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Cytochrome c Release Is Upstream to Activation of Caspase-9, Caspase-8, and Caspase-3 in the Enhanced Apoptosis of Anaplastic Thyroid Cancer Cells Induced by Manumycin and Paclitaxel

Section of Endocrine Neoplasia and Hormonal Disorders, Department of Internal Medicine Specialties (J.P., G.X., S.-C.J.Y.), and Section of General Internal Medicine, Department of Internal Medicine Specialties (S.-C.J.Y.), University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

Address all correspondence and requests for reprints to: S. Jim Yeung, M.D., Ph.D., University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 40, Houston, Texas 77030. E-mail: syeung{at}notes.mdacc.tmc.edu

Abstract

We previously demonstrated that the combination of a farnesyltransferase inhibitor, manumycin A, and paclitaxel had a synergistic antineoplastic effect on anaplastic thyroid cancer. In this study we investigated the apoptosis pathway involved. In ARO and KAT-4 cells, manumycin- plus paclitaxel-induced DNA fragmentation was blocked by the inhibitors of caspase-9, caspase-8, and caspase-3. The drug combination enhanced the activation of caspase-9, caspase-8, and caspase-3 and cytochrome c release into the cytosol. Cytochrome c release was not affected by the inhibitors of caspase-9, caspase-8 and caspase-3. In a cell-free reconstitution assay, DNA fragmentation occurred after incubating nuclei purified from untreated KAT-4 cells with deoxy-ATP, exogenous cytochrome c and S-100 extracts from control KAT-4 cells, and also after incubation of purified KAT-4 nuclei with S-100 extracts from KAT-4 cells treated with manumycin-plus-paclitaxel. In both cases, the DNA fragmentation was blocked by the inhibitors of caspase-9, caspase-8 and caspase-3. We concluded that the cytochrome c release was upstream of the activation of caspase-9, caspase-8, and caspase-3 in the enhanced apoptosis of anaplastic thyroid cancer cells treated with manumycin plus paclitaxel, and that the interaction between manumycin and paclitaxel occurred at or upstream of cytochrome c in the apoptosis regulatory pathway in anaplastic thyroid cancer cells.

ALTHOUGH ANAPLASTIC thyroid cancer (ATC) accounts for about 5% of all thyroid cancers (1, 2), it is one of the most aggressive solid tumors. Current therapy does not significantly improve survival rates (3, 4, 5, 6). Paclitaxel, an inhibitor of tubulin depolymerization (7, 8), has been shown to have antineoplastic activity against ATC (9). A clinical trial of paclitaxel against ATC demonstrated a response rate of 53% in 19 patients (10). Thus, investigating new therapeutic modalities against ATC is necessary.

Our previous work demonstrated that the combination of paclitaxel and manumycin, a farnesyltransferase inhibitor (FTI), resulted in enhanced antineoplastic activity against ATC cells (11). FTIs are a novel group of compounds with potential chemotherapeutic effects (12, 13, 14). Although the mechanism of the antineoplastic action of FTIs is complex and probably multimodal, one widely accepted view is that interference of the Ras signaling pathway plays a role. Recent evidence has implicated Rho B in the mechanism of action of FTIs as well (15, 16). Another possible mechanism for FTI-induced apoptosis of cancer cells is through inhibition of the PI3K/Akt2-mediated cell survival pathway (17, 18). The Akt kinase may be the link between Ras and Pak and, subsequently, the cell survival signals (19). Manumycin A, a natural product of Streptomyces parvulus (20), inhibits farnesyltransferase by competition with farnesyl pyrophosphate groups (21). Manumycin has shown antitumor activity in several experimental systems (11, 22, 23, 24, 25). The enhanced inhibitory effect on ATC cells in vitro by the combination of paclitaxel and manumycin was due to enhanced induction of apoptosis (11). The present study sought to identify the apoptosis pathway involved in the ATC cells treated with manumycin plus paclitaxel by tracing backward the regulatory pathway of apoptosis.

Numerous hierarchical models of the caspase cascade have been proposed (26, 27, 28). Caspase-8 is thought to be the key upstream regulatory caspase in the death [TNF/Fas (CD95)] receptor signaling pathway (27, 29). Based on whether cytochrome c release is required, two pathways of Fas death receptor signals have been identified (30, 31). One pathway involves direct activation of caspase-3 by caspase-8 without cytochrome c release (pathway 1, dotted arrows in Fig. 1). The other pathway involves the release of cytochrome c by activated caspase-8 via Bid (pathway 2, dashed arrows in Fig. 1). As for drug-induced apoptosis, cytochrome c release is upstream to caspase activation, and caspase-9 is activated by cytochrome c via Apaf-1 (solid arrows in Fig. 1) (26, 27). Overall, the current understanding of apoptosis regulation can be summarized in these three convergent pathways.

View larger version (21K): [in this window] [in a new window]   Figure 1. Three major apoptosis pathways. The type I death receptor pathway is represented by the dotted arrows. The type II death receptor pathway is represented by the dashed arrows. The drug- induced apoptosis pathway is represented by the solid arrows.

