Involvement of caspase-9 in autophagy-mediated cell survival pathway

Biochimica et Biophysica Acta 1813 (2011) 80–90

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a m c r

Involvement of caspase-9 in autophagy-mediated cell survival pathway Hyo-Soon Jeong 1, Hye Yeon Choi 1, Eung-Ryoung Lee, Jung-Hyun Kim, Kilsoo Jeon, Hyun-Joo Lee, Ssang-Goo Cho ⁎ Department of Animal Biotechnology (BK21), Animal Resources Research Center, and RCTCP, Konkuk University, Seoul, South Korea

a r t i c l e

i n f o

Article history: Received 17 January 2010 Received in revised form 19 September 2010 Accepted 23 September 2010 Available online 1 October 2010 Keywords: NSAIDs Caspase Apoptosis Autophagy MCF-7

a b s t r a c t Nonsteroidal anti-inflammatory drugs (NSAIDs) have been considered for use in the prevention and treatment of cancer malignancy. FR122047 (FR) is known to have an anti-inflammatory effect, but the anticancer activity of the chemical has not yet been identified. In the present study, we could find that treatment of breast cancer MCF-7 cells with FR led to apoptosis accompanying with apparent activation of caspases. Treatment of caspase-specific inhibitors revealed that FR-induced apoptosis was caspase-8dependent and inhibition of caspase-9 activity resulted in unexpected, marked enhancement of cell death. Knockdown of caspase-9 expression by specific siRNA caused increased susceptibility to FR-induced cell death, consistent with the results obtained with treatment of caspase-9 inhibitor. Inhibition of caspase-9 blocked the autophagic process by modulating lysosomal pH and acid-dependent cathepsin activities and augmented cell death due to blockage of cytoprotective autophagy. MCF-7 cells treated with sulforaphane, an autophagy-inducing drug, also showed marked accumulation of LC3-II, and co-treatment with caspase-9 inhibitor brought about increased susceptibility to sulforaphane-induced cell death. Different from the cases with FR or sulforaphane, etoposide- or doxorubicin-induced cell death was suppressed with co-treatment of caspase-9 inhibitor, and the drugs failed to induce significant autophagy in MCF-7 cells. Taken together, our data originally suggest that inhibition of caspase-9 may block the autophagic flux and enhance cell death due to blockage of cytoprotective autophagy. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Breast cancer is one of the most common malignancies in women globally and the second leading cause of cancer mortality in females. Nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit the enzyme cyclooxygenase (COX), have been considered for use in the prevention and treatment of malignancy [1–4]. Epidemiological studies have reported that long-term use of NSAIDs reduces the risk of cancer of the breast, prostate, colon, lung, and other gastrointestinal organs [5–10]. However, the molecular mechanisms underlying NSAID-induced apoptosis in breast cancer are poorly understood. FR122047, a selective and potent inhibitor of COX-1, is known to have an anti-inflammatory effect in animal models of chronic inflammation [11,12], but the anticancer activity of the chemical has not yet been identified. Apoptosis is a well-characterized process of non-necrotic cell death, which is triggered by activation of both the mitochondrial (intrinsic) and death receptor (extrinsic) pathways [13]. Although apoptotic cell death is generally known to require caspase activity and can be ⁎ Corresponding author. Department of Animal Biotechnolgy, Konkuk University, Seoul 143-701, Korea. Tel.: + 82 2 450 4207; fax: + 82 2 455 1044. E-mail address: [email protected] (S.-G. Cho). 1 Both authors contributed equally. 0167-4889/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2010.09.016

suppressed by specific caspase inhibitors, recent evidence suggests that caspase inhibitors can also promote necrotic cell death and enhance even stress-induced apoptosis [14,15]. A comprehensive analysis of the functions of caspases, extending beyond the findings of previous studies, remains to be done. In recent years, autophagy has been shown to induce caspaseindependent cell death without the morphological changes characteristic of apoptosis, such as membrane blebbing, cell shrinkage, and chromatin condensation [16]. Autophagy is an evolutionarily conserved mechanism by which long-lived proteins and damaged organelles are digested in lysosomes [17,18]. Autophagy also promotes cancer cell survival under conditions of stress, such as starvation, and functions as a defense mechanism in response to various anticancer agents [19,20]. Despite of a lot of studies on the relationship between autophagy and apoptosis, the functional relationship between caspases and autophagy is not well understood. Here, we report that treatment of MCF-7 breast cancer cells with the NSAID FR122047 (FR) leads to the activation of a linear cascade of caspases. We originally found that inhibition of caspase-9 may unpredictably enhance cell death due to blockage of cytoprotective autophagy. Our data provide new insights into the understanding of the relationship between caspases and cytoprotective autophagy and suggest the possibility that caspase-9 may be involved in the cytoprotective autophagic process.

H.-S. Jeong et al. / Biochimica et Biophysica Acta 1813 (2011) 80–90

81

2. Materials and methods

2.5. Preparation of mitochondrial and cytoplasmic fractions

2.1. Reagents

After the indicated treatments, cells were washed three times in cold PBS and scraped from the dishes. Mitochondrial and cytoplasmic fractions were prepared using a mitochondrial fractionation kit (Active Motif, CA, USA), according to the manufacturer's protocol.

FR122047, a nonsteroidal anti-inflammatory drug (NSAID), bafilomycin A1, pepstatin A methyl ester, and E-64D were purchased from Calbiochem (La Jolla, CA, USA) and dissolved in dimethyl sulfoxide (DMSO) and 3,3′dihexyloxacarbocyanine iodide (DiOC6(3)), were from Molecular Probes (Eugene, OR, USA). Electrophoresis reagents and BioRad protein assay kit were obtained from Bio-Rad (Hercules, CA, USA). Rapamycin, chloroquine, etoposide, doxorubicin, and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Pancaspase inhibitor (z-VAD-FMK), caspase8 inhibitor (z-IETD-FMK), and caspase-9 inhibitor (z-LEHD-FMK) were from BD Biosciences (San Diego, CA, USA). D,L-Sulforaphane (purity N 99%) was from LKT Laboratories (St. Paul, MN, USA).

2.2. Cell culture and transfection MCF-7 breast cancer cells were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum and 100 U/ml penicillin/ streptomycin (Life Technologies, Inc., Gaithersburg, MD, USA). Plasmid GFP-LC3 was kindly provided by Dr. Tamotsu Yoshimori (Research Institute for Microbial Diseases, Osaka University, Japan). The transfection was carried out using Lipofectamine (Invitrogen, Paisley, UK) according to the manufacturer's protocol, and stable cell lines were selected by growth in presence of 800 μg/ml G418. MCF-7 cells expressing GFP-tagged LC3 were used to demonstrate induction of autophagy. To produce Bcl-2-overexpressing MCF-7 cells, cells were transfected with pcDNA-Bcl-2 using Lipofectamine 2000 and stable cell lines were selected by growth in the presence of G418. Cells were maintained under a humidified atmosphere of 95% air 5% CO2 at 37 °C.

