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A Crucial Role of Caspase 3 and Caspase 8 in Paclitaxel-Induced Apoptosis
Molecular Cell Biology Research Communications 2, 36 – 41 (1999) Article ID mcbr.1999.0146, available online at http://www.idealibrary.com on
A Crucial Role of Caspase 3 and Caspase 8 in Paclitaxel-Induced Apoptosis Haruki Oyaizu,* ,† Yasushi Adachi,* Shigeru Taketani,‡ Rikio Tokunaga,‡ Shirou Fukuhara,† and Susumu Ikehara* ,1 *First Department of Pathology, †First Department of Internal Medicine, and ‡Department of Hygiene, Kansai Medical University, Moriguchi, Osaka, Japan, 570-0055
Received June 16, 1999
reports about the mechanism underlying apoptosis induced by paclitaxel, in which not only the activation of p34 cdc2 kinase and cyclin B1 kinase but also the inactivation of bcl-2 secondary to drug-induced phosphorylation are described [4 – 6]. Caspase 3, designated as CPP32 or Apopain, is a key protease involved in Fasmediated apoptosis [7], and caspase 3 has been reported as the downstream enzyme of caspase 8 [8, 9]. The cleavage of DFF45/ICAD by caspase 3 results in the activation of CAD, which degradates DNA [10]. The role of caspase 3, caspase 8, DFF45/ICAD and mitochondrial transmembrane potential (Dc m) in paclitaxelinduced apoptosis remains to be clarified. In this report, we investigate the correlation of mitosis arrest and apoptosis in various concentrations of paclitaxel, and the mechanism underlying apoptosis induced by paclitaxel.
The anticancer drug paclitaxel is well known as an inhibitor of microtubule depolymerization, resulting in mitosis arrest. We investigated the mechanism underlying antitumor effects of paclitaxel on the lung adenocarcinoma cell line LC-2-AD. Less than 10 mg/ml paclitaxel induced mitosis arrest upon LC-2-AD, followed by apoptosis, but more than 30 mg/ml paclitaxel induced apoptosis without mitosis arrest. LC-2-AD with less than 1 mg/ml paclitaxel showed a loss of mitochondrial transmembrane potential (Dc m), which correlated with antitumor effects. However, LC-2-AD with more than 10 mg/ml paclitaxel showed slight changes in the loss of Dc m in spite of its ability to induce apoptosis significantly. The cleavage of caspase 3, caspase 8, and DFF45/ICAD was also observed in paclitaxel-induced apoptosis, and the inhibitor of caspase 3 and caspase 8 inhibited both antitumor effects and apoptosis induced by paclitaxel. These results suggest that activation of caspase 3 and caspase 8 plays a crucial role in paclitaxel-induced apoptosis under any concentrations of paclitaxel.
MATERIALS AND METHODS Reagents and antibodies. Paclitaxel (Taxol) was kindly donated by Bristol–Myers Squibb (Tokyo, Japan). Anti-Fas antibody (clone CH-11), FITC-labeled anti-Fas antibody (clone UB-2), anti-DFF45/ICAD, DEVD-FMK (caspase 3 inhibitor) and IETD-FMK (caspase 8 inhibitor) were obtained from MBL (Nagoya, Japan). Anti-caspase 3 antibody and anti-caspase 8 antibody were obtained from Pharmingen (San Diego, CA). HRP-labeled anti-mouse IgG, HRP-labeled antirabbit IgG, and PVDF membrane were purchased from Bio-Rad Laboratories (Hercules, CA).
© 1999 Academic Press
Key Words: paclitaxel; lung cancer; caspase; apoptosis; ICAD.
The anticancer drug paclitaxel (Taxol), which is an alkaloid isolated from the pacific yew Taxus brevifolia, is generally used as an effective anti-tumor agent for the therapy of lung, head, ovarian and breast cancers [1]. Paclitaxel is an inhibitor of microtubule depolymerization, stabilizing the spindle and causing mitotic arrest at the metaphase [2]. Paclitaxel also has the ability to induce apoptotic cell death [3]. There are several
Cell culture. LC-2-AD, a human lung cancer cell line, was obtained from Japanese Cancer Resources Cell Bank. LC-2-AD was adjusted to 2 3 10 5 cells/ml and cultured in RPMI 1640 (Nikken Biological Laboratories, Kyoto, Japan) containing 10% fetal calf serum (FCS) with or without paclitaxel. For an inhibition assay, 2 3 10 5 cells/ml LC-2-AD were cultured with or without the caspase inhibitor at the concentration of 10
Abbreviations used: DFF45, DNA fragmentation factor 45; CAD, caspase 3-activated death factor; ICAD, inhibitor of CAD; PI, propidium iodide; ED, effective dose; MTT, 3-(4,5-dimethyl-thiazole2yl)-2,5-diphenyl tetrazolium bromide. 1 To whom correspondence should be addressed. Fax: 81-6-69948283. E-mail: [email protected]. 1522-4724/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Anti-tumor effect of paclitaxel upon lung cancer cell line, LC-2-AD. (A) 2 3 10 5/ml LC-2-AD was cultured for 72 h with or without various concentrations of paclitaxel. TetraColor One was added to the cultured cells, and incubated at 37°C in a CO 2 incubator for 1 h, followed by the measurement of OD with a microplate reader. Results shown are representative data of two individual studies. (B) Representative data of PI staining pattern of LC-2-AD. 2 3 10 5/ml LC-2-AD were cultured with various concentrations of paclitaxel for a maximum of 72 h. Results shown are representative data of five individual studies. (C) Representative data of CMXRos staining pattern of LC-2-AD. 2 3 10 5/ml LC-2-AD, which were cultured with various concentrations of paclitaxel for 24 h, were stained with CMXRos to detect the loss of mitochondrial transmembrane potential. Results shown are representative data of two individual studies. 37
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FIG. 2. Activities of caspase 3 and caspase 8 induced by paclitaxel. 2 3 10 5/ml LC-2-AD was cultured for 24, 48, or 72 h with 0.1 mg/ml (E), 1 mg/ml (h), 10 mg/ml (‚), and 30 mg/ml (3) of paclitaxel or without paclitaxel (■). The activities of caspase 3 (A) and caspase 8 (B) were estimated, as described under Materials and Methods. Results shown are representative data of two individual studies.