We hypothesized that the combination of manumycin and paclitaxel enhanced apoptosis of ATC cells through the drug-induced apoptosis pathway, which implied that release of cytochrome c was upstream of activation of caspase-8, -9, and -3. This report examined the effect of the combination of manumycin and paclitaxel on cytochrome c and caspases in ATC cells. Using different caspase inhibitors and an in vitro reconstitution assay, we defined the hierarchical relationship between cytochrome c and the caspases and provided evidence to support the hypothesis that the release of cytochrome c was upstream of caspase activation in the enhanced apoptosis induced by combining manumycin and paclitaxel.

Materials and Methods

Materials

Paclitaxel, tissue culture grade dimethylsulfoxide (DMSO), bovine cytochrome c, Igepal CA-630, sodium deoxycholate, Tween 40, Tween 20, Triton X-100, and digitonin were purchased from Sigma (St. Louis, MO). All tissue culture media and additives were purchased from Life Technologies, Inc. (Gaithersburg, MD). Monoclonal antiactin antibody and anticaspase-3 antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The anticaspase-8 antibody and anticaspase-9 antibody were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Antirabbit and antimouse IgG-peroxidase conjugates were purchased from Roche (Indianapolis, IN). Anticytochrome c oxidase subunit II was purchased from Molecular Probes, Inc. (Eugene, OR). Inhibitor of caspase-3 (Ac-DEVD-CHO), inhibitor of caspase-8 (Ac-IETD-CHO), inhibitor of caspase-6 (Ac-VEID-CHO), and anticytochrome c antibody were purchased from BD PharMingen (San Diego, CA). Manumycin A, caspase-1 inhibitor (benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone), and caspase-9 inhibitor (Ac-LEHD-CHO) were purchased from Alexis Biochemicals Corp. (San Diego, CA).

Cell lines and tissue culture

Human ATC cell lines ARO (34) and KAT-4 (9) were used in this study. The cells were cultured in RPMI 1640 with heat-treated bovine serum (10%), penicillin (50 U/ml), streptomycin (50 µg/ml), MEM nonessential amino acids (1x), pyruvate (1 mM), glutamine (2 mM), and amphotericin (2.5 µg/ml) at 37 C in a water-saturated atmosphere with 5% CO₂. Manumycin and paclitaxel were dissolved in DMSO. The stock solutions of these drugs were then diluted in complete culture medium with 10% bovine serum (as described above) to appropriate concentrations. The doses of manumycin and paclitaxel chosen were close to their respective 75% inhibitory concentrations based on a cell viability assay using the WST-1 dye (11). The final concentration of DMSO in the culture medium was 0.1% (vol/vol) or less.

DNA fragmentation ELISA

Quantitative measurement of DNA fragmentation was performed using the cellular DNA fragmentation ELISA kit from Roche. Equal numbers of cells were plated into each well in a tissue culture plate. Each experimental group or the control group consists of three wells. 5'-Bromo-2'-deoxyuridine (BrdU) was used to label the nuclear DNA. In apoptotic cells, the BrdU-labeled DNA was fragmented and released into the cytosol. The labeled DNA in cytosolic extracts was detected and quantitated using a monoclonal antibody directed against BrdU in ELISA according to the manufacturer’s protocol.

Fluorogenic caspase-8, -9, -6, and -1 activity assays

The activity of caspase-8 in ARO cells after drug treatments for 6 h was measured using a kit from CLONTECH Laboratories, Inc. (Palo Alto, CA), with a fluorogenic substrate (IETD tetrapeptide conjugated to 7-amino-4-trifluoromethyl coumarin). We examined four treatment groups: control [treated with 0.1% (vol/vol) DMSO], manumycin (54 µM), paclitaxel (22 µM), and manumycin plus paclitaxel. Fluorescence was measured using a microplate reader (SpectroFluor Plus, Tecan, Durham, NC). The amount of product in the presence of a specific inhibitor of caspase-8 (IETD-fmk) was subtracted from the amount of fluorescent cleavage product in the absence of the inhibitor after incubation of the substrate with cell lysate samples to measure the degree of caspase-8-specific proteolytic cleavage. The formation of fluorescent cleavage product was measured after incubation of the substrate with cell lysate samples (equal amount of protein loaded) in the presence and absence of the caspase-8 inhibitor (IETD-fmk) under the same conditions. Fluorogenic caspase-9 substrate (Ac-LEHD-AMC; A. G. Scientific, Inc., San Diego, CA) was used to measure the activity of caspase-9. Fluorogenic caspase-6 substrate (Ac-VEID-AFC) and fluorogenic caspase-1 substrate (AC-YVAD-AMC), both purchased from BD PharMingen, were used to determine the activities of caspase-6 and caspase-1, respectively. The same buffers used in the caspase-8 assay were used to prepare the samples for the assays of caspase-9, -6 and -1. After incubation of a substrate with a cell lysate sample, the amount of fluorescent cleavage product was measured using the fluorescence microplate reader. All of these experiments were performed in triplicate wells.

