Clinical Cancer Research Prevention Award Advances in Breast Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Huisman, C.
Right arrow Articles by Giaccone, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Huisman, C.
Right arrow Articles by Giaccone, G.
Clinical Cancer Research Vol. 8, 596-606, February 2002
© 2002 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Paclitaxel Triggers Cell Death Primarily via Caspase-independent Routes in the Non-Small Cell Lung Cancer Cell Line NCI-H4601

Cynthia Huisman2, Carlos G. Ferreira2, Linda E. Bröker, Jose A. Rodriguez, Egbert F. Smit, Pieter E. Postmus, Frank A. E. Kruyt and Giuseppe Giaccone3

Departments of Medical Oncology [C. H., C. G. F., L. E. B., J. A. R., F. A. E. K., G. G.] and Pulmonary Diseases [C. H., E. F. S., P. E. P.], University Hospital Vrije Universiteit, 1081 HV Amsterdam, the Netherlands


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Here we report on the role of mitochondria, death receptors (DRs), and caspases in exerting the cytotoxic effect of clinically relevant concentrations of paclitaxel in the non-small cell lung cancer cell line NCI-H460.

Experimental Design: We have characterized paclitaxel-induced cell death with annexin V, propidium iodide staining, and poly(ADP-ribose) polymerase cleavage assays. The involvement of the mitochondria pathway was studied by monitoring cytochrome c release and using H460 cells stable in overexpressing Bcl-2 or Bcl-xL. DR dependency was analyzed in FADD