mM for 1 h, and the cells were cultured with or without 10 mg/ml paclitaxel.
Measurement of activity of caspase 8 and caspase 3. For evaluation of caspase 8 activity, we used the “FLICE/Caspase 8 Colorimetric Protease Assay Kits” (MBL, Nagoya, Japan), while for estimation of caspase 3 activity, we used the “Cpp32/Caspase 3 Colorimetric Protease Assay Kits” (MBL, Nagoya, Japan). The main peptide amino acid sequences of “FLICE/Caspase 8 Colorimetric Protease Assay Kits” and “Cpp32/Caspase 3 Colorimetric Protease Assay Kits” are IETD and DEVD, respectively.
Estimation of living cell numbers. The number of living cells was estimated using TetraColor One (Seikagaku Co., Tokyo, Japan), as previously described [11]. Briefly, LC-2-AD was cultured in 96-well culture plates (Corning, NY) with or without Taxol. After incubation, TetraColor One was added to cultured cells 1 h before measurement of OD with microplate reader (Bio-Rad Laboratories).
Western blotting. Cells were harvested and lysed in lysis buffer. The samples were sonicated and boiled. Proteins were adjusted and analyzed by SDS–polyacrylamide gel electrophoresis, and then transferred to a polyvinylidene difluoride membrane. Immunoblotting was performed using monoclonal antibodies (antiFLICE/Caspase 8 and anti-DFF45/ICAD) or polyclonal antibody (anti-CPP32/Caspase 3).
DNA fragmentation analyses. DNA fragmentation analysis were performed, as described previously [12]. Cytofluorometric analyses of mitochondrial parameter. To measure the Dc m, a cationic lipophilic fluorochrome, chloromethyl X-rosamine (CMXRos) (Molecular Probes, Eugene, OR) was utilized, as previously described [13]. Estimation of percentage apoptosis using PI staining. Cultured cells were harvested by using 0.05% trypsin, washed in PBS and suspended in 70% ethanol at 4°C for 1 h, followed by resuspension in 500 ml PBS, 250 ml RNase (1 mg/ml, Sigma Chemicals, St. Louis, MO) and 250 ml propidium iodide (100 mg/ml, Molecular Probes). The percentage of apoptotic cells was measured using the sub G0/G1 peak in PI staining.
RESULTS AND DISCUSSION Apoposis induced by paclitaxel. Since paclitaxel has been reported to have antitumor activity, we estimated its antitumor effect on the human lung adenocarcinoma cell line, LC-2-AD, by measuring living cell numbers using TetraColor One [11] (Fig. 1A). The antitumor effect of paclitaxel appeared from 0.001 mg/ml, 38
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FIG. 3. Western blot analyses for caspase 3, caspase 8, and DFF45/ICAD. LC-2-AD was harvested after 72 hour-culture period with various concentrations of paclitaxel (A), and was harvested at 8, 24, 48, and 72 h after stimulation with 10 mg/ml paclitaxel (B). Western blot analyses were performed with anti-caspase 3, anti-caspase 8 and anti-DFF45/ICAD, as described under Materials and Methods. Results shown are representative data of two individual studies.
