in multidrug resistance (MDR) leukemic cell lines (810), differing in this behavior from many xenobiotics widely used in cancer chemotherapy and known to stimulate MDR (1416).

Unlike {alpha}-TQ, significant amounts of {gamma}-TQ are not identified in animal tissues for several reasons. The concentration of precursor {gamma}-T is low in most tissues because the liver synthesizes a specific transport protein for {alpha}-T (17). Furthermore, large amounts of {gamma}-T are selectively metabolized to {gamma}-[2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxy chroman] ({gamma}-CEHC) and excreted in the urine (18). On the other hand, {gamma}-T is widely distributed and abundant in vegetable oils (19). It is plausible that the inability of the tissues to retain {gamma}-T represents an evolutionary advantage by allowing the production of only small amounts of its mutagenic metabolite {gamma}-TQ (20). Finally, {gamma}-TQ is an arylating electrophile rapidly converted to the glutathion-S-yl and other nucleophile derivatives (8) that, unlike {alpha}-TQ, would not be identified by conventional assay methods. It has been suggested that, even though scarce, the possible tissue production of {gamma}-TQ may induce a state of tolerance toward it, that would explain the ability of this quinone to escape MDR (8).

Recently it has been shown that the effect of {gamma}-TQ, similarly to different cytotoxic quinones used in chemotherapy, including doxorubicin (21), mitomycin C (22,23) and menadione (24), is mainly related to its ability to induce apoptosis in leukemia cells and in breast cancer cells (10). In the present investigation, we have evaluated the apoptogenic potential of {gamma}-TQ in WiDr colonic adenocarcinoma, HL-60 leukemia and murine tumor thymocyte cells, and studied pathways through which {gamma}-TQ mediates the expression of different proteins involved in the apoptotic cascade. We found the activation of the mitochondrial death pathway characterized by the disruption of mitochondrial transmembrane potential, release of cytochrome c and activation of caspase-9.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cells
Two human cell lines were used: HL-60 cells, a promyelocytic leukemia and WiDr cells, a colon adenocarcinoma (American Type Culture Collection, Manassas, VA). We also used a murine ascites thymoma grown in Balb/c mice originally provided by the Institute of Pathology, University of Perugia, Italy (25). HL-60, and WiDr cells were maintained in RPMI 1640 (Sigma, St Louis, MO) supplemented with 10% (v/v) fetal calf serum (FCS, GIBCO-Invitrogen Corporation, San Diego, CA), 2 mM L-glutamine, 1% penicillin-streptomycin in a humidified atmosphere containing 5% CO2 at 37°C. HL-60 cells were seeded at 3x105 cells/ml twice a week to maintain log phase growth. WiDr cells were split by trypsinization and plated at 3x105 cells/ml. Experiments were performed 1 day after trypsinization. Thymoma cells were grown by weekly intraperitoneal (i.p.) transplantation in Balb/c mice. Cells were harvested after 7–8 days, washed twice and resuspended in Ringer-HEPES, (130 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1.3 mM CaCl2; 50 mM HEPES, pH 7.4). For morphological experiments, thymoma cells were incubated at 37°C under O2 for variable length of time at a cell density of 2x106 cells/ml.

{gamma}-TQ was synthesized and purified as described previously (8,9). {gamma}-TQ, which was dissolved in ethanol, was diluted to the final concentration in the appropriate culture medium.

Evaluation of apoptosis
Morphological evaluation. Cell suspensions were stained with a mixture of chromatin dyes, the membrane permeant dye acridine-orange (100 µg/ml) and membrane impermeant ethidium bromide (100 µg/ml). Stained cells were examined by fluorescence microscopy at x400 magnification. Necrotic cells (damaged plasma membrane and non-condensed nuclei) and apoptotic cells (condensed or fragmented nuclei) were scored manually. At least 200 cells/time point were scored (26). When indicated, 200 µM AC-LEHD-CHO or Ac-IETD-CHO (Alexis Biochemical Italia, FI, Italy), specific inhibitors of caspase-9 and caspase-8 activity, respectively, were added to the incubation medium.

Cytofluorimetric evaluation. Apoptotic cells were also identified using the Apo-alert Annexin V-FITC apoptosis detection kit (Calbiochem, La Jolla, CA) according to the manufacturer’s instructions. This allows the detection of phosphatidyl-serine on the external cell membrane early in apoptotic cell death. Increased permeability to propidium iodide (PI) is noted in the late phase of apoptosis. {gamma}-TQ-induced apoptosis was thereafter quantified by flow cytometry (Coulter Epics XL-MLC flow cytometer, Miami, FL).