Differential detergent extraction

Differential detergent extraction was performed according to the method described by Ramsby and Makowski (35). In accordance with the differential detergent extraction technique, the first fraction was the digitonin extract, which corresponded to cytosolic proteins. The second fraction was the Tween 40/deoxycholate extract, which corresponded to the proteins in organelles (including mitochondria) and most membrane proteins. The third fraction was the Triton X-100 extract, which corresponded to the soluble nuclear proteins. The fourth fraction was the SDS extract, which corresponded to the cytoskeleton, insoluble nuclear proteins, and other hydrophobic proteins. After experimental or control treatments, cultured cells (both floating and attached) were washed twice with ice-cold PBS and then extracted sequentially with 0.5 ml/25 cm² of each of the following ice-cold buffers in the same order: digitonin buffer [10 mM PIPES (pH 6.8), 0.015% (wt/vol) digitonin, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl₂, 5 mM EDTA, and 1 mM phenylmethylsulfonylfluoride (PMSF)], Triton X-100 buffer [10 mM PIPES (pH 7.4), 0.5% (vol/vol) Triton X-100, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl₂, 5 mM EDTA, and 1 mM PMSF], Tween 40/deoxycholate buffer [10 mM PIPES (pH 7.4), 1% (vol/vol) Tween 40, 0.5% (wt/vol) deoxycholate, 1 mM MgCl₂, and 1 mM PMSF], and SDS buffer [5% (wt/vol) SDS, 10 mM sodium phosphate (pH 7.4), and 10% (vol/vol) 2-mercaptoethanol]. The cells were incubated on ice and gently rocked in each buffer for 10 min. The digitonin, Triton X-100, and Tween 40/deoxycholate extracts were mixed 5:1 with 6 x SDS sample buffer before SDS-PAGE.

Preparation of total cell lysates

After experimental treatments, cells floating in the culture medium were pelleted by centrifugation. Cells that attached to the well were rinsed with PBS. Both the cell pellet and the cells attached to the well were then lysed in a total of 300 µl RIPA buffer [1 x PBS, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 mg/ml PMSF, and Complete Protease Inhibitor Mix (Roche), one tablet per 50 ml]. The DNA in the lysate was sheared by rapidly passing the lysate five times through a 23-gauge needle or by sonication with eight 1-sec bursts at medium power.

SDS-PAGE and immunoblotting

SDS-PAGE was performed using standard methods. The protein concentrations of samples were measured using a modified Lowry method (protein assay, Bio-Rad Laboratories, Inc., Hercules, CA). Equal amounts of total protein from each sample were loaded onto the SDS-polyacrylamide gel. Kaleidoscope Prestained Standards (Bio-Rad Laboratories, Inc.) were used for mol wt calibration. Immunoblotting was performed using polyvinylidine fluoride membranes (Hybond-P, Amersham Pharmacia Biotech, Piscataway, NJ). Kodak X-AR film (Eastman Kodak Co., Rochester, NY) was used to record the image generated by enhanced chemiluminescence using the ECL kit (Amersham Pharmacia Biotech) or the SuperSignal West Dura extended duration substrate (Pierce Chemical Co., Rockford, IL).

Preparation of S-100 extracts

After experimental and control treatments for the specified duration, the floating cells were harvested by centrifugation at 500 x g for 10 min at 4 C and washed twice with ice-cold PBS. The adherent cells were also washed twice with ice-cold PBS, and then 0.25 ml/25 cm² ice-cold buffer A (20 mM HEPES [4-(2-hydroxyethyl)-1-piperazineethane-sufonic acid]-KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, and 0.1 mM PMSF) supplemented with protease inhibitors (5 µg/ml pepstatin A, 10 µg/ml leupeptin, and 2 µg/ml aprotinin) was added, and the cells were scraped into a Dounce glass homogenizer (Kontes Co., Vineland, NJ) with the pellet of floating cells. After incubation on ice for 15 min, the cells were disrupted by 15 up and down strokes with pestle B. The nuclei and debris were removed by centrifugation at 10,000 x g at 4 C. Then the supernatant was centrifuged at 100,000 x g at 4 C for 1 h. The resulting supernatant (S-100) was divided into aliquots, snap-frozen in liquid nitrogen, and stored at -80 C until use.

Purification of nuclei

Untreated cultured cells were rinsed with ice-cold PBS three times and homogenized in buffer B [10 mM HEPES-KOH (pH 7.6), 1.7 M sucrose, 15 mM KCl, 2 mM EDTA, 0.15 mM spermine, 0.15 mM spermidine, 0.5 mM dithiothreitol, 0.5 mM PMSF, and 10% (vol/vol) glycerol; density, 1.24 g/ml] by 20 up-and-down strokes of a motor-driven Teflon pestle in a glass homogenization tube. The homogenates were centrifuged through a 1-ml cushion buffer B in a swinging bucket rotor at 100,000 x g at 4 C for 1 h. The nuclear pellet was resuspended in buffer C [10 mM PIPES (pH 7.4), 80 mM KCl, 20 mM NaCl, 5 mM EDTA, 250 mM sucrose, 1 mM dithiothreitol, and 10% (vol/vol) glycerol] at 8.5 x 10⁷ nuclei/ml. The nuclei were used immediately or snap-frozen in aliquots with liquid nitrogen and stored at -80 C.