reaching a plateau from 0.01 mg/ml to 10 mg/ml, which is around ED 50. Therefore, this cell line is relatively more resistant to paclitaxel than other cell lines [5, 14]. More than 10 mg/ml paclitaxel showed a significantly stronger antitumor effect. Previously, it has been reported that paclitaxel induces apoptosis in several cancer cell lines [15–17], while TetraColor One assay, which, like the MTT assay, indicates only living cell numbers, can not differentiate apoptosis from growth inhibition. Therefore, to confirm that paclitaxel induces apoptosis of LC-2-AD, we performed DNA analyses and PI staining. 180 –200 base pair DNA fragmentations were observed at various concentrations of paclitaxel (data not shown). Since it is difficult to estimate to what degree apoptosis occurs using DNA fragmentation analyses, we next performed PI staining in which the sub-G0/G1 population was estimated as apoptosis (Fig. 1B). Less than 10 mg/ml paclitaxel augmented G2/M arrested cancer cells, reduced G0/G1 cells, and induced apoptosis. Apoptotic cells increased slightly in a dose-dependent manner in these concentrations. In more than 30 mg/ml paclitaxel, however, the G2/M arrested cells of LC2-AD decreased, and the apoptotic cells increased significantly as compared with less than 10 mg/ml paclitaxel, especially on day 3. These results suggest that the mechanism underlying apoptosis induction of less than 10 mg/ml paclitaxel could be different from that in
more than 30 mg/ml paclitaxel. Therefore, we examined the disruption of mitochondrial transmembrane potential (Dc m), which has been reported to precede nuclear apoptosis and to locate the upstream of caspases [13] (Fig. 1C). Interestingly, more than 30% of LC-2-AD with 0.01 to 1 mg/ml paclitaxel showed the loss of Dc m, while less than 10% of LC-2-AD with more than 10 mg/ml paclitaxel showed the loss of Dc m in spite of significant % apoptosis. Activation of caspase 3 and caspase 8. To examine the signal transduction pathway of apoptosis leading to DNA fragmentation, we estimated the activity of caspase 3 and caspase 8 (Fig. 2). Caspase 8 has been reported to be cleaved by the activated CD95 deathinducing signaling complex and to change to the active form [18]. It has been reported that activated caspase 8 cleaves caspase 3 to the active form [19], and that activated caspase 3 cleaves DFF45/ICAD, resulting in activation of CAD [14,20]. The activities of caspase 3 and caspase 8 in LC-2-AD under the stimulation of paclitaxel increased in time- and dose-dependent manners. Even though low percentages of apoptosis were shown in less than 1 mg/ml paclitaxel on day 1, the activities of caspase 3 and 8 were already detected. Degradation of caspase 3, caspase 8, and DFF45/ ICAD. To confirm the cleavages of caspases, western blot analyses were performed. It has been reported 39
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that both caspase 3 and caspase 8 are cleaved into smaller active forms [18 –20]. We also examined the expression of DFF45/ICAD, since it has been shown that the active form of caspase 3 cleaves DFF45/ICAD and inactivates its CAD-inhibitory effect [19, 20]. First, we fixed the analysis date on day 3 when apoptosis was clearly defined, and applied various concentrations of paclitaxel to LC-2-AD (Fig. 3A). The expression of caspase 3, caspase 8, and DFF45/ICAD decreased slightly from 0.1 to 3 mg/ml paclitaxel, but decreased dramatically with more than 10 mg/ml paclitaxel. Next, we fixed the analysis dosage at 10 mg/ml, and were performed time course analysis. As shown in Fig. 3B, the expression of caspase 3 and DFF45/ICAD remained unchanged until day 2 of culture, but both decreased significantly on day 3. On the other hand, the expression of caspase 8 began to decrease from 2 day of culture. These results were compatible with previous reports in which caspase 8 locates the upstream of caspase 3 [8, 9]. To confirm the effect of caspases on paclitaxelinduced apoptosis and its anti-tumor ability, we performed an inhibition assay using caspase-specific inhibitors (Fig. 4). DEVD-FMK and IETD-FMK are well known as the caspase 3 and caspase 8 inhibitors, respectively. Both DEVD-FMK and IETD-FMK inhibited the anti-tumor effect, and the percentage apoptosis of paclitaxel was reduced to almost the paclitaxeluntreated level. These results suggest that caspases 3 and 8 play a critical role in the induction of apoptosis by paclitaxel. No correlation between paclitaxel-induced apoptosis and signaling through Fas. It was previously reported that paclitaxel augments CD95 ligand-induced apoptosis of malignant glioma cells [14]. Therefore, we examined whether paclitaxel upregulates Fas and whether paclitaxel augments Fas-induced apoptosis. The Fas-expression of LC-2-AD treated with paclitaxel remained unchanged as compared with untreated controls (data not shown). The ratios of apoptotic cells remained unchanged between the untreated controls and anti-Fas-treated LC-2-AD, and between only paclitaxel-treated LC-2-AD and paclitaxel 1 anti-Fas Ab-treated LC-2-AD (data not shown). These results suggest that paclitaxel neither upregulates the Fasexpression nor upregulates the sensitivity to Fasstimulation in our system. In this study, we examined the involvement of caspase 3 and 8 in the effects of paclitaxel on the lung adenocarcinoma cell line, LC-2-AD. The low concentration of paclitaxel-induced apoptosis of LC-2-AD through G2/M arrest, and significant numbers of cells showed low Dc m, whereas high concentrations of paclitaxel induced apoptosis without G2/M arrest, and small numbers of cells showed low Dc m. Both caspase 3 and caspase 8 were activated when apoptosis was in-
FIG. 4. Inhibition assay for anti-tumor effect and apoptosis inducing ability of paclitaxel using caspase-specific inhibitors. (A) 2 3 10 5/ml LC-2-AD was cultured with or without DEVD-FMK or IETDFMK, which are inhibitors for caspase 3 and caspase 8, respectively. DEVD-FMK and IETD-FMK were dissolved in DMSO, and the same volume of DMSO was then added in control wells. After 2 h, a final concentration (10 mg/ml) of paclitaxel was added and the cells were cultured for another 48 h. After culture, cell numbers were estimated using TetraColor One. Results shown are representative data of two individual studies. N.S., not significant. (B) After 2 3 10 5/ml LC2-AD was cultured with or without DEVD-FMK or IETD-FMK for 2 h, a final concentration (10 mg/ml) of paclitaxel was added. The cells were cultured for another 72 h. Cultured cells were harvested, and PI staining was performed to estimate percentage apoptosis. Results shown are representative data of two individual studies.