2.3. DAPI staining DAPI (4′,6-diamidino-2-phenylindole ) staining of DNA was performed as described previously [42]. Briefly, cells were fixed in ice-cold methanol/acetone (1:1; v/v) for 15 min and then stained with the DNAspecific fluorochrome DAPI in the dark. Morphological changes in the apoptotic cells were observed and photographed using a Zeiss Axiovert 200 microscope. Apoptotic cells were identified by condensation and nuclear fragmentation, and the percentage of apoptotic cells was calculated as the ratio of apoptotic cells to total cells counted.

2.6. Western blot analysis To prepare whole-cell extracts, cells were washed three times in cold PBS, scraped from the dishes, and suspended in extraction buffer (1% Triton X-100, 100 mM Tris–HCl, pH 8.8, 10 mM NaCl, 10% glycerol, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 1 mM pnitrophenyl phosphate, and 1 mM phenylmethylsulfonyl fluoride). After incubation on ice for 30 min, lysates were centrifuged and proteins in the supernatants were quantified using the Bradford Protein Assay Reagent (Bio-Rad). An equal amount of proteins was then separated on a 10–15% SDS-PAGE gel followed by electrophoretic transfer to nitrocellulose membranes. Membranes were blocked in 5% nonfat dry milk and 0.1% Tween-20 in Tris-buffered saline and probed with primary antibody. Antibodies against caspase-7, actin, Bcl-2, BclxL, Bax, VDAC, cytochrome c, and cleaved poly (ADP-ribose) polymerase (PARP) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against TRADD, Bid, caspase-8, and caspase-9 were from Cell Signaling (Beverly, MA, USA). LC3 antibody was purchased from Novus Biologicals (Littleton, CO, USA). Antibody– antigen complexes were incubated with goat anti-mouse IgG- or goat anti-rabbit IgG-peroxidase conjugates, followed by detection using an enhanced chemiluminescence (ECL) kit (Amersham Biosciences, Uppsala, Sweden). 2.7. RT-PCR analysis Total RNA was extracted from the cells using Trizol reagent (Gibco BRL) according to the manufacturer's protocol, and the extracted RNA samples were subsequently treated with MMLV reverse transcriptase (Promega). PCRs were performed using specific primers for LC3B: forward, 5′-CGGAGAAGACCTTCAAGCAG-3′ and reverse, 5′CTGGGAGGCATAGACCATGT-3′; ATG7: forward, 5′-ACCCAGAAGAAGCTGAACGA-3′ and reverse, 5′-AGACAGAGGGCAGGATAGCA-3′; and GAPDH: forward, 5′-GGGAAGAGTCAACGGATTTGGTCGT-3′ and reverse, 5′-GGGAATTGATTTTGGAGGGATCTCG-3′. The cycling conditions were 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 s, at 58 °C for 30 s, and at 72 °C for 1 min and a final extension at 72 °C for 10 min. The PCR products were analyzed by electrophoresis on 1% agarose gel containing ethidium bromide and photographed under ultraviolet light.

2.4. Flow cytometry analysis 2.8. Fluorescence microscopy For detection of sub-diploid population, cells were detached from plates by the addition of 0.25% trypsin–EDTA, washed in phosphatebuffered saline (PBS), fixed in 70% ethanol, and stained with propidium iodide (PI) for 15 min at room temperature in the dark. Analysis of cells with sub-G0/G1 DNA content (subdiploid cells) was performed on 10,000 cells on a FACScan flow cytometer using the CellQuest analysis program (Becton-Dickinson, Cowley, UK), as described previously [43]. PI-exclusion assay was used to determine the percentage of viable cells. Briefly, after treatment, cells were collected, incubated for 10 min on ice in the dark in PBS containing 20 μg/ml PI, and then analyzed with FACScan flow cytometer. For determination of mitochondrial membrane potential, cells were collected after exposure to 30 μM FR122047 for 24 h. Cells were washed and resuspended in 1 ml of PBS containing 2 μM DiOC6(3), incubated at 37 °C in the dark for 15 min, and then washed and resuspended in PBS. Mitochondrial membrane potential (Δψm) was assessed by flow cytometry [44].

Cells were treated with 30 μM FR122047 for 24 h and incubated with 50 μM MDC, a selective fluorescent marker for autophagic vacuoles, at 37 °C for 60 min as described previously [26]. The cells were examined under a fluorescence microscope (Carl Zeiss). To detect acidic vesicular organelles (AVOs), cells were stained with 5 μg/ml acridine orange for 15 min and washed with PBS. Formation of AVOs was examined under a fluorescence microscope. For LysoTracker staining, cells were incubated for 30 min in PBS with 75 nM LysoTracker Red (Lonza, PA-3015) and observed under a confocal microscope (Olympus). 2.9. Assay for caspase-9 or cathepsin B activity After the indicated treatments, cells were collected and lysed on ice for 10 min. Caspase-9 activity was measured using a colorimetric assay kit (Calbiochem, La Jolla, CA, USA) and LEDH-pNA as substrate. The amount of free pNA was determined by measuring absorbance at

82

H.-S. Jeong et al. / Biochimica et Biophysica Acta 1813 (2011) 80–90

405 nm. Cathepsin B activity was analyzed using a cathepsin B activity assay kit (Calbiochem, La Jolla, CA, USA) and z-Arg-Arg-AMC as substrate. Activity was determined by measuring fluorescence of free AMC on a fluorescence plate reader at an excitation wavelength of 370 nm and emission wavelength of 450 nm.

2.10. Transmission electron microscopy Cells were fixed with 2.5% glutaraldehyde in 0.1 M PBS, pH 7.4, for 2 h and then washed three times in 0.1 M PBS. Cells were post-fixed in 2 mM osmium tetroxide in 0.1 M PBS for 1 h, dehydrated in an