Internucleosomal DNA fragmentation assay

For the in vitro reconstitution assay, 50 µl KAT-4 cell S-100 extract and 6 µl purified KAT-4 cell nuclei were incubated at 30 C for 2 h with 1 mM MgCl₂ in the absence or presence of 1 mM deoxy-ATP and/or 0.2 µg/ml bovine heart cytochrome c. In experiments involving caspase inhibitors, the S-100 extracts were preincubated with 100 µM of one of the caspase inhibitors (Ac-DEVD-CHO, a caspase-3 inhibitor; Ac-IETD-CHO, a caspase-8 inhibitor; or Ac-LEHD-CHO, a caspase-9 inhibitor) for 1 h before the addition of nuclei and other components. After incubation with the nuclei, the samples were processed as described by Liu et al. (36), with minor modifications. Five hundred microliters of buffer D [100 mM Tris-HCl (pH 8.5), 5 mM EDTA, 0.2 M NaCl, 0.2% (wt/vol) SDS, and 0.2 mg/ml proteinase K] were added to each reaction, and incubation was continued overnight at 37 C. Then NaCl was added to a final concentration of 1.5 M. After 10 min at room temperature, the mixtures were centrifuged at 10,000 x g at 4 C for 20 min. The DNA in the supernatant was precipitated by mixing with an equal volume of absolute ethanol. The DNA precipitate was washed once with 70% ethanol, resuspended in 10 mM Tris and 1 mM EDTA (pH 8.0) buffer with 200 µg/ml deoxyribonuclease-free ribonuclease A, and incubated at 37 C for an additional 2 h.

For detecting internucleosomal DNA fragmentation in intact cells after drug treatments, the treated cells were detached by trypsin and EDTA treatment, washed twice with ice-cold PBS, and pelleted by centrifugation at 400 x g for 5 min. The cell pellet was lysed in 1 ml buffer containing 10 mM Tris-HCl (pH 8.0), 10 mM EDTA, 75 mM NaCl, 0.5% (wt/vol) SDS, 400 µg/ml proteinase K, and 20 µg/ml ribonuclease A. After incubation at 50 C for 3–6 h, the samples were centrifuged at 10,000 x g for 20 min at 4 C. One half of a milliliter of supernatant was then mixed with 150 µl 5 M NaCl and 1 ml absolute ethanol. After incubation on ice for 30 min, the precipitated DNA was pelleted by centrifugation at 10,000 x g for 20 min at 4 C. The pellet was washed once with 1 ml 70% ethanol and then dissolved in 50 µl TE buffer by incubating at 65 C for 60 min.

The isolated DNA samples from in vitro reconstitution or intact cells were subjected to electrophoresis at 50 V for 4–5 h in a 2% agarose gel in 40 mM Tris, 0.5 mM EDTA, and glacial acetic acid to adjust pH to 7.5. Ethidium bromide-stained DNA was visualized by transillumination with UV light, and the images were captured using a digital video gel documentation system (Foto/Analyst Visionary gel documentation system, Fotodyne, Inc., Hartland, WI).

Statistics

Comparison in experiments was performed using one-way ANOVA with Tukey’s test or t test to assess the statistical significance of difference between groups. Differences with P < 0.05 were considered significant. To assess the significance of interaction between two treatments, two-way ANOVA was performed with the F test. Interaction was considered significant at P < 0.05.

Results

Enhanced activation of caspase-8 and caspase-9 by manumycin plus paclitaxel in ARO and KAT-4 cells

We measured the enzyme activities of two regulatory caspases, caspase-8 and caspase-9, in both ARO and KAT-4 cells. Four treatment groups of each cell line were treated for 6 h with DMSO (0.1%, vol/vol), manumycin (54 µM), paclitaxel (22 µM), or manumycin plus paclitaxel. The 6-h treatment duration was chosen to ensure an easily detectable activation of caspases based on the time course of caspase-8 activation presented later in Fig. 10B. Each treatment group had three samples. The combination of manumycin and paclitaxel enhanced the activation of caspase-8-specific enzyme activity in ARO cells (by two-way ANOVA, F test for interaction, P < 0.05; Fig. 2A) and KAT-4 cells (by two-way ANOVA, F test for interaction, P < 0.05; Fig. 2C) and of caspase-9 activity in ARO cells (by two-way ANOVA, F test for interaction, P < 0.05; Fig. 2B) and KAT-4 cells (by two-way ANOVA, F test for interaction, P < 0.05; Fig. 2D).

View larger version (41K): [in this window] [in a new window]   Figure 10. Time course of drug-induced cytochrome c release and the time course of activation of caspase-8. A, The immunoblot demonstrated the time course for the release of cytochrome c into the cytosolic fractions (digitonin extracts). The drug treatments and the duration of treatment are labeled above each lane of the gels. The locations of the cytochrome c band and the actin band are indicated on the right. B, The amount of fluorescent products produced by caspase-8, measured in relative fluorescent units, was plotted against the duration of drug treatment of the KAT-4 cell samples. Each time point of caspase-8 activity consisted of three independent measurements. The error bars represent 95% confidence intervals. *, Significant difference from control (by t test, P < 0.05).