duced in LC-2-AD by paclitaxel. Caspase 3, caspase 8, and DFF45/ICAD were degradated and activated by paclitaxel in accordance with augmentation of apoptotic cells. Apoptotic cells, which paclitaxel induced, were reduced by the inhibitors of caspase 3 or caspase 8. These results strongly suggest that caspase 3 and caspase 8 play a central role in paclitaxel-induced apoptosis. Milross et al. described that the antitumor effect of paclitaxel correlated with paclitaxel-induced apoptosis 40
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but not with mitotic arrest in the in vivo experiment [17]. They concluded that apoptosis could be an important mechanism underlying cell death in response to paclitaxel treatment. In our data, the inhibitor of caspase 3 or caspase 8 significantly reduced apoptotic cells. Simultaneously, they reduced the anti-tumor effects of paclitaxel. Our in vitro results support the in vivo data of Milross et al. and suggest that DNA fragmentation followed by the activation of caspase 3 and caspase 8, the degradation of DFF45/ICAD and the activation of CAD play a crucial role in apoptosis induction. The activation of cyclin B1 kinase and inactivation of bcl-2 secondary to drug-induced phosphorylation through the activation of MAP kinase have been described as playing an important role in Taxol-induced mitotic arrest and apoptosis [5, 6]. It has also been described that caspases directly inactivate DFF45/ ICAD, followed by the activation of CAD, which digests DNA. Therefore, cyclin B1 kinase and MAP kinase could locate the upstream enzyme of the caspase system. Ling et al. reported that all taxol-induced cellular effects were abrogated by both the protein synthesis inhibitor, cycloheximid, and the RNA synthesis inhibitor, actinomycin D [5]. The caspase-CAD system is believed not to need new protein synthesis, because caspase and ICAD already exist and their activation occurs as a result of degradation. These phenomena suggest that other factors also exist upstream of the caspase–CAD system, but that they do not correlate with the Fas–Fas ligand system, at least in our system. In conclusion, caspase 3 and caspase 8 play a crucial role in apoptosis induction by paclitaxel on the human lung adenocarcinoma cell line, LC-2-AD, and the apoptosis induction correlates with the antitumor effect of paclitaxel.
REFERENCES 1. McGuire, W. P., Rowinsky, E. K., Rosenheim, N. B., Grumbine, F. C., Ettinger, D. S., Amstrong, D. K., and Donehower, R. C. (1998) Ann. Intern. Med. 111, 273–279. 2. Tishler, R. B., Geard, C. R., Hall, E. J., and Schiff, P. B. (1992) Cancer Res. 52, 3495–3497. 3. Milross, C. G., Mason, K. A., Hunter, N. R., Chung, W., Peter, L. J., and Milas, L. (1996) J. Natl. Cancer Inst. 88, 1308 –1314. 4. Donaldson, K. L., Goolsby, G., Kiener, P. A., and Wahl, A. F. (1994) Cell Growth Differ. 5, 1041–1050. 5. Ling, Y-H., Consoli, U., Tronos, C., Andreeff, M., and PerezSoler, R. (1998) Int. J. Cancer 75, 925–932. 6. Blagosklonny, M. V., Schulte, T., Nguyen, P., Trepel, J., and Neckers, L. M. (1996) Cancer Res. 56, 1851–1854. 7. Schlegel, J., Peters, I., Orrenius, S., Miller, D. K., Thornberry, N. A., Yamin, T. T., and Nicholson, D. W. (1996) J. Biol. Chem. 271, 1841–1844. 8. Fulda, S., Susin, S. A., Kroemer, G., and Debatin, K-D. (1998) Cancer Res. 58, 4453– 4460. 9. Li, H., Zhu, H., Xu, C-J., and Yuan, J. (1998) Cell 94, 491–500. 10. Sakahira, H., Enari, M., and Nagata, S. (1998) Nature 391, 96 –99. 11. Ishiyama, M., Miyazono, M., Sasamoto, K., Ohkura, Y., and Ueno, K. (1997) Talanta 44, 1299 –1305. 12. Gaucher, E. R., Piche, L., Lemieux, G., and Lemieux, R. (1996) Cancer Res. 56, 1451–1456. 13. Castedo, M., Hirsh, T., Susin, S. A., Zamzami, N., Marchetti, P., Macho, A., and Kroemer, G. (1996) J. Immunol. 157, 512–521. 14. Roth, W., Wagenknecht, B., Grimmel, C., Dichgans, J., and Weller, M. (1998) Br. J. Cancer 77, 404 – 411. 