Fig. 1. Treatment of breast cancer MCF-7 cells with FR122047 induces apoptosis and caspase activation. (A) Cells were treated with 10, 20, or 30 μM FR122047 (FR) or DMSO alone (0 μM) for 24 h, fixed, and stained with DAPI. Images were acquired using a fluorescence microscope. Cells were counted and percentage of apoptotic cells expressed as the ratio of apoptotic cells to total cells. Values represent the mean ± SE from three independent experiments. *P b 0.05 versus the control (DMSO). (B) Cells were treated with 0, 10, 20, or 30 μM FR for 24 h and lysed, and proteins were separated by SDS-polyacrylamide gel electrophoresis. Western blot analysis was performed using antibody against cleaved PARP or actins (loading control), and proteins were visualized using the ECL detection system. Representative blots from three independent experiments were shown. (C) Percentage of subdiploid cells was determined by flow cytometry after treatment with 30 μM FR or DMSO alone for 24 h. (D) Cells were treated with 30 μM FR for the indicated time period, and cell lysates were analyzed by Western blotting with antibodies to caspase-8, caspase-7, caspase-9, and actin (loading control). Representative data from three independent experiments were presented. (E) Cells were incubated with 30 μM FR for the indicated time period. The expression of Bid, Bcl-2, Bcl-xL, or Bax was analyzed by Western blotting, and actin was used as loading control. Representative blots from three independent experiments were shown. (F) Control MCF-7 and Bcl-2-overexpressing MCF-7 cells were treated with 30 μM FR for 24 h, and caspase-9 activation was analyzed by Western blotting with anti-caspase-9 antibody. Representative blots from three independent experiments were shown. (G) MCF-7 cells were cultured in 30 μM FR or 50 μM carbonyl cyanide m-chloro-phenylhydrazone (CCCP; positive control) for 24 h, stained with 2 μM DiOC6(3), and mitochondrial membrane potential (ΔΨm) was analyzed by FACScan. Values represent the mean ± SE from three independent experiments. *P b 0.05 versus the control (DMSO). (H) Following treatment with 30 μM FR for 24 h, an accumulation of cytochrome c (Cyto c) in the cytoplasmic fraction was accessed by Western blotting with antibody to cytochrome c. Representative blots from three independent experiments were shown. (I) Cells were treated with 30 μM FR for 24 h, and the level of caspase-9 activity in cell lysates was analyzed using a colorimetric cleavage assay and LEHD-pNA as substrate. Values represent the mean ± SE from three independent experiments. *P b 0.05 versus the control (DMSO).

H.-S. Jeong et al. / Biochimica et Biophysica Acta 1813 (2011) 80–90

ascending series (50–100%) of ethanols, and embedded using the EMbed 812 kit (Electron Microscopy Sciences, Hatfield, PA, USA). Sections of 70 nm were double-stained with 3% uranyl acetate and lead citrate and examined using a transmission electron microscope (H-7600s, Hitachi, Japan) at an acceleration voltage of 80 kV.

83

Lafayette, CO, USA) using LipofectAMINE 2000 reagent (Invitrogen, Paisley, UK) according to the manufacturer's protocol. A control siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as negative control. Knockdown of caspase-9 expression by siRNA was assessed by Western blot analysis.

2.11. Knockdown of caspase-9 expression by small interfering RNA 2.12. Statistical analysis Cells were seeded in 60-mm dishes and incubated for 24 h. The next day, cells were transfected with human caspase-9 siRNA directed against caspase-9 (siGENOME SMART pool reagents, Dharmacon,

The Student's t-test was used for testing statistical significance between groups and P b 0.05 was considered significant.

Fig. 2. Blockage of caspase-9 function significantly enhances FR122047-induced cell death. (A) MCF-7 cells were incubated in the presence or absence of pan-caspase inhibitor z-VAD-FMK (z-VAD), caspase-8 inhibitor z-IETD-FMK (Cas8-inh), or caspase-9 inhibitor z-LEHD-FMK (Cas9-inh) for 1 h before the addition of 30 μM FR. Cells were incubated for another 24 h and then analyzed by FACScan. Values represent the mean± SE from three independent experiments. *P b 0.05 versus FR alone. (B) Cells were incubated in the presence or absence of 30 μM FR and 20 μM caspase-9 inhibitor z-LEHD-FMK (Cas9-inh) for 24 h, and cell viability was determined by PI-exclusion assay. The gated cells represent viable cells. Representative data from three independent experiments were presented. (C) Cells were preincubated with 20 μM caspase- 9 inhibitor, z-LEHD-FMK (Cas9-inh) for 1 h prior to treatment with 30 μM FR for 24 h. Western blot analyses were conducted with antibodies to caspase-8, caspase-7, cleaved PARP, and actin. Representative blots from three independent experiments were shown. (D) Results of densitometric analyses of cleaved caspase-8, cleaved caspase-7, and cleaved PARP levels. (E) Cells were pretreated with 20 μM caspase-9 inhibitor, z-LEHD-FMK (Cas9-inh) for 1 h followed by addition of 100 μM sulforaphane (SFN), 400 μM etoposide (Etopo), or 10 μM doxorubicin (Doxo) and incubated for another 24 h. Subdiploid cells were analyzed by FACScan. Values represent the mean ± SE from three independent experiments. *P b 0.05 versus the drug alone.

84

H.-S. Jeong et al. / Biochimica et Biophysica Acta 1813 (2011) 80–90

3. Results 3.1. Treatment with the NSAID FR122047 promotes apoptosis and caspase activation in MCF-7 cells To determine whether the NSAID FR122047 (FR) induces apoptotic cell death in MCF-7 cells, we examined changes in nuclear morphology in FR-treated cells by DAPI staining. Treatment with FR resulted in pronounced apoptotic chromatin condensation and a significant increase in the percentage of apoptotic cells (Fig. 1A). We also examined changes in PARP cleavage as a marker for apoptosis and the proportion of apoptotic cells with subdiploid DNA content. FR treatment elicited an increase in PARP cleavage (Fig. 1B) and apparent enhancement in the proportion of apoptotic cells with subdiploid DNA content (Fig. 1C). Moreover, time-dependent increases in caspase-8, caspase-7, and caspase-9 cleavages were detected in caspase-3-deficient MCF-7 cells following FR treatment [21] (Fig. 1D). Treatment of FR also induced the expression of FADD (Fas-associated death domain protein) and TRADD (TNFR-1-associated death domain protein), components of DISC (death-inducing signaling complex) in MCF-7 cells (Fig. S1). To investigate whether FR treatment would trigger the recruitment of

caspase-8 into FADD in FR122047-treated MCF-7 cells, lysates from FRtreated MCF-7 cells were immunoprecipitated with anti-FADD antibody. The results from immunoprecipitation experiments showed obvious direct association between FADD and caspase-8 in FR-treated MCF-7 cells, indicating that caspase-8 activation correlates with components of DISC (Fig. S2). Next, we analyzed the expression level of the Bcl-2 family proteins in FR-treated MCF-7 cells. Treatment of MCF7 cells with FR resulted in induction of Bid cleavage into truncated form (tBid) and down-regulation of Bcl-2 expression with no change in expression of Bcl-xL or Bax, leading to an increase in the Bax/Bcl-2 ratio (Fig. 1E). We could confirm that overexpression of Bcl-2 exhibited the inhibition of FR-induced caspase-9 activation (Fig. 1F). In addition, treatment with FR induced an alteration of the mitochondrial membrane potential (ΔΨm) of MCF-7 cells as detected with the ΔΨm—sensitive dye DiOC6(3) (Fig. 1G). As this event results in accumulation of cytochrome c in the cytosol and a subsequent activation of caspase-9 [22], we investigated effect of FR on cytochrome c release and analyzed caspase-9 activity by assaying cleavage of the substrate LEHD-pNA in lysates of FRtreated cells. We could find that treatment of MCF-7 cells with FR triggered significant release of mitochondrial cytochrome c (Fig. 1H) and caspase-9 activation (Fig. 1I).