View larger version (32K): [in this window] [in a new window]   Figure 2. Enhancement of caspase-8 and caspase-9 enzyme activities after drug treatment for 6 h. ARO cells: A, results of the caspase-8 activity assay using fluorogenic substrate IETD-AFC; B, results of the caspase-9 activity assay using fluorogenic substrate Ac-LEHD-AMC. KAT-4 cells: C, results of the caspase-8 activity assay using fluorogenic substrate IETD-AFC; D, results of the caspase-9 activity assay using fluorogenic substrate Ac-LEHD-AMC. Each experimental or control treatment was performed in triplicate, and the error bars in all four graphs represent the 95% confidence intervals. In all four datasets, enhancement was significant by testing the interaction by two-way ANOVA (by F test for interaction, P < 0.05).

Using immunoblotting, we documented that the combination of manumycin and paclitaxel enhanced activation of caspase-3 and caspase-8 in ATC cells treated with control medium [0.1% (vol/vol) DMSO], manumycin (54 µM), paclitaxel (22 µM), or manumycin plus paclitaxel for 4, 8, 12, or 18 h. After electrophoresis, anticaspase-3 and anticaspase-8 immunoblotting of the cell lysates revealed a time-dependent decrease in procaspase-3 and procaspase-8 produced by manumycin plus paclitaxel treatment (Fig. 3). Therefore, the combination of manumycin and paclitaxel enhanced the activation of both caspase-3 and caspase-8 in ARO and KAT-4 cells. This decrease in procaspase-8 induced by the combination of manumycin and paclitaxel confirmed the data from enzymatic assay of caspase-8 in ARO and KAT-4 cells presented above.

View larger version (38K): [in this window] [in a new window]   Figure 3. Enhanced cleavage of procaspase-3 and procaspase-8 induced by the combination of manumycin and paclitaxel. The anticaspase-3 immunoblot, the anticaspase-8 immunoblot, and the antiactin immunoblot were shown as labeled. The top set was from ARO cells, and the bottom set was from KAT-4 cells. Each protein sample was total lysate from cells treated for a certain length of time as indicated at the top.

Four treatment groups of each cell line were treated for 18 h with control medium (0.1% DMSO in complete culture medium), manumycin (54 µM), paclitaxel (22 µM), or manumycin plus paclitaxel. Five additional treatment groups of each cell line were pretreated for 1 h with a caspase inhibitor (inhibitor of caspase-3, caspase-8, caspase-9, caspase-6, or caspase-1) at a concentration of 100 µM and then treated with manumycin plus paclitaxel for 18 h in the continued presence of the respective caspase inhibitor. After drug treatments, the cells were analyzed for the presence of fragmented DNA in the cytosol using the DNA fragmentation ELISA. Manumycin plus paclitaxel significantly increased DNA fragmentation compared with the control (by one-way ANOVA, Tukey test for multiple pairwise comparison, P < 0.05), and inhibition of caspase-3, -8, or -9 reversed this effect in both KAT-4 and ARO cells (Fig. 4). In contrast, inhibitors of caspase-1 and caspase-6 did not reverse the apoptosis induced by manumycin and paclitaxel.

View larger version (24K): [in this window] [in a new window]   Figure 4. Effects of caspase inhibitors to block manumycin- plus paclitaxel- induced DNA fragmentation. The vertical axes indicate the OD at 485 nm obtained using an ELISA of DNA fragmentation. A, KAT-4 cells. B, ARO cells. The treatment groups are labeled. The error bars in both graphs represent the 95% confidence intervals (n = 3). In both cell lines, the groups treated with manumycin plus paclitaxel, manumycin plus paclitaxel plus caspase-6 inhibitor, and manumycin plus paclitaxel plus caspase-1 inhibitor were significantly different from the control group (by one-way ANOVA, Tukey test for multiple pairwise comparison, P < 0.05).

View larger version (31K): [in this window] [in a new window]   Figure 5. Effectiveness of caspase-1 and caspase-6 inhibitors. The panels on the left are ARO cells, and the panels on the right are KAT-4 cells. The upper panels show the caspase-1 enzyme activity of various treatment groups, and the lower panels show the caspase-6 enzyme activity of various treatment groups. The treatments are labeled at the bottom of each chart. The error bars in all charts represent the 95% confidence intervals (n = 3). *, Significant difference (by t test, P < 0.05).

Differential detergent extraction is a technique that can fractionate proteins from different subcellular compartments without ultracentrifugation and requires fewer cells than the traditional ultracentrifugation fractionation techniques (35). Using differential detergent extraction, SDS-PAGE, and immunoblotting with anticytochrome c antibody, we demonstrated that a 6-h incubation of KAT-4 or ARO cells with manumycin (54 µM) elicited the release of cytochrome c from the Triton X-100 fraction (i.e. proteins of the organelles, mitochondria, and membranes) into the digitonin fraction (i.e. the cytosolic proteins). Cytochrome c was not present at detectable levels in the Tween 40/deoxycholate fraction and the SDS fraction, indicating that this method provides good separation of proteins from various intracellular compartments (data not shown).

Enhanced release of cytochrome c into the cytosol by manumycin plus paclitaxel

Cultured cells were divided into four treatment groups: control [treated with 0.1% (vol/vol) DMSO], manumycin (54 µM), paclitaxel (22 µM), and manumycin plus paclitaxel. Within each treatment group, separate cell samples were treated for 3, 6, 9, or 12 h. Using the digitonin buffer described above, cytosolic fractions were prepared from the KAT-4 and ARO cells. For comparison, ARO cell mitochondrial protein fractions obtained by differential centrifugation were loaded in separate lanes on the polyacrylamide gel. In the ARO cells, the anticytochrome c immunoblot showed that paclitaxel did not release a detectable amount of cytochrome c into the cytosol after 12 h, and that, compared with manumycin alone, the combination of manumycin and paclitaxel enhanced the release of cytochrome c (Fig. 6). The cytochrome c oxidase subunit II is a mitochondrial protein. The anticytochrome c oxidase subunit II immunoblot showed that the digitonin extracts were not contaminated by mitochondrial debris.