Caspase activity. To measure the activity of caspases-3, -8 and -9 in HL-60 cells, a fluorimetric assay was used according to the instruction of manufacturer (27). Briefly, untreated and {gamma}-TQ-treated cells (2x106) were collected, resuspended in cold lysis buffer (50 mM Tris–HCl, pH 7.5, containing 0.5 mM EDTA, 0.5% Igepal and 150 mM NaCl) and incubated for 30 min on ice. After centrifugation (1000 g, for 10 min) cell lysates (50 µl) were incubated for 2 h at 37°C with fluorogenic substrates, Ac-DEVD-AMC (7-amido-4-methylcoumarin) (caspase-3), Ac-IETD-AMC (caspase-8) and Ac-LEHD-AMC (caspase-9) (Alexis Biochemical Italia), in a reaction buffer [10 mM HEPES, pH 7.5, containing 50 mM NaCl and 2.5 mM dithiothreitol (DTT)]. The release of fluorocrome AMC (7-amido-4-methylcoumarin) was measured at 380 nm excitation and 480 nm emission using a fluorescence spectrophotometer (CytofluorTM 2300/2350, Millipore, Bedford, MA).

Quantification of mitochondrial transmembrane potential. Mitochondrial transmembrane potential ({Delta}{Psi}m) was measured using a cationic fluorochrome 3,3'-dihexyloxacarbocyanide iodide [DiOC6(3)] as described by Zamzami et al. (28). Briefly, the cells (1x106 in 0.5 ml of phosphate buffered saline, PBS) were stained with 50 µl of DiOC6(3) (40 nM final concentration) and transferred in a water bath kept at 37°C. After 15–20 min of incubation the cells were placed on ice and then analyzed cytofluorimetrically (488 excitation, 525 emission). As a negative control, in each experiment aliquots of cells were labeled in the presence of an uncoupling agent [the protonophore carbonyl cyanide m-chlorophenyl hydrazone (mClCCP) 50 mM final concentration] that abolishes the {Delta}{Psi}m.

Western blot analysis. Cytosolic extracts for cytochrome c determination were prepared according to Park et al. (29). Briefly, HL-60 cells (4x106) were washed with PBS, resuspended in cold lysis buffer (250 mM sucrose, 1 mM EDTA, 20 mM Tris–HCl, pH 7.2, 1.0 mM DTT, 10 mM KCl, 1.5 mM MgCl2, 10 µg/ml leupeptin, 2 µg/ml aprotinin), and incubated for 2 min on ice. After centrifugation (12 000 g for 10 min) the supernatants were collected and stored at –80°C until used. Total cell extracts for BID and Bcl-2 determination were prepared by resuspending cells in cold lysis buffer (1 mM MgCl2, 350 mM NaCl, 20 mM HEPES, 0.5 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM Na4P2O7, 1 mM PMSF, 1 mM aprotinin, 1.5 mM leupeptin, 20% glycerol, 1% NP-40) (30). After incubation for 30 min at 4°C, the cells were centrifuged (10 000 g for 10 min at 4°C) and the supernatants were stored at –80°C until used. The proteins were separated through electrophoresis on 12% SDS–PAGE gel and transferred onto nitrocellulose membranes for western blotting analysis. The membrane was blocked overnight at 4°C in 5% dried milk (w/v) in PBS plus 0.05% Tween-20, and then incubated with polyclonal primary antibody to cytochrome c, BID or Bcl-2 (Santa Cruz Laboratories, Santa Cruz, CA) for 2 h at room temperature. After incubation with secondary antibody the immunocomplexes were visualized using ECL-Plus Detection System (Amersham, Pharmacia Italia, Milano, Italy) according to the instruction of the manufacturer and quantified by densitometric scanning.