Fig. 3. FR122047 treatment induces autophagy in MCF-7 cells. (A) MCF-7 cells were treated with 30 μM FR122047 (FR) or DMSO alone for 18 h and processed for transmission electron microscopy. Arrows and arrowheads indicate autophagosomes and autophagolysosomes, respectively. Scale bars represent 2 μm (8000×) and 500 nm (12,000×). (B) Cells were treated with 30 μM FR for the indicated times, and cell lysates were analyzed by Western blotting for levels of LC3-I (18 kDa) and LC3-II (16 kDa). Representative blots from three independent experiments were shown. (C) Cells were treated with 30 μM FR for the indicated times, and the mRNA level of LC3B or ATG 7 was analyzed by RT-PCR. Expression levels relative to GAPDH are presented. The experiments were repeated independently at least three times. (D) Cells transfected with GFP-LC3 were treated for 24 h with 30 μM FR or DMSO alone and examined by confocal microscopy. Nuclei were stained with TOPRO-3 (blue). Scale bars represent 20 μm. Images were representative of the results of three independent experiments. (E) Cells were treated with 30 μM FR or DMSO alone for 24 h and labeled with MDC. Images were acquired using fluorescence microscopy. Scale bars represent 20 μm. Right, enlarged images of areas indicated by squares. Images representative of the results of three different experiments were presented. (F) Cells were treated with 30 μM FR or DMSO alone for 24 h, and formation of AVOs (red fluorescence) was examined under a fluorescence microscope.

H.-S. Jeong et al. / Biochimica et Biophysica Acta 1813 (2011) 80–90

3.2. Blockage of caspase-9 activation significantly augments FR122047-induced cell death Next, we examined whether the caspase inhibitors would suppress FR-induced cell death in MCF-7 cells. Addition of the pan-caspase inhibitor z-VAD-FMK or the caspase-8 inhibitor z-IETD-FMK significantly blocked the accumulation of a subdiploid fraction in FR-treated cells (Fig. 2A).

85

Unpredictably, co-treatment with the caspase-9 inhibitor, z-LEHD-FMK, however, resulted in a marked increase in cell death (Fig. 2A) and a significant reduction in cell viability compared to FR alone (Fig. 2B). Addition of the caspase-9 inhibitor showed inhibition of caspase-7 and PARP cleavages but did not affect caspase-8 cleavage in FR-treated MCF-7 cells (Fig. 2C and D), suggesting that caspase-9-mediated alternative pathway may be involved in regulation of FR-induced cell death in the

Fig. 4. Treatment with caspase-9 inhibitor suppresses FR122047-induced autophagy. (A) Cells were pretreated with 20 nM rapamycin (RA) or 20 μM chloroquine (CQ) for 1 h before the addition of 30 μM FR122047 (FR). The cells were incubated for another 24 h and then analyzed by FACScan. Values represent the mean ± SE from three independent experiments. *P b 0.05 versus FR alone. Bottom panel: cells were preincubated with 20 nM rapamycin (RA) for 1 h prior to treatment with 30 μM FR for 24 h and Western blot analysis was conducted with antibodies to LC3 and actin. (B) Cells were pretreated with 20 μM chloroquine (CQ) or 20 μM caspase-9 inhibitor, z-LEHD-FMK (Cas9-inh) for 1 h before the addition of 30 μM FR. Cells were incubated for another 24 h, and cell lysates were analyzed by Western blotting. Images representative of the results of three different experiments were presented. (C) Cells were pretreated with 100 nM bafilomycin A1 or cathepsin inhibitors (E64d + Pepstatin A methyl ester (E/P), 10 μg/ml for each) for 1 h before the addition of 30 μM FR. Cells were incubated for another 24 h, and cell lysates were analyzed by Western blotting. (D) Cells were pretreated with 20 μM chloroquine (CQ), 20 nM rapamycin (RA), 20 μM caspase-9 inhibitor, z-LEHD-FMK (Cas9-inh), or 100 nM bafilomycin A1 for 1 h before the addition of 30 μM FR. Cells were incubated for another 24 h, and formation of AVOs (red fluorescence) was examined under a fluorescence microscope. (E) Cathepsin B activity assay was performed after treatment for 12 h with 30 μM FR alone or in combination with 20 μM chloroquine or 20 μM caspase-9 inhibitor. Values represent the mean ± SE of from three independent experiments. *P b 0.05 versus the control (DMSO), **P b 0.01 versus FR alone. (F) GFP-LC3-expressing cells were pretreated with 20 μM caspase-9 inhibitor, z-LEHD-FMK (Cas9-inh) for 1 h before the addition of 30 μM FR. Cells were incubated for another 24 h, stained with Lysotracker Red dye (Lysotracker), and observed under a confocal microscope. Scale bars represent 50 μm.

86

H.-S. Jeong et al. / Biochimica et Biophysica Acta 1813 (2011) 80–90

Fig. 4 (continued).

cells. We next raised the question of whether enhancement of cell death by caspase-9 inhibitor occurs only in concert with the action of FR. MCF-7 cells were treated with sulforaphane, etoposide, or doxorubicin, in the presence and absence of caspase-9 inhibitor. Co-treatment with caspase-9 inhibitor resulted in increased susceptibility to sulforaphane-induced cell death, consistent with the results obtained with FR. Distinct from its effect with FR or sulforaphane, co-treatment of caspase-9 inhibitor led to a decrease in etoposide- or doxorubicin-induced cell death of MCF-7 cells (Fig. 2E).