15. Ling, Y., Yang, Y., Tornos, C., Singh, B., and Perez-Soler, R. (1998) Cancer Res. 58, 3633–3640. 16. Milross, C. G., Peters, L. J., Hunter, N. R., Mason, K. A., and Milas, L. (1995) Int. J. Cancer 35, 297–303. 17. Medema, J. P., Scaffidi, C., Kischkel, F. C., Shevchenko, A. N. J., Mann, M., Krammer, P. H., and Peter, M. E. (1997) EMBO J. 14, 5579 –5588. 18. Stennicke, H. R., Jurgensmeier, J. M., Shin, H., Deveraux, Q., Wolf, B. B., Yang, X., Zhou, Q., Ellerby, H. M., Ellerby, L. M., Beredesen, D., Green, D. R., Reed, J. C., Froelich, C. J., and Salvesen, G. S. (1998) J. Biol. Chem. 273, 27084 –27090. 19. Sakahira, H., Enari, M., and Nagata, S. (1998) Nature 391, 96 –99. 20. Enari, M., Sakahira, H., Yokoyama, H., Iwamatsu, A., and Nagata, S. (1998) Nature 391, 43–50.
ACKNOWLEDGMENTS The authors thank Ms. Mari Shinkawa and Ms. Yoko Tokuyama for their expert technical assistance and Ms. Keiko Ando for her help in the preparation of the manuscript. This work was supported by a Grant-in-Aid for Scientific Research of Japan (09877051).
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A Crucial Role of Caspase 3 and Caspase 8 in Paclitaxel-Induced Apoptosis Haruki Oyaizu,* ,† Yasushi Adachi,* Shigeru Taketani,‡ Rikio Tokunaga,‡ Shirou Fukuhara,† and Susumu Ikehara* ,1 *First Department of Pathology, †First Department of Internal Medicine, and ‡Department of Hygiene, Kansai Medical University, Moriguchi, Osaka, Japan, 570-0055
Received June 16, 1999
reports about the mechanism underlying apoptosis induced by paclitaxel, in which not only the activation of p34 cdc2 kinase and cyclin B1 kinase but also the inactivation of bcl-2 secondary to drug-induced phosphorylation are described [4 – 6]. Caspase 3, designated as CPP32 or Apopain, is a key protease involved in Fasmediated apoptosis [7], and caspase 3 has been reported as the downstream enzyme of caspase 8 [8, 9]. The cleavage of DFF45/ICAD by caspase 3 results in the activation of CAD, which degradates DNA [10]. The role of caspase 3, caspase 8, DFF45/ICAD and mitochondrial transmembrane potential (Dc m) in paclitaxelinduced apoptosis remains to be clarified. In this report, we investigate the correlation of mitosis arrest and apoptosis in various concentrations of paclitaxel, and the mechanism underlying apoptosis induced by paclitaxel.
The anticancer drug paclitaxel is well known as an inhibitor of microtubule depolymerization, resulting in mitosis arrest. We investigated the mechanism underlying antitumor effects of paclitaxel on the lung adenocarcinoma cell line LC-2-AD. Less than 10 mg/ml paclitaxel induced mitosis arrest upon LC-2-AD, followed by apoptosis, but more than 30 mg/ml paclitaxel induced apoptosis without mitosis arrest. LC-2-AD with less than 1 mg/ml paclitaxel showed a loss of mitochondrial transmembrane potential (Dc m), which correlated with antitumor effects. However, LC-2-AD with more than 10 mg/ml paclitaxel showed slight changes in the loss of Dc m in spite of its ability to induce apoptosis significantly. The cleavage of caspase 3, caspase 8, and DFF45/ICAD was also observed in paclitaxel-induced apoptosis, and the inhibitor of caspase 3 and caspase 8 inhibited both antitumor effects and apoptosis induced by paclitaxel. These results suggest that activation of caspase 3 and caspase 8 plays a crucial role in paclitaxel-induced apoptosis under any concentrations of paclitaxel.
MATERIALS AND METHODS Reagents and antibodies. Paclitaxel (Taxol) was kindly donated by Bristol–Myers Squibb (Tokyo, Japan). Anti-Fas antibody (clone CH-11), FITC-labeled anti-Fas antibody (clone UB-2), anti-DFF45/ICAD, DEVD-FMK (caspase 3 inhibitor) and IETD-FMK (caspase 8 inhibitor) were obtained from MBL (Nagoya, Japan). Anti-caspase 3 antibody and anti-caspase 8 antibody were obtained from Pharmingen (San Diego, CA). HRP-labeled anti-mouse IgG, HRP-labeled antirabbit IgG, and PVDF membrane were purchased from Bio-Rad Laboratories (Hercules, CA).