Statistical analysis
The results are presented as the mean ± SEM. The data were analyzed using one-way analysis of variance (ANOVA). Post hoc comparisons of means were made using Fisher’s test (significance P < 0.05). Differences were analyzed using Minitab Software (Minitab, State College, PA).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Morphological evaluation of apoptosis
The morphological examination of cells stained with the fluorescent dye acridine-orange revealed that {gamma}-TQ caused a concentration- and time-dependent increase in the percentage of apoptotic cells in all the strains of cells evaluated (Figure 1Go). WiDr cells were less sensitive to the pro-apoptogenic effect of {gamma}-TQ than either HL-60 or thymoma cells after 4 h exposure. Whereas 50 µM {gamma}-TQ induced apoptosis in ~60% of HL-60 cells and neoplastic thymocytes (Figure 1A and BGo), the maximal percentage of apoptotic WiDr cells, obtained with 200 µM {gamma}-TQ, was only 25% (Figure 1CGo). Neoplastic thymocytes were the most responsive to the pro-apoptogenic effect of {gamma}-TQ, showing a significant percentage of apoptotic cells already at 2 h of {gamma}-TQ treatment (Figure 1BGo). The other two cell strains had an initial lag phase of 2 h before showing the morphological changes of apoptosis.



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Fig. 1. Effect of {gamma}-TQ on the percentage of apoptotic cells. Apoptosis was measured by morphological examination of HL-60 (A), thymoma (B) and WiDr (C) cells stained with acridine-orange. Cells were exposed to indicated amounts of {gamma}-TQ for 4 h. Values were the mean ± SEM of six to eight experiments. *Significantly different from control (P < 0.05).

 
Translocation of cell membrane phosphatidyl serine
The apoptosis-inducing effect of {gamma}-TQ was confirmed in HL-60 cells (Figure 2Go) by flow cytometric evaluation of Annexin V binding, which measures phosphatidyl serine on the external leaflet of the plasma membrane, an event characteristic of early apoptosis. Four hour treatment with 50 µM {gamma}-TQ increased the percentage of apoptotic cells from 5.4 ± 0.5 (control condition) to 56.9 ± 5.4%.



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Fig. 2. Representative dot plot showing the effect of {gamma}-TQ on apoptosis on HL-60 cells. Proportion of apoptotic cells in a population was measured by the bivariate Annexin V/PI flow cytometry in control cells and cells treated with 50 µM {gamma}-TQ for 4 h. One representative plot of three similar experiments is shown.

 
Involvement of caspase-3, -8 and -9
As a family of aspartate-specific cysteinyl proteases (caspases) plays a pivotal role in the execution of programmed cell death (3134), we tested whether treatment of HL-60 cells with {gamma}-TQ resulted in activation of two upstream caspases-8 and -9 and of the downstream caspase-3 (Figure 3Go). In particular, we evaluated caspase-8 activity because it represents the apical caspase in the death receptor (extrinsic) pathway (32) and caspase-9, as it serves as the apical caspase of the mitochondrial (intrinsic) pathway (33). Moreover, we analyzed caspase-3 because it has been shown to be one of the most important cell executioners for apoptosis (34). To determine whether activation of caspase-3, -8 and -9 plays a role in {gamma}-TQ-induced apoptosis, HL-60 cells were incubated with {gamma}-TQ and the cleavage of the fluorocrome AMC from the specific fluorogenic peptide substrates (Ac-DEVD-AMC, Ac-IETD-AMC and Ac-LEHD-AMC for caspase-3, -8 and -9, respectively) was analyzed fluorimetrically (Figure 3Go). A marked time-dependent increase in the activities of caspase-3, -8 and -9 was observed in HL-60 cells treated with 50 µM {gamma}-TQ (Figure 3AGo). However, the temporal pattern of activation of the three caspases was markedly different. There was significant activation of caspase-9 as early as 2 h of treatment, and the activity continued to increase at 3 and 4 h of the assay. On the other hand, significant activation of caspase-8 was not found until after 3 h. Caspase-3 activity was very low at 2 h and then increased abruptly. The early activation of caspase-9 suggests that the {gamma}-TQ pro-apoptotic effect may be elicited mainly via the mitochondrial pathway. Figure 3Go(B–D) shows that {gamma}-TQ-induced activation of caspases is both dose-dependent and time-dependent.



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Fig. 3. Time-dependent and dose-dependent activation of caspase-9, -8 and -3 by {gamma}-TQ in HL-60 cells. (A) Cells were treated with 50 µM {gamma}-TQ for the indicated times. The activation was measured as the increase in activity (%) with respect to control cells (100%). The control basal value was 640 ± 31, 635 ± 32 and 57 ± 4 arbitrary fluorescence units for caspase-3, -8 and -9, respectively. In (BD), cells were treated with increasing concentration of {gamma}-TQ for the indicated times. Values were the mean ± SEM of five experiments.