3.3. FR treatment leads to induction of autophagy in MCF-7 cells As sulforaphane has been reported to induce autophagy [23], we investigated whether autophagy, which corresponds to type II programmed cell death, occurs in FR-treated MCF-7 cells. Transmission electron microscopy revealed a pronounced increase in the autophagic compartments in FR-treated cells (Fig. 3A). Moreover, there was a time-dependent increase in the level of LC3-II in FRtreated MCF-7 cells (Fig. 3B), although there was no apparent timedependent change in the mRNA level of LC3 (Fig. 3C). We could also detect a significant time-dependent increase in the mRNA level of ATG 7 in FR-treated cells (Fig. 3C). Previous studies have reported that the LC3-II form is primarily localized to autophagosomal membranes [24]. As expected, fluorescence microscopic analysis showed that, in GFPLC3-expressing MCF-7 cells, GFP was distributed in a punctuate pattern after FR treatment, indicating translocation of LC3-II to autophagosomes (Fig. 3D). In contrast, GFP-LC3 fluorescence appeared in a diffuse cytoplasmic pattern in DMSO-treated control MCF-7 cells. When cells were stained with monodansylcadaverine (MDC), a spontaneously fluorescent dye that selectively labels autophagosomes and autolysosomes [25,26], FR-treated MCF-7 cells exhibited strong staining compared to DMSO-treated cells (Fig. 3E). As formation of acidic vesicular organelles (AVOs) correlates with autophagy [23], we could also confirm that treatment of FR resulted in apparent formation of AVOs (red fluorescence) in MCF-7 cells (Fig. 3F). These findings apparently reveal that autophagy is induced in the response of MCF-7 cells to FR treatment.

3.4. Co-treatment with caspase-9 inhibitor results in suppression of FR-induced autophagy Next, we investigated whether FR-induced autophagy is involved in cell death or in cell survival. Treatment of rapamycin, a specific mTOR inhibitor that promotes autophagy [27,28], led to protection of FR-induced cell death (Fig. 4A). The elicitation of autophagy by rapamycin treatment was confirmed by the conversion of LC3-I to LC3-II (Fig. 4A, bottom panel). In contrast, treatment of chloroquine, a lysosomal enzyme inhibitor [19,29], markedly enhanced FR-induced cell death (Fig. 4A), implying that autophagy would play a defensive role in FR-induced cell death. Since chloroquine is known to be a lysosomal enzyme inhibitor [19,29] and blocks the lysosomal degradation of autophagosomes [30], we assessed the effect of chloroquine on FRinduced LC3-II accumulation. Chloroquine alone induced an increase in the level of LC3-II and had an additive effect on FR-induced accumulation of LC3-II in MCF-7 cells (Fig. 4B). Interestingly, treatment with caspase-9 inhibitor also showed an increase of both LC3-I and LC3II levels in FR-treated MCF-7 cells (Fig. 4B). Treatment with another autophagy inhibitor, such as bafilomycin A1 or pepstatin A methyl ester and E-64D, also led to an increase of both LC3-I and LC3-II accumulation in FR-treated MCF-7 cells (Fig. 4C). Moreover, we analyzed the autophagic flux upon co-treatment with rapamycin, chloroquine, caspase-9 inhibitor, or bafilomycin A1 in FR-treated MCF-7 cells using acridine orange staining. As expected, co-treatment with chloroquine, caspase-9 inhibitor, or bafilomycin A1 inhibited formation of acidic vesicular organelles (AVOs) in FR-treated MCF-7 cells, but formation of AVOs (red fluorescence) was observed in rapamycin alone-treated cells as well as in FR and rapamycin-treated MCF-7 cells (Fig. 4D). To confirm the suppressive effect of caspase-9 in autophagic process, we examined whether inhibition of caspase-9 had an effect on cathepsin activity, because cathepsins are lysosomal enzymes and are closely involved in autophagosomal degradation [30] and also the accumulation of LC3-II may be related to the activity. Addition of caspase-9 inhibitor efficiently suppressed FR-induced cathepsin activity and chloroquine had a similar effect, as expected (Fig. 4E). Additionally, we conducted lysosome staining analysis using LysoTracker Red (LTR), which is a lysosomotropic fluorescence dye that accumulates within intact acidic lysosomes

H.-S. Jeong et al. / Biochimica et Biophysica Acta 1813 (2011) 80–90

exhibiting red fluorescence. Interestingly, we found that inhibition of caspase-9 affected vesicular pH in acidic organelles (Fig. 4F) and cotreatment with caspase-9 inhibitor did not suppress FR-induced GFPLC3 puncta formation in stable GFP-LC3 transformants. However, addition of caspase-9 inhibitor resulted in the emergence of cells with weak red fluorescence in FR-treated cells. Taken together, these data suggest that caspase-9 may be correlated with acid-dependent activities of cathepsins and inhibition of caspase-9 may suppress autophagic flux in FR-treated MCF-7 cells. 3.5. siRNA knockdown of caspase-9 expression sensitizes MCF-7 cells to FR122047 or sulforaphane-induced cell death Although the induction of autophagy by sulforaphane treatment was also confirmed by marked accumulation of LC3-II, treatment with etoposide or doxorubicin failed to induce significant accumulation of LC3-II in MCF-7 cells (Fig. 5A). To confirm that the caspase-9 inhibitormediated sensitization of MCF-7 cells to FR-induced cell death was due to the specific suppression of caspase-9 activity, we knocked down the caspase-9 gene expression using caspase-9-specific siRNA. MCF-7 cells transfected with the caspase-9 siRNA showed markedly

87

reduced expression of caspase-9 compared to control (Fig. 5B). Knockdown of caspase-9 expression resulted in increased susceptibility of cells to FR or sulforaphane-induced cell death (Fig. 5C) and significant suppression of FR- or sulforaphane-induced cathepsin B activity (Fig. 5D), strongly indicating that caspase-9 may be involved in specific drug-induced autophagy. 4. Discussion Since COXs are highly expressed in tumors and enhance tumorigenesis, lots of studies have focused on evaluating the possibility of using COX inhibitors for cancer prevention or treatment. For this reason, recently, clinical trials are being carried out to investigate the use of COX inhibitors such as celecoxib as an anticancer agent [4]. Since NSAIDs, inhibitors of COXs inhibit the enzyme COXs, which convert arachidonic acid to prostaglandins (PGs), the inhibition of PGs formation accounts for part of the anticancer effect of NSAIDs. Several studies have shown that cell growth inhibition caused by treatment with a COX-2 inhibitor was reversed by addition of PGs [31,32]. However, compelling evidence suggests that NSAIDs also regulate through COXs-independent mechanisms. For example, NSAIDs reduced cell survival in COX-deficient cell

Fig. 5. siRNA knockdown of caspase-9 expression sensitizes MCF-7 cells to FR122047-induced cell death. (A) Cells were treated with 100 μM sulforaphane (SFN), 400 μM etoposide (Etopo), or 10 μM doxorubicin (Doxo) for 24 h and levels of LC3-I and LC3-II in lysates were analyzed by Western blotting. Blots representative of the results of three independent experiments were presented. (B) MCF-7 cells were transfected with control (Con-si) or caspase-9 (Cas9-si) siRNA. Whole-cell lysates were harvested for Western blot analysis 24 and 48 h after transfection. Blots representative of the results of three independent experiments were presented. (C) At 24 h post-transfection, MCF-7 cells were treated with 30 μM FR122047 (FR) or 100 μM sulforaphane (SFN) for a further 24 h, and the proportion of subdiploid cells was analyzed by FACScan. Values represent the mean ± SE from three independent experiments. *P b 0.05 versus FR or SFN alone. (D) Cathepsin B activity assay was performed after treatment for 12 h with 30 μM FR or 100 μM SFN at 24 h posttransfection. Values represent the mean ± SE of from three independent experiments. *P b 0.05 versus the control (DMSO), **P b 0.05 versus FR or SFN alone.