View larger version (28K): [in this window] [in a new window]   Figure 6. Enhanced cytochrome c release by the combination of manumycin and paclitaxel in ARO cells. The upper image shows the anticytochrome c immunoblot using digitonin extract. The lower image shows the anticytochrome c oxidase subunit II immunoblot, which was included as a quality control indicator to confirm that the cytosolic fractions were not contaminated by mitochondrial debris. The mitochondria/organelle fraction (M) is indicated at the right edge of each immunoblot.

View larger version (53K): [in this window] [in a new window]   Figure 7. Enhanced cytochrome c release by the combination of manumycin and paclitaxel in KAT-4 cells. The treatment groups and the treatment duration are labeled on top. The upper anticaspase-3 blot was a short exposure to show increased cleavage of procaspase-3 induced by manumycin and paclitaxel. The lower anticaspase-3 blot was a long exposure to show the inversely correlated appearance of the 19-kDa subunit of activated caspase-3.

Treatment of intact KAT-4 cells with the combination of manumycin and paclitaxel for 18 h enhanced internucleosomal fragmentation of DNA (Fig. 8, left panel). This finding agreed with our previous observation in ARO cells (11). Using the in vitro cell-free reconstitution assay, we studied the role of the release of cytochrome c into cytosol in apoptosis. Internucleosomal fragmentation of DNA in purified intact KAT-4 nuclei was used as the indicator (or read-out) of the assay. Electron microscopy of the nuclei purified from untreated KAT-4 cells, performed at the Electron Microscopy Core Facility of our institution, confirmed that the purified nuclear pellet consisted of more than 80% intact nuclei (data not shown). Incubation of purified intact KAT-4 nuclei with S-100 extracts isolated from KAT-4 cells that had undergone 12-h treatment with DMSO, manumycin, paclitaxel, or manumycin plus paclitaxel revealed a pattern of enhanced DNA fragmentation similar to that of intact KAT-4 cells (Fig. 8, right panel).

View larger version (54K): [in this window] [in a new window]   Figure 8. Enhanced internucleosomal DNA fragmentation by manumycin plus paclitaxel. Both panels showed UV fluorescent images of the ethidium bromide-stained agarose gels. Left panel: Lane 1, DNA sample from KAT-4 cells treated with control; lane 2, with manumycin (54 µM, 12 h); lane 3, with paclitaxel (22 µM, 12 h); lane 4, with manumycin plus paclitaxel. The lane to the right of lane 4 shows the 100-bp DNA size markers. Right panel: Lane 1, DNA from intact KAT-4 nuclei incubated with control KAT-4 cell S-100 extract; lane 2, with S-100 extract from cells treated with manumycin (54 µM, 12 h); lane 3, with S-100 extract from cells treated with paclitaxel (22 µM, 12 h); lane 4, with S-100 extract from cells treated with manumycin plus paclitaxel.

View larger version (68K): [in this window] [in a new window]   Figure 9. Inhibition of caspase enzyme activity blocked internucleosomal DNA fragmentation induced by cytosolic cytochrome c. The UV fluorescent image of the ethidium bromide-stained agarose gel is shown. The gel contained DNA samples from intact KAT-4 nuclei incubated with S-100 extract either from cells treated with manumycin plus paclitaxel or from control cells. The presence (+) or absence (-) of deoxy-ATP, exogenous bovine cytochrome c, caspase-3 inhibitor, caspase-9 inhibitor, and caspase-8 inhibitor is indicated above the gel. The far left lane shows the 100-bp DNA size markers.

As caspase-8 has been known to be able to release cytochrome c from mitochondria via BH3 interacting domain death agonist, we examined the time course of the two events to determine which event occurred first. By immunoblotting, cytochrome c was detectable in the cytosolic fraction (digitonin extracts) as early as 1 h after treatment with the combination of manumycin (54 µM) and paclitaxel (20 µM; Fig. 10A). The inclusion of antiactin antibody in immunoblotting excluded bias in sample loading. Using fluorescent detection of cleaved fluorogenic caspase-8 substrate (a highly sensitive method of detecting caspase-8 enzyme activity), a significant increase in caspase-8 enzymatic product was first detected after 3 h of treatment with manumycin (54 µM) and paclitaxel (20 µM) compared with the corresponding control samples (P < 0.05, by t test; Fig. 10B). Each data point consisted of triplicate samples. Therefore, release of cytochrome c occurred before activation of caspase-8 enzyme activity after treatment with manumycin plus paclitaxel.