 
To further evaluate whether caspase-9 mitochondrial pathway was primarily induced by {gamma}-TQ, we investigated to which extent its activation may be responsible for apoptosis induced by the quinone. Figure 4Go shows the percentage of apoptotic HL-60 cells after treatment with 50 µM {gamma}-TQ for 4 h in the absence or in the presence of 200 µM AC-LEHD-CHO or Ac-IETD-CHO, specific caspase-9 and -8 inhibitors, respectively. Apoptosis was evaluated morphologically using acridine-orange to stain the cells. The specific inhibition of caspase-9 led to the complete suppression of {gamma}-TQ-induced apoptotsis. Lower concentrations of the inhibitor (10–20 µM AC-LEHD-CHO) had less of an effect (data not shown). In contrast, there was a much smaller decrease in the percentage of apoptotic cells when cells were incubated with the caspase-8 inhibitor. A similar limited inhibition of {gamma}-TQ-induced apoptosis was observed in cells treated with a higher concentration (300 µM) of caspase-8 inhibitor (data not shown). These findings provide evidence that {gamma}-TQ induces apoptosis through the mitochondrial pathway.



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Fig. 4. Effect of caspase-9 and -8 inhibition on {gamma}-TQ-induced apoptosis in HL-60 cells. {gamma}-TQ was added at a concentration of 50 µM with or without 200 µM Ac-LEDH-CHO (caspase-9 inhibitor) or Ac-IETD-CHO (caspase-8 inhibitor) for 4 h and apoptosis was evaluated morphologically after acridine-orange staining. Values were the mean ± SEM of three experiments. *Significantly different from control (P < 0.05).

 
Cytochrome c release from mitochondria
To confirm the involvement of the mitochondrial pathway of apoptosis, we measured the induction of cytochrome c release from the mitochondria by {gamma}-TQ. It is known that cytochrome c released from mitochondria into the cytosol binds to the apoptotic protease activating factor (Apaf) complex and triggers the activation of pro-caspase-9 to the active caspase-9 (35). As shown in Figure 5Go, a marked fraction of the cytochrome c was released from the mitochondria of {gamma}-TQ-treated cells at 2 h, and the release was more pronounced at 3 h. This increase in the release of cytochrome c was in agreement with the data showing the continued increase in caspase-9 activity after 2 and 3 h of treatment (Figure 3AGo). The close association of the release of cytochrome c from mitochondria with the concurrent increase in caspase-9 provide evidence that {gamma}-TQ induces apoptosis in HL-60 cells through the mitochondrial pathway.



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Fig. 5. Effect of {gamma}-TQ treatment on mitochondrial cytochrome c release in HL-60 cells. Cytochrome c protein content of the cytosolic fraction was measured by western blotting. Cells were treated with 20 or 50 µM {gamma}-TQ for 2 and 3 h. (A) One representative of five similar experiments is shown. (B) Values were the mean ± SEM of five experiments.

 
{Delta}{Psi}m loss
Cytochrome c may be released from mitochondria into the cytosol by opening a pore during membrane permeability transition, and changes in the opening of this pore have been postulated to play a role in cellular events leading to apoptosis of certain types of cells (36). Figure 6Go presents the data of FACS analysis of the incorporation of fluorochrome DiOC6(3) used to test for changes in the membrane potential of intracellular mitochondria in intact cells treated with {gamma}-TQ. HL-60 cells were incubated with 0, 20 and 50 µM {gamma}-TQ for 2 and 3 h. A time- and dose-dependent loss of mitochondrial membrane potential ({Delta}{Psi}m) was observed. {Delta}{Psi}m decreased after 2 h of {gamma}-TQ treatment by 24.7 ± 3.0% at the highest concentration tested (50 µM). After 3 h, DiOC6(3) incorporation decreased by 20.6 ± 3.2 and 55.0 ± 5.1% in HL-60 cells treated with 20 and 50 µM {gamma}-TQ, respectively. The data suggest that the early {Delta}{Psi}m decrease may trigger cytochrome c release and its subsequent activation of caspase-9.