88

H.-S. Jeong et al. / Biochimica et Biophysica Acta 1813 (2011) 80–90

lines [33,34] and also did exert anti-tumorigenic effect through other cellular pathway, independent of COX inhibition [35]. In the present study, we could find that the NSAID FR122047 induces COX activityindependent cell death in breast cancer MCF-7 cells (data not shown). After FR treatment, we could observe an alteration of mitochondrial membrane potential, indicative of a well-established intrinsic apoptotic pathway (Fig. 1G), as well as recruitment of caspase-8 into FADD, which correlates with the extrinsic apoptotic pathway (Fig. S2). FR treatment also resulted in induction of Bid cleavage into truncated form (tBid) (Fig. 1E), an increase in the Bax/Bcl-2 ratio (Fig. 1E), and significant release of mitochondrial cytochrome c (Fig. 1H). Based on our observations, we could propose the involvement of caspase-9 in the mechanism of FR-induced cell death and actually detected caspase-9 activation (Fig. 1I). However, treatment of the caspase-9 inhibitor z-LEHD-FMK was unable to block but it rather enhanced FRinduced cell death (Fig. 2A), although the inhibitor suppressed the cleavage of PARP and caspase-7 (Fig. 2C). Furthermore, siRNAmediated knockdown of caspase-9 expression in MCF-7 cells caused increased susceptibility of cells to FR-induced cell death (Fig. 5C), consistent with the results obtained with caspase-9 inhibitor. These data led us to hypothesize that caspase-9 might be involved in another pathway specifically regulating the typical apoptotic signaling pathway. We could observe hallmarks of autophagy, e.g., autophagosomes and autophagolysosomes, in FR-treated MCF-7 cells. More recent studies suggest that autophagy can be either a process that contributes to cell death or a defense mechanism. For example, inhibition of autophagy blocked TNF α-induced apoptosis in human T-lymphoblastic leukaemic cells [36], while autophagy protected cells from sulforaphane-induced apoptosis in human prostate cancer cells [23]. In our study, autophagy was shown to be cytoprotective as co-treatment of the cells with rapamycin, a well-known inducer of autophagy [27], suppressed FR-induced cell death in MCF-7 cells (Fig. 4A). We examined whether the caspase-9 inhibitor had any effect on the autophagic process and could find compelling experimental evidence that treatment with the inhibitor suppresses the autophagic event. First of all, we originally could find that inhibition of caspase-9 blocks the FRinduced increase in activity of cathepsin. Several previous studies have reported that a relationship may exist between caspase-9 activation and cathepsin activity. Gyrd-Hansen et al. reported that caspase-8-activated

caspase-9 triggers lysosomal membrane permeabilization, leading to cell death [37]. They also showed that caspase-9-deficient cells exhibit decreased cathepsin activity in lysosomal cell death compared to parental cells. However, they did not provide any data showing the involvement of caspase-9 activity in autophagic process. In our study, to confirm the suppressive effect of caspase-9 in autophagic process, we introduced the acridine orange staining and lysosome staining analyses. Our data showed that inhibition of caspase-9 affected vesicular pH in acidic organelles (Fig. 4F) and co-treatment with caspase-9 inhibitor did not suppress FR-induced GFP-LC3 puncta formation in stable GFP-LC3 transformants. However, addition of caspase-9 inhibitor resulted in the emergence of cells with weak red fluorescence in FR-treated cells, indicating that inhibition of caspase-9 evidently influenced vesicular pH in acidic lysosomes. These results strongly suggest that caspase-9 may be involved in regulation of lysosomal pH and acid-dependent cathepsin activities. Moreover, co-treatment of FR and caspase-9 inhibitor significantly reduced the quantity of acridine orange-positive vesicles compared to FR alone treatment (Fig. 4D), confirming that the autophagosomes produced by FR undergo the maturation process with the fusion with lysosomes. Indeed, in the caspase-9 inhibitortreated cells, LC3-II levels remain elevated despite of a marked reduction of acidic vesicles, indicating that the earlier stages of autophagy prior to lysosomal fusion were not affected by caspase-9 inhibitor. In contrast, caspase-8 inhibitor had no effect on both LC3-II accumulation and caspase-9 activation in FR-treated MCF-7 cells, even though it inhibited caspase-8-dependent cleavage of BID (Fig. S3), implying that caspase8 activation seems to be not directly involved in FR-induced autophagy of MCF-7 cells. These data originally suggest that inhibition of caspase-9 may block the autophagic flux and enhance cell death due to blockage of cytoprotective autophagy (Fig. 6). We then asked whether enhancement of cell death in MCF-7 cells by co-treatment with caspase-9 inhibitor is limited to FR. Previous studies had shown that etoposide and doxorubicin induce cell death by activation of caspase-9 [38]. As expected, caspase-9 inhibition was found to have an inhibitory effect on etoposide- and doxorubicininduced cell death in MCF-7cells (Fig. 2E). Moreover, caspase8 inhibition had no effect on doxorubicin-induced cell death in the MCF-7 cells (Fig. S4). Interestingly, neither etoposide nor doxorubicin induced significant autophagic event such as conversion of LC3-I to LC3-

Fig. 6. Treatment with FR122047 triggers the activation of both caspase-9 and caspase-8 in MCF-7 cells. Blockade of caspase-8-mediated apoptosis results in apparent suppression of FR-induced cell death. In contrast, inhibition of caspase-9 sensitizes MCF-7 cells to FR-induced cell death. It seems that the cell death enhanced by caspase-9 inhibitor correlates with suppression of cytoprotective autophagy during FR-induced cell death. Although treatment of anticancer drugs such as etoposide or doxorubicin results in apparent apoptotic cell death, it does not lead to significant autophagy. Cell death induced by these drugs is significantly blocked by addition of caspase-9 inhibitor. It appears that the caspase-9-dependent apoptotic pathway may be important in the cell death induced by these drugs.