Inhibitors of caspases did not inhibit release of cytochrome c

If a caspase is upstream of cytochrome c release, then inhibiting the caspase is expected to block or attenuate the release of cytochrome c. To investigate whether caspase-3, caspase-8, or caspase-9 is upstream of cytochrome c, ARO and KAT-4 cells were treated with control medium, manumycin (54 µM), paclitaxel (22 µM), or manumycin plus paclitaxel in the presence or absence of the caspase inhibitors. Incubation with caspase inhibitors began 30 min before the 6-h manumycin and paclitaxel treatments. Immunoblots of digitonin extracts (cytosolic fractions) of the treated KAT-4 and ARO cells showed that the release of cytochrome c by manumycin plus paclitaxel was not inhibited by the inhibitor of caspase-3, caspase-8, or caspase-9 (100 µM; Fig. 11). Inclusion of actin antibody in immunoblotting demonstrated that there was no bias in sample loading. Therefore, release of cytochrome c did not require activation of caspase-3, caspase-8, or caspase-9, and cytochrome c was upstream to the three caspases tested. These results together with the data presented above supported the conclusion that manumycin and paclitaxel increased apoptosis in ATC cells by enhancing the release of mitochondrial cytochrome c, and that this event of cytochrome c release occurred upstream of the caspase cascade in the hierarchical pathway of apoptosis.

View larger version (51K): [in this window] [in a new window]   Figure 11. Cytochrome c release not affected by caspase inhibitors. The immunoblot demonstrated the presence or absence of cytochrome c in the cytosolic fractions (digitonin extracts) of various cell samples treated with manumycin and paclitaxel and various caspase inhibitors. The drug treatments and the presence or absence of caspase inhibitors are labeled above each lane of the gel. The locations of the cytochrome c band and the actin band are indicated on the left.

We found that the combination of manumycin and paclitaxel enhanced apoptosis and determined that the point of interaction between manumycin and paclitaxel was at the release of cytochrome c or further upstream in the apoptosis regulatory pathway. In ATC cells treated with manumycin plus paclitaxel, but not in the control cells or cells treated with either drug alone, we previously observed the following terminal events of apoptosis: internucleosomal DNA fragmentation (as documented both by the appearance of a DNA ladder upon agarose gel electrophoresis and a DNA fragmentation ELISA) (11), specific cleavage of poly (ADP-ribose) polymerase (as demonstrated by immunoblotting) (11), and nuclear morphological changes characteristic of apoptosis (as observed by fluorescent microscopy after staining with the Hoechst 33342 dye; our unpublished data). The present study showed that the early events in apoptosis, such as activation of caspase-8, caspase-9, and cytochrome c release, were also enhanced by the combination of manumycin and paclitaxel.

Among most of the numerous proposed hierarchical models of the caspase cascade (26, 27, 28), caspase-3 is probably the most important effector caspase. The enhanced activation of caspase-3 in ATC cells by manumycin plus paclitaxel is evidenced by increased caspase-3 enzyme activity (11), specific cleavage of poly (ADP-ribose) polymerase (11), cleavage of procaspase-3, and formation of the 19-kDa (p19) subunit of activated caspase-3. Our data of the time course of cleavage of procaspase-8 and procaspase-3 and caspase-3 inhibitor experiments in intact cells and cell-free assays are consistent with the effector role of caspase-3 in the apoptosis pathway activated by manumycin and paclitaxel in ATC cells.

Caspase-8, caspase-9, and cytochrome c are upstream regulatory elements in apoptosis pathways (Fig. 1). Our hypothesis is that the combination of manumycin and paclitaxel induces apoptosis via the drug-induced apoptosis pathway. In this pathway, release of cytochrome c from the mitochondria is a key regulatory event. The drug treatments release cytochrome c from mitochondria in a manner independent of caspase activation. Caspase-9 is activated by the interaction of apoptosis protein-activating factor-1 and cytochrome c (29, 37). Caspase-9 activates caspase-3. Caspase-3 activates deoxyribonucleases to cause DNA fragmentation. Although caspase-8 is thought to be the key upstream regulatory element in the death receptor apoptosis pathway (27, 29), activation of caspase-8 does not necessarily imply involvement of the death receptor apoptosis pathway, because drug- induced apoptosis does not require synthesis of death ligands or interaction with Fas, and caspase-8 can be activated in the absence of a death receptor signal (32, 38). In ATC cells, enhanced activation of caspase-8 by manumycin plus paclitaxel has been demonstrated by measurement of caspase-8-specific enzyme activity and the cleavage of procaspase-8. The reason for the requirement of caspase-8 activity in drug-induced apoptosis is not clear. One possible explanation is that caspase-8 is downstream of caspase-9. As the biological half-lives of the activated caspases are very short (Nicholson, D. W., Merck Frost Center for Therapeutic Research, Dorval, Canada, personal communication), it is also possible that a positive feedback loop involving caspase-8 is required to amplify the apoptosis signal and maintain the signal for a duration long enough to execute the terminal apoptotic events. This interpretation is consistent with the finding of Engels et al. (39) that caspase-9 is cleaved in MCF7 cells (a breast cancer cell line) lacking caspase-3 when treated with anticancer drugs, and caspase-8 activation is only observed in caspase-3-transfected MCF7 cells.