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Fig. 6. Effect of {gamma}-TQ on {Delta}{Psi}m in control and treated HL-60 cells. Representative histograms showing {Delta}{Psi}m of control and treated cells. {Delta}{Psi}m was measured by flow-cytometry as DiOC6(3) intensity in cells treated with 20 µM {gamma}-TQ for 2 (A) and 3 h (B), and with 50 µM {gamma}-TQ for 2 (C) and 3 h (D). The histogram in the presence of the uncoupler mClCCPP is also shown in (A). The effect of {gamma}-TQ on {Delta}{Psi}m loss (percent of total cell number) in the presence of 20 and 50 µM {gamma}-TQ is shown in (E), and the values were the mean ± SEM of three experiments.

 
Alterations in the expression of cellular Bcl-2 and BID
Caspase-8 activation, when triggered downstream of mitochondrial pathway of apoptosis, may further amplify caspase-9 and -3 activation through cleavage of the pro-apoptotic protein BID (37,38). Cleaved BID binds to mitochondria, antagonizes anti-apoptotic proteins of the Bcl-2 family and causes a further efflux of cytochrome c into the cytosol (39). Therefore, we investigated whether {gamma}-TQ elicits the cleavage of BID, thus activating an amplifying loop for caspase-9 and -3. Figure 7Go shows that there was a significant decrease of uncleaved BID in cells treated with 20 or 50 µM {gamma}-TQ for 3 h (25.4 and 42.0% decrease, respectively, as compared with control). Interestingly, the level of uncleaved BID was similar to controls at 2 h when caspase-8 had not yet been activated (data not shown). Thus, it is possible to speculate that caspase-8 activation and BID cleavage could trigger a feedback amplification pathway for caspase-9 activation.



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Fig. 7. Effect of {gamma}-TQ on the expression of BID (A and C) and Bcl2 (B and D) in HL-60 cells. One representative of five similar western blot analyses is shown for BID (A) and Bcl-2 (B). (C) Measures BID as percent of control. (D) Measures Bcl-2 as percent of control. 50 µM {gamma}-TQ was added for 3 h. Values were the mean ± SEM of five experiments. Bars with different letters are significantly different (P < 0.05).

 
We also studied the effect of {gamma}-TQ on Bcl-2. The anti-apoptotic protein Bcl-2 is an integral membrane protein located mainly on the outer membrane of mitochondria (40). As some observations suggest that Bcl-2 can be involved in the release of cytochrome c from mitochondria (41), we measured the expression of Bcl-2 in HL-60 cells treated with 20 and 50 µM {gamma}-TQ. A small but significant decrease in Bcl-2 was observed after 3 h of treatment with {gamma}-TQ, which indicated that Bcl-2 was involved in {gamma}-TQ-induced release of cytochrome c from mitochondria.


    Discussion
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 References
 
A number of studies have implicated apoptosis as an important mechanism by which chemotherapeutic agents kill susceptible cells (42,43). The results shown in this report demonstrate that {gamma}-TQ, an oxidative metabolite of {gamma}-tocopherol (810), is able to induce apoptosis in different strains of tumor cells of human (leukemia HL-60 and colonic adenocarcinoma WiDr cells) and murin origin (Balb/c neoplastic thymocytes). Moreover, they provide some insights into the signaling mechanisms that underlie {gamma}-TQ-induced apoptosis. Recently, a potential chemotherapeutic role for {gamma}-TQ has been hypothesized, which was shown to exert cytotoxic effects more powerful than the widely studied chemotherapeutic drugs doxorubicin and vinblastine in human leukemia cell lines (8). Moreover, it was recently reported that this compound is able to induce apoptosis in breast cancer and leukemia cells (10). {gamma}-TQ was also shown to be able to escape drug resistance, exerting its cytotoxic and pro-apoptotic effects also in MDR leukemia cells (810). This property suggests a high tolerance of the organism toward this compound, which has been related to the molecular structure of {gamma}-TQ (8).

{gamma}-TQ induces apoptosis in a concentration- and time-dependent manner in cells of different origin and the various cell types show different sensitivities with a more powerful pro-apoptogenic action toward HL-60 and thymoma cells, than toward the colonic cell line. Our previous studies (810) and the present finding suggest that cells of leukocytic origin may represent the preferential target of the possible chemotherapeutic action of {gamma}-TQ. Furthermore, the morphological features of apoptosis were detectable very early during the treatment of all the types of cells (2–3 h), and the pro-apoptotic effect of {gamma}-TQ was confirmed by the cytofluorimetric analysis of Annexin V binding in HL-60 cells.