H.-S. Jeong et al. / Biochimica et Biophysica Acta 1813 (2011) 80–90

II in MCF-7 cells (Fig. 5A). These data suggest the possibility that caspase-9 may have autophagy-involved another function in druginduced cell death accompanied by significant autophagy. Further experiments are required to analytically examine this possibility and currently underway. In recent years, there has been increasing evidence that caspase inhibition by specific caspase inhibitor can induce events independent of blocking cell death or apoptosis. Loss of caspase-8 has been reported to promote dissemination of neuroblastoma cells [39], and inhibition of caspase-3 activity resulted in attenuated neurite extension and cellular remodeling during differentiation [40]. Treatment of pan-caspase inhibitor, z-VAD-FMK, has been shown to induce necrotic cell death in L929 murine fibrosarcoma cells [41]. Moreover, treatment of a caspase-9 inhibitor was reported to augment stress-induced apoptosis in B-lineage cells [15], enhance the generation of ROS, and induce necrosis in RAW 264 macrophage cells infected with Mycobacterium tuberculosis [14]. These findings raise the possibility that there may be more diverse functions of caspases than thought. In conclusion, our data show that treatment of breast cancer MCF-7 cells with the NSAID FR122047 led to caspase-mediated apoptosis and simultaneously stimulated cytoprotective autophagy. We could observe hallmarks of intrinsic pathway-mediated apoptosis, but inhibition of caspase-9 enhanced FR-induced cell death by blocking autophagy. Our data provide new insights into the understanding of the function of caspase-9 during the process of cytoprotective autophagy. Acknowledgements We thank Dr. T. Yoshimori (Research Institute for Microbial Diseases, Osaka University, Japan) for providing the GFP-LC3 plasmid and Dr. Seunghwa Park (Konkuk University, South Korea) for helping with the transmission electron microscopy. This work was supported in part by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (No. 2009-0083694 and No. 2010-0001348) and by a grant from Korea Health 21 R&D Project, Ministry for Health, Welfare, and Family Affairs (A08-4065), a grant from BioGreen 21 Program, Rural Development Administration (Code # 20070501-034-001-009-02-00), and Top Brand Project grant from Korea Research Council of Fundamental Science & Technology and KRIBB Initiative program (KGM3110912). We acknowledge a graduate fellowship provided by the Korean government (MEST) through the Brain Korea 21 project, Republic of Korea. Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10.1016/j.bbamcr.2010.09.016. References [1] S.J. Baek, T.E. Eling, Changes in gene expression contributes to cancer prevention by COX inhibitors, Prog. Lipid Res. 45 (2006) 1–16. [2] A. Lupulescu, Prostaglandins, their inhibitors and cancer, Prostaglandins Leukot. Essent. Fatty Acids 54 (1996) 83–94. [3] N. Kundu, A.M. Fulton, Selective cyclooxygenase (COX)-1 or COX-2 inhibitors control metastatic disease in a murine model of breast cancer, Cancer Res. 62 (2002) 2343–2346. [4] D. Mazhar, R. Ang, J. Waxman, COX inhibitors and breast cancer, Br. J. Cancer 94 (2006) 346–350. [5] R.E. Harris, K. Namboodiri, S.D. Stellman, E.L. Wynder, Breast cancer and NSAID use: heterogeneity of effect in a case–control study, Prev. Med. 24 (1995) 119–120. [6] R.E. Harris, R.T. Chlebowski, R.D. Jackson, D.J. Frid, J.L. Ascenseo, G. Anderson, A. Loar, R.J. Rodabough, E. White, A. McTiernan, Breast cancer and nonsteroidal antiinflammatory drugs: prospective results from the Women's Health Initiative, Cancer Res. 63 (2003) 6096–6101. [7] D.M. Schreinemachers, R.B. Everson, Aspirin use and lung, colon, and breast cancer incidence in a prospective study, Epidemiology 5 (1994) 138–146. [8] J.A. Baron, R.S. Sandler, Nonsteroidal anti-inflammatory drugs and cancer prevention, Annu. Rev. Med. 51 (2000) 511–523. [9] W.H. Wang, J.Q. Huang, G.F. Zheng, S.K. Lam, J. Karlberg, B.C. Wong, Non-steroidal anti-inflammatory drug use and the risk of gastric cancer: a systematic review and meta-analysis, J. Natl Cancer Inst. 95 (2003) 1784–1791.