© 1999 Academic Press
Key Words: paclitaxel; lung cancer; caspase; apoptosis; ICAD.
The anticancer drug paclitaxel (Taxol), which is an alkaloid isolated from the pacific yew Taxus brevifolia, is generally used as an effective anti-tumor agent for the therapy of lung, head, ovarian and breast cancers [1]. Paclitaxel is an inhibitor of microtubule depolymerization, stabilizing the spindle and causing mitotic arrest at the metaphase [2]. Paclitaxel also has the ability to induce apoptotic cell death [3]. There are several
Cell culture. LC-2-AD, a human lung cancer cell line, was obtained from Japanese Cancer Resources Cell Bank. LC-2-AD was adjusted to 2 3 10 5 cells/ml and cultured in RPMI 1640 (Nikken Biological Laboratories, Kyoto, Japan) containing 10% fetal calf serum (FCS) with or without paclitaxel. For an inhibition assay, 2 3 10 5 cells/ml LC-2-AD were cultured with or without the caspase inhibitor at the concentration of 10
Abbreviations used: DFF45, DNA fragmentation factor 45; CAD, caspase 3-activated death factor; ICAD, inhibitor of CAD; PI, propidium iodide; ED, effective dose; MTT, 3-(4,5-dimethyl-thiazole2yl)-2,5-diphenyl tetrazolium bromide. 1 To whom correspondence should be addressed. Fax: 81-6-69948283. E-mail: [email protected]. 1522-4724/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Anti-tumor effect of paclitaxel upon lung cancer cell line, LC-2-AD. (A) 2 3 10 5/ml LC-2-AD was cultured for 72 h with or without various concentrations of paclitaxel. TetraColor One was added to the cultured cells, and incubated at 37°C in a CO 2 incubator for 1 h, followed by the measurement of OD with a microplate reader. Results shown are representative data of two individual studies. (B) Representative data of PI staining pattern of LC-2-AD. 2 3 10 5/ml LC-2-AD were cultured with various concentrations of paclitaxel for a maximum of 72 h. Results shown are representative data of five individual studies. (C) Representative data of CMXRos staining pattern of LC-2-AD. 2 3 10 5/ml LC-2-AD, which were cultured with various concentrations of paclitaxel for 24 h, were stained with CMXRos to detect the loss of mitochondrial transmembrane potential. Results shown are representative data of two individual studies. 37
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FIG. 2. Activities of caspase 3 and caspase 8 induced by paclitaxel. 2 3 10 5/ml LC-2-AD was cultured for 24, 48, or 72 h with 0.1 mg/ml (E), 1 mg/ml (h), 10 mg/ml (‚), and 30 mg/ml (3) of paclitaxel or without paclitaxel (■). The activities of caspase 3 (A) and caspase 8 (B) were estimated, as described under Materials and Methods. Results shown are representative data of two individual studies.
mM for 1 h, and the cells were cultured with or without 10 mg/ml paclitaxel.
Measurement of activity of caspase 8 and caspase 3. For evaluation of caspase 8 activity, we used the “FLICE/Caspase 8 Colorimetric Protease Assay Kits” (MBL, Nagoya, Japan), while for estimation of caspase 3 activity, we used the “Cpp32/Caspase 3 Colorimetric Protease Assay Kits” (MBL, Nagoya, Japan). The main peptide amino acid sequences of “FLICE/Caspase 8 Colorimetric Protease Assay Kits” and “Cpp32/Caspase 3 Colorimetric Protease Assay Kits” are IETD and DEVD, respectively.
Estimation of living cell numbers. The number of living cells was estimated using TetraColor One (Seikagaku Co., Tokyo, Japan), as previously described [11]. Briefly, LC-2-AD was cultured in 96-well culture plates (Corning, NY) with or without Taxol. After incubation, TetraColor One was added to cultured cells 1 h before measurement of OD with microplate reader (Bio-Rad Laboratories).
Western blotting. Cells were harvested and lysed in lysis buffer. The samples were sonicated and boiled. Proteins were adjusted and analyzed by SDS–polyacrylamide gel electrophoresis, and then transferred to a polyvinylidene difluoride membrane. Immunoblotting was performed using monoclonal antibodies (antiFLICE/Caspase 8 and anti-DFF45/ICAD) or polyclonal antibody (anti-CPP32/Caspase 3).
DNA fragmentation analyses. DNA fragmentation analysis were performed, as described previously [12]. Cytofluorometric analyses of mitochondrial parameter. To measure the Dc m, a cationic lipophilic fluorochrome, chloromethyl X-rosamine (CMXRos) (Molecular Probes, Eugene, OR) was utilized, as previously described [13]. Estimation of percentage apoptosis using PI staining. Cultured cells were harvested by using 0.05% trypsin, washed in PBS and suspended in 70% ethanol at 4°C for 1 h, followed by resuspension in 500 ml PBS, 250 ml RNase (1 mg/ml, Sigma Chemicals, St. Louis, MO) and 250 ml propidium iodide (100 mg/ml, Molecular Probes). The percentage of apoptotic cells was measured using the sub G0/G1 peak in PI staining.