Some of the molecular and biochemical pathways involved in {gamma}-TQ-induced apoptosis (44) were investigated in HL-60 cells. We found that {gamma}-TQ induced apoptosis by the activation of the downstream caspase-3, which has been shown to play an important role in apoptosis induced by several conditions (4547), and to be necessary in determining the nuclear alteration of apoptosis (34). In our model, caspase-3 activation was preceded by the activation of caspase-9, the apical caspase of the intrinsic mitochondrial pathway of apoptosis (occurring at the second hour of treatment). On the other hand, the activation of caspase-8, the apical caspase of the extrinsic pathway, became evident only later (at the third hour), concomitantly to the activity of caspase-3. This finding suggests that caspase-9 may play the main role in the initial triggering of the cleavage and activation of pro-caspase-3 and that caspase-8 activation may represent a downstream event after the activation of caspase-9. This hypothesis is in agreement with recent evidences (37,38) suggesting that activation of caspase-8 may also occur as a consequence of the activation of caspase-9, even though traditionally it was associated with Fas receptor-induced apoptosis. It is conceivable that the lipophilic quinone permeates the membrane and activates the intrinsic mitochondrial pathway, leading to the activation of caspase-9, thus supporting the hypothesis that caspase-9 represents the most apical caspase in chemical induced apoptosis (48). The dose- and time-dependent decrease in {Delta}{Psi}m, occurring after only 2 h of {gamma}-TQ treatment, further indicates the early activation of the intrinsic mitochondrial pathway (33). Similarly, {gamma}-TQ induced the release of cytochrome c into the cytosol after 2 h, and this release markedly increased after 3 h. The primary involvement of the mitochondrial pathway in {gamma}-TQ-induced apoptosis was confirmed by the observation that a specific inhibitor of caspase-9 (Ac-LEHD-CHO) completely inhibited {gamma}-TQ-induced apoptosis in HL-60 cells, whereas specific inhibitor of caspase-8 (Ac-IETD-CHO) caused only a small, but significant decrease in apoptosis. The data support the hypothesis that caspase-8 activation is secondary to the activation of caspase-9 and functions to amplify caspase-9 activation in {gamma}-TQ-induced apoptosis. Recent reports (37,38) suggest that caspase-8 activation, when triggered downstream of the mitochondrial pathway of apoptosis, may amplify caspase-9 activation through the cleavage of the pro-apoptotic protein BID, which, in turn, elicits a further efflux of cytochrome c from mitochondria (39). We hypothesize that this amplification loop for caspase-9, involving the caspase-8/BID pathway, may take place in our model. In agreement, we found that, after 3 h of treatment, when caspase-8 became activated, a significant caspase-8-dependent BID cleavage occurred in HL-60 cells treated with {gamma}-TQ.

Our data show a slight but significant reduction in the expression of the anti-apoptogenic protein Bcl-2 following {gamma}-TQ treatment, which suggests that this protein is involved in the release of cytochrome c from mitochondria.

The biochemical and molecular mechanisms underlying the pro-apoptotic and cytotoxic effects of {gamma}-TQ were recently clarified by Jones et al. (10). They put forward an ‘arylation hypothesis’ based on the notion that {gamma}-TQ, as other alkylating and arylating quinones, is detoxified through the formation of glutathione (GSH) adducts (10). They hypothesized that the consequent depletion of intracellular GSH, known to be a common event in damage-induced apoptosis (49), may be essential in triggering {gamma}-TQ-induced apoptosis.

In conclusion, {gamma}-TQ-induced apoptosis appears to be primarily associated with the early activation of caspase-9 through the mitochondrial pathway, as demonstrated by the early {Delta}{Psi}m loss and cytochrome c release from mitochondria. The activation of the mitochondrial pathway may in turn activate caspase-3, -8 and BID cleavage, triggering a feedback amplification loop for caspase-9. These results, together with previous findings concerning the high cytotoxic and the pro-apoptogenic effects of the quinone toward tumor cells suggest a potential role of {gamma}-TQ as a powerful chemotherapeutic agent.


    Notes
 
4 To whom correspondence should be addressed Email: g.calviello{at}rm.unicatt.it Back


    Acknowledgments
 
The authors dedicate this work to the memory of Prof. G.M.Bartoli who started it with great enthusiasm. This work was supported by a grant from MIUR, Ministero Istruzione Università e Ricerca Scientifica, Italy.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 15, 2002; revised October 28, 2002; accepted November 26, 2002.