Our hypothesis that manumycin and paclitaxel induced apoptosis through the drug-induced apoptosis pathway can be tested by examining the hierarchical relationship between cytochrome c and the caspases. If cytochrome c is upstream of the caspases, then inhibition of caspases is expected to block the late apoptotic events induced by cytochrome c. The combination of manumycin and paclitaxel enhanced the release of cytochrome c from the mitochondria into the cytosol of ATC cells. Using DNA fragmentation as the indicator, cell-free reconstitution assay experiments have shown that the addition of exogenous cytochrome c to control S-100 extract can mimic the effect of S-100 extract from cells treated with manumycin plus paclitaxel. Experimentation with caspase-3, caspase-9, and caspase-8 inhibitors has shown that these caspases are downstream from cytochrome c release, because the inhibitors of these caspases block DNA fragmentation, and these caspase inhibitors cannot block the release of cytochrome c induced by manumycin and paclitaxel. Caspase-8 has been known to activate BH3 interacting domain death agonist, which then releases cytochrome c from the mitochondria. The time courses of the two events, cytochrome c release and activation of caspase-8, have also demonstrated the sequential order of cytochrome c release before activation of caspase-8. Therefore, we conclude that cytochrome c is upstream of the caspases (including caspase-8) in the apoptotic pathway induced by the manumycin plus paclitaxel in ATC cells.

As far as we know, activation of caspase-8 by an FTI has not been reported, although caspase-8 activation by several other chemotherapy drugs (daunorubicin, doxorubicin, etoposide, and mitomycin C) has been observed in Jurkat leukemic T cells (32). Paclitaxel has also been reported to induce apoptosis in a colon cancer cell line (HT-29-D4) (40) and a lung carcinoma cell line (LC-2-AD) (41) by caspase-8 activation. Park et al. (42) demonstrated that mitomycin C- induced apoptosis in SNU-16 cells (a gastric adenocarcinoma cell line) is mediated by caspase-8, caspase-9, and caspase-3 activation and cytochrome c release. Our findings in ATC cells parallel the findings of Park et al. in gastric carcinoma cells, and we have also demonstrated that the release of cytochrome c is independent of caspase-8.

Recent evidence that the PI-3 kinase/Akt2-mediated cell survival pathway (17, 18) is involved in FTI-induced apoptosis raises the relevance of serum concentration in the culture medium in cultured cell experiments. We performed all of the experiments in the presence of 10% bovine serum in the culture medium. Jiang et al. (17) reported that a high serum level prevents FTI-induced apoptosis in H-Ras- but not Akt2-transformed cells, and constitutively active Akt2 rescues cancer cells from FTI-induced apoptosis. The fact that the combination of manumycin and paclitaxel induces apoptosis in ATC cells in the presence of a high serum level underscores the effectiveness of this drug combination against ATC cells. However, the presence of a high level of serum may be a confounding factor in the evaluation of apoptosis mechanisms. The kinases of the Akt family promote cellular survival by phosphorylating and inhibiting apoptosis-inducing proteins, e.g. apoptosis signal-regulating kinase 1 (43), and inhibition of the PI3K/Akt pathway has been reported to enhance drug-induced apoptosis (44, 45). Although Akt certainly affects some important upstream elements regulating apoptosis, whether it can change the choice of apoptosis execution pathways (i.e. death receptor apoptosis pathways or drug-induced apoptosis pathways) is unknown.

Ali et al. (46) have shown that 10 µM manumycin can cause the mitochondria of COS cells to distribute in a perinuclear pattern and become swollen. Perhaps direct mitochondrial damage by manumycin may play a role in enhancing apoptosis induced by the combination of manumycin and paclitaxel. Nevertheless, manumycin and paclitaxel interact at the release of cytochrome c from the mitochondria or at steps further upstream (Fig. 12). To continue the attempt to identify the mechanism of interaction between manumycin and paclitaxel by tracing the apoptosis pathway backward, we plan to determine the effects of these two drugs on members of the Bcl-2 family of apoptosis inhibitors as well as on mitochondrial structure and function.

View larger version (17K): [in this window] [in a new window]   Figure 12. Summary. In ATC cells, manumycin and paclitaxel interacted at a step upstream of cytochrome c release to enhance apoptosis. The enhanced apoptosis was executed through the drug-induced apoptosis pathway with caspase-9, caspase-8, and caspase-3 downstream of cytochrome c release.

Acknowledgments

The KAT-4 cell line was kindly provided by Dr. K. Ain. The ARO cell line was kindly provided by Dr. J. A. Fagin. We thank Alexander Bamiagis for excellent technical assistance.

Footnotes

This work was supported by a grant from the Physician Referral Service of the University of Texas M. D. Anderson Cancer Center and in part by a grant (to S.J.Y.) from the American Cancer Society (RPG-99–154-01-CDD). The core facilities at the University of Texas M. D. Anderson Cancer Center are partially supported by the Cancer Center Support Grant (CORE, CA16672).

¹ Present address: Department of Head and Neck Surgery, Cancer Center, Sun Yat-Sen University of Medical Sciences, Guangzhou, People’s Republic of China.

Abbreviations: ATC, Anaplastic thyroid cancer; BrdU, 5'-bromo-2'-deoxyuridine; DMSO, dimethylsulfoxide; FTI, farnesyltransferase inhibitor; PMSF, phenylmethylsulfonylfluoride.

Received November 29, 2000.

Accepted May 9, 2001.

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