RESULTS AND DISCUSSION Apoposis induced by paclitaxel. Since paclitaxel has been reported to have antitumor activity, we estimated its antitumor effect on the human lung adenocarcinoma cell line, LC-2-AD, by measuring living cell numbers using TetraColor One [11] (Fig. 1A). The antitumor effect of paclitaxel appeared from 0.001 mg/ml, 38
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FIG. 3. Western blot analyses for caspase 3, caspase 8, and DFF45/ICAD. LC-2-AD was harvested after 72 hour-culture period with various concentrations of paclitaxel (A), and was harvested at 8, 24, 48, and 72 h after stimulation with 10 mg/ml paclitaxel (B). Western blot analyses were performed with anti-caspase 3, anti-caspase 8 and anti-DFF45/ICAD, as described under Materials and Methods. Results shown are representative data of two individual studies.
reaching a plateau from 0.01 mg/ml to 10 mg/ml, which is around ED 50. Therefore, this cell line is relatively more resistant to paclitaxel than other cell lines [5, 14]. More than 10 mg/ml paclitaxel showed a significantly stronger antitumor effect. Previously, it has been reported that paclitaxel induces apoptosis in several cancer cell lines [15–17], while TetraColor One assay, which, like the MTT assay, indicates only living cell numbers, can not differentiate apoptosis from growth inhibition. Therefore, to confirm that paclitaxel induces apoptosis of LC-2-AD, we performed DNA analyses and PI staining. 180 –200 base pair DNA fragmentations were observed at various concentrations of paclitaxel (data not shown). Since it is difficult to estimate to what degree apoptosis occurs using DNA fragmentation analyses, we next performed PI staining in which the sub-G0/G1 population was estimated as apoptosis (Fig. 1B). Less than 10 mg/ml paclitaxel augmented G2/M arrested cancer cells, reduced G0/G1 cells, and induced apoptosis. Apoptotic cells increased slightly in a dose-dependent manner in these concentrations. In more than 30 mg/ml paclitaxel, however, the G2/M arrested cells of LC2-AD decreased, and the apoptotic cells increased significantly as compared with less than 10 mg/ml paclitaxel, especially on day 3. These results suggest that the mechanism underlying apoptosis induction of less than 10 mg/ml paclitaxel could be different from that in
more than 30 mg/ml paclitaxel. Therefore, we examined the disruption of mitochondrial transmembrane potential (Dc m), which has been reported to precede nuclear apoptosis and to locate the upstream of caspases [13] (Fig. 1C). Interestingly, more than 30% of LC-2-AD with 0.01 to 1 mg/ml paclitaxel showed the loss of Dc m, while less than 10% of LC-2-AD with more than 10 mg/ml paclitaxel showed the loss of Dc m in spite of significant % apoptosis. Activation of caspase 3 and caspase 8. To examine the signal transduction pathway of apoptosis leading to DNA fragmentation, we estimated the activity of caspase 3 and caspase 8 (Fig. 2). Caspase 8 has been reported to be cleaved by the activated CD95 deathinducing signaling complex and to change to the active form [18]. It has been reported that activated caspase 8 cleaves caspase 3 to the active form [19], and that activated caspase 3 cleaves DFF45/ICAD, resulting in activation of CAD [14,20]. The activities of caspase 3 and caspase 8 in LC-2-AD under the stimulation of paclitaxel increased in time- and dose-dependent manners. Even though low percentages of apoptosis were shown in less than 1 mg/ml paclitaxel on day 1, the activities of caspase 3 and 8 were already detected. Degradation of caspase 3, caspase 8, and DFF45/ ICAD. To confirm the cleavages of caspases, western blot analyses were performed. It has been reported 39
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that both caspase 3 and caspase 8 are cleaved into smaller active forms [18 –20]. We also examined the expression of DFF45/ICAD, since it has been shown that the active form of caspase 3 cleaves DFF45/ICAD and inactivates its CAD-inhibitory effect [19, 20]. First, we fixed the analysis date on day 3 when apoptosis was clearly defined, and applied various concentrations of paclitaxel to LC-2-AD (Fig. 3A). The expression of caspase 3, caspase 8, and DFF45/ICAD decreased slightly from 0.1 to 3 mg/ml paclitaxel, but decreased dramatically with more than 10 mg/ml paclitaxel. Next, we fixed the analysis dosage at 10 mg/ml, and were performed time course analysis. As shown in Fig. 3B, the expression of caspase 3 and DFF45/ICAD remained unchanged until day 2 of culture, but both decreased significantly on day 3. On the other hand, the expression of caspase 8 began to decrease from 2 day of culture. These results were compatible with previous reports in which caspase 8 locates the upstream of caspase 3 [8, 9]. To confirm the effect of caspases on paclitaxelinduced apoptosis and its anti-tumor ability, we performed an inhibition assay using caspase-specific inhibitors (Fig. 4). DEVD-FMK and IETD-FMK are well known as the caspase 3 and caspase 8 inhibitors, respectively. Both DEVD-FMK and IETD-FMK inhibited the anti-tumor effect, and the percentage apoptosis of paclitaxel