89

[10] I. Cheng, X. Liu, S.J. Plummer, L.M. Krumroy, G. Casey, J.S. Witte, COX2 genetic variation, NSAIDs, and advanced prostate cancer risk, Br. J. Cancer 97 (2007) 557–561. [11] T. Ochi, Y. Motoyama, T. Goto, The analgesic effect profile of FR122047, a selective cyclooxygenase-1 inhibitor, in chemical nociceptive models, Eur. J. Pharmacol. 391 (2000) 49–54. [12] T. Ochi, T. Goto, Differential effect of FR122047, a selective cyclo-oxygenase-1 inhibitor, in rat chronic models of arthritis, Br. J. Pharmacol. 135 (2002) 782–788. [13] M.C. Abraham, S. Shaham, Death without caspases, caspases without death, Trends Cell Biol. 14 (2004) 184–193. [14] R. Uchiyama, I. Kawamura, T. Fujimura, M. Kawanishi, K. Tsuchiya, T. Tominaga, T. Kaku, Y. Fukasawa, S. Sakai, T. Nomura, M. Mitsuyama, Involvement of caspase-9 in the inhibition of necrosis of RAW 264 cells infected with Mycobacterium tuberculosis, Infect. Immun. 75 (2007) 2894–2902. [15] N. Shah, R.J. Asch, A.S. Lysholm, T.W. Lebien, Enhancement of stress-induced apoptosis in B-lineage cells by caspase-9 inhibitor, Blood 104 (2004) 2873–2878. [16] A. Thorburn, Apoptosis and autophagy: regulatory connections between two supposedly different processes, Apoptosis 13 (2008) 1–9. [17] T. Yorimitsu, D.J. Klionsky, Autophagy: molecular machinery for self-eating, Cell Death Differ. 12 (Suppl 2) (2005) 1542–1552. [18] P. Codogno, A.J. Meijer, Autophagy and signaling: their role in cell survival and cell death, Cell Death Differ. 12 (Suppl 2) (2005) 1509–1518. [19] R.K. Amaravadi, D. Yu, J.J. Lum, T. Bui, M.A. Christophorou, G.I. Evan, A. ThomasTikhonenko, C.B. Thompson, Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma, J. Clin. Invest. 117 (2007) 326–336. [20] M.J. Abedin, D. Wang, M.A. McDonnell, U. Lehmann, A. Kelekar, Autophagy delays apoptotic death in breast cancer cells following DNA damage, Cell Death Differ. 14 (2007) 500–510. [21] R.U. Janicke, M.L. Sprengart, M.R. Wati, A.G. Porter, Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis, J. Biol. Chem. 273 (1998) 9357–9360. [22] H.A. Ferguson, P.M. Marietta, C.L. Van Den Berg, UV-induced apoptosis is mediated independent of caspase-9 in MCF-7 cells: a model for cytochrome c resistance, J. Biol. Chem. 278 (2003) 45793–45800. [23] A. Herman-Antosiewicz, D.E. Johnson, S.V. Singh, Sulforaphane causes autophagy to inhibit release of cytochrome c and apoptosis in human prostate cancer cells, Cancer Res. 66 (2006) 5828–5835. [24] Y. Kabeya, N. Mizushima, T. Ueno, A. Yamamoto, T. Kirisako, T. Noda, E. Kominami, Y. Ohsumi, T. Yoshimori, LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing, EMBO J. 19 (2000) 5720–5728. [25] H. Yanagisawa, T. Miyashita, Y. Nakano, D. Yamamoto, HSpin1, a transmembrane protein interacting with Bcl-2/Bcl-xL, induces a caspase-independent autophagic cell death, Cell Death Differ. 10 (2003) 798–807. [26] F. Akdemir, R. Farkas, P. Chen, G. Juhasz, L. Medved'ova, M. Sass, L. Wang, X. Wang, S. Chittaranjan, S.M. Gorski, A. Rodriguez, J.M. Abrams, Autophagy occurs upstream or parallel to the apoptosome during histolytic cell death, Development 133 (2006) 1457–1465. [27] C. Cao, T. Subhawong, J.M. Albert, K.W. Kim, L. Geng, K.R. Sekhar, Y.J. Gi, B. Lu, Inhibition of mammalian target of rapamycin or apoptotic pathway induces autophagy and radiosensitizes PTEN null prostate cancer cells, Cancer Res. 66 (2006) 10040–10047. [28] B. Ravikumar, Z. Berger, C. Vacher, C.J. O'Kane, D.C. Rubinsztein, Rapamycin pretreatment protects against apoptosis, Hum. Mol. Genet. 15 (2006) 1209–1216. [29] G.O. Ivy, F. Schottler, J. Wenzel, M. Baudry, G. Lynch, Inhibitors of lysosomal enzymes: accumulation of lipofuscin-like dense bodies in the brain, Science 226 (1984) 985–987. [30] W. Bursch, The autophagosomal–lysosomal compartment in programmed cell death, Cell Death Differ. 8 (2001) 569–581. [31] H. Sheng, J. Shao, J.D. Morrow, R.D. Beauchamp, R.N. DuBois, Modulation of apoptosis and Bcl-2 expression by prostaglandin E2 in human colon cancer cells, Cancer Res. 58 (1998) 362–366. [32] S. Lanza-Jacoby, A.P. Dicker, S. Miller, F.E. Rosato, J.T. Flynn, S.N. Lavorgna, R. Burd, Cyclooxygenase (COX)-2-dependent effects of the inhibitor SC236 when combined with ionizing radiation in mammary tumor cells derived from HER-2/ neu mice, Mol. Cancer Ther. 3 (2004) 417–424. [33] R. Hanif, A. Pittas, Y. Feng, M.I. Koutsos, L. Qiao, L. Staiano-Coico, S.I. Shiff, B. Rigas, Effects of nonsteroidal anti-inflammatory drugs on proliferation and on induction of apoptosis in colon cancer cells by a prostaglandin-independent pathway, Biochem. Pharmacol. 52 (1996) 237–245. [34] S.H. Kim, S.H. Song, S.G. Kim, K.S. Chun, S.Y. Lim, H.K. Na, J.W. Kim, Y.J. Surh, Y.J. Bang, Y.S. Song, Celecoxib induces apoptosis in cervical cancer cells independent of cyclooxygenase using NF-kappaB as a possible target, J. Cancer Res. Clin. Oncol. 130 (2004) 551–560. [35] I. Tegeder, J. Pfeilschifter, G. Geisslinger, Cyclooxygenase-independent actions of cyclooxygenase inhibitors, FASEB J. 15 (2001) 2057–2072. [36] L. Jia, R.R. Dourmashkin, P.D. Allen, A.B. Gray, A.C. Newland, S.M. Kelsey, Inhibition of autophagy abrogates tumour necrosis factor alpha induced apoptosis in human T-lymphoblastic leukaemic cells, Br. J. Haematol. 98 (1997) 673–685. [37] M. Gyrd-Hansen, T. Farkas, N. Fehrenbacher, L. Bastholm, M. Hoyer-Hansen, F. Elling, D. Wallach, R. Flavell, G. Kroemer, J. Nylandsted, M. Jaattela, Apoptosomeindependent activation of the lysosomal cell death pathway by caspase-9, Mol. Cell. Biol. 26 (2006) 7880–7891.

90

H.-S. Jeong et al. / Biochimica et Biophysica Acta 1813 (2011) 80–90

[38] K. Sato, U. Aytac, T. Yamochi, T. Yamochi, K. Ohnuma, K.S. McKee, C. Morimoto, N. H. Dang, CD26/dipeptidyl peptidase IV enhances expression of topoisomerase II alpha and sensitivity to apoptosis induced by topoisomerase II inhibitors, Br. J. Cancer 89 (2003) 1366–1374. [39] D.G. Stupack, T. Teitz, M.D. Potter, D. Mikolon, P.J. Houghton, V.J. Kidd, J.M. Lahti, D.A. Cheresh, Potentiation of neuroblastoma metastasis by loss of caspase-8, Nature 439 (2006) 95–99. [40] P. Fernando, S. Brunette, L.A. Megeney, Neural stem cell differentiation is dependent upon endogenous caspase 3 activity, FASEB J. 19 (2005) 1671–1673. [41] Y.T. Wu, H.L. Tan, Q. Huang, Y.S. Kim, N. Pan, W.Y. Ong, Z.G. Liu, C.N. Ong, H.M. Shen, Autophagy plays a protective role during zVAD-induced necrotic cell death, Autophagy 4 (2008) 457–466.

[42] E.R. Lee, Y.J. Kang, J.H. Kim, H.T. Lee, S.G. Cho, Modulation of apoptosis in HaCaT keratinocytes via differential regulation of ERK signaling pathway by flavonoids, J. Biol. Chem. 280 (2005) 31498–31507. [43] B.W. Kim, E.R. Lee, H.M. Min, H.S. Jeong, J.Y. Ahn, J.H. Kim, H.Y. Choi, H. Choi, E.Y. Kim, S.P. Park, S.G. Cho, Sustained ERK activation is involved in the kaempferolinduced apoptosis of breast cancer cells and is more evident under 3-D culture condition, Cancer Biol. Ther. 7 (2008) 1080–1089. [44] M.A. Kern, A.M. Haugg, A.F. Koch, T. Schilling, K. Breuhahn, H. Walczak, B. Fleischer, C. Trautwein, C. Michalski, H. Schulze-Bergkamen, H. Friess, W. Stremmel, P.H. Krammer, P. Schirmacher, M. Muller, Cyclooxygenase-2 inhibition induces apoptosis signaling via death receptors and mitochondria in hepatocellular carcinoma, Cancer Res. 66 (2006) 7059–7066.