was reduced to almost the paclitaxeluntreated level. These results suggest that caspases 3 and 8 play a critical role in the induction of apoptosis by paclitaxel. No correlation between paclitaxel-induced apoptosis and signaling through Fas. It was previously reported that paclitaxel augments CD95 ligand-induced apoptosis of malignant glioma cells [14]. Therefore, we examined whether paclitaxel upregulates Fas and whether paclitaxel augments Fas-induced apoptosis. The Fas-expression of LC-2-AD treated with paclitaxel remained unchanged as compared with untreated controls (data not shown). The ratios of apoptotic cells remained unchanged between the untreated controls and anti-Fas-treated LC-2-AD, and between only paclitaxel-treated LC-2-AD and paclitaxel 1 anti-Fas Ab-treated LC-2-AD (data not shown). These results suggest that paclitaxel neither upregulates the Fasexpression nor upregulates the sensitivity to Fasstimulation in our system. In this study, we examined the involvement of caspase 3 and 8 in the effects of paclitaxel on the lung adenocarcinoma cell line, LC-2-AD. The low concentration of paclitaxel-induced apoptosis of LC-2-AD through G2/M arrest, and significant numbers of cells showed low Dc m, whereas high concentrations of paclitaxel induced apoptosis without G2/M arrest, and small numbers of cells showed low Dc m. Both caspase 3 and caspase 8 were activated when apoptosis was in-
FIG. 4. Inhibition assay for anti-tumor effect and apoptosis inducing ability of paclitaxel using caspase-specific inhibitors. (A) 2 3 10 5/ml LC-2-AD was cultured with or without DEVD-FMK or IETDFMK, which are inhibitors for caspase 3 and caspase 8, respectively. DEVD-FMK and IETD-FMK were dissolved in DMSO, and the same volume of DMSO was then added in control wells. After 2 h, a final concentration (10 mg/ml) of paclitaxel was added and the cells were cultured for another 48 h. After culture, cell numbers were estimated using TetraColor One. Results shown are representative data of two individual studies. N.S., not significant. (B) After 2 3 10 5/ml LC2-AD was cultured with or without DEVD-FMK or IETD-FMK for 2 h, a final concentration (10 mg/ml) of paclitaxel was added. The cells were cultured for another 72 h. Cultured cells were harvested, and PI staining was performed to estimate percentage apoptosis. Results shown are representative data of two individual studies.
duced in LC-2-AD by paclitaxel. Caspase 3, caspase 8, and DFF45/ICAD were degradated and activated by paclitaxel in accordance with augmentation of apoptotic cells. Apoptotic cells, which paclitaxel induced, were reduced by the inhibitors of caspase 3 or caspase 8. These results strongly suggest that caspase 3 and caspase 8 play a central role in paclitaxel-induced apoptosis. Milross et al. described that the antitumor effect of paclitaxel correlated with paclitaxel-induced apoptosis 40
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but not with mitotic arrest in the in vivo experiment [17]. They concluded that apoptosis could be an important mechanism underlying cell death in response to paclitaxel treatment. In our data, the inhibitor of caspase 3 or caspase 8 significantly reduced apoptotic cells. Simultaneously, they reduced the anti-tumor effects of paclitaxel. Our in vitro results support the in vivo data of Milross et al. and suggest that DNA fragmentation followed by the activation of caspase 3 and caspase 8, the degradation of DFF45/ICAD and the activation of CAD play a crucial role in apoptosis induction. The activation of cyclin B1 kinase and inactivation of bcl-2 secondary to drug-induced phosphorylation through the activation of MAP kinase have been described as playing an important role in Taxol-induced mitotic arrest and apoptosis [5, 6]. It has also been described that caspases directly inactivate DFF45/ ICAD, followed by the activation of CAD, which digests DNA. Therefore, cyclin B1 kinase and MAP kinase could locate the upstream enzyme of the caspase system. Ling et al. reported that all taxol-induced cellular effects were abrogated by both the protein synthesis inhibitor, cycloheximid, and the RNA synthesis inhibitor, actinomycin D [5]. The caspase-CAD system is believed not to need new protein synthesis, because caspase and ICAD already exist and their activation occurs as a result of degradation. These phenomena suggest that other factors also exist upstream of the caspase–CAD system, but that they do not correlate with the Fas–Fas ligand system, at least in our system. In conclusion, caspase 3 and caspase 8 play a crucial role in apoptosis induction by paclitaxel on the human lung adenocarcinoma cell line, LC-2-AD, and the apoptosis induction correlates with the antitumor effect of paclitaxel.
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ACKNOWLEDGMENTS The authors thank Ms. Mari Shinkawa and Ms. Yoko Tokuyama for their expert technical assistance and Ms. Keiko Ando for her help in the preparation of the manuscript. This work was supported by a Grant-in-Aid for Scientific Research of Japan (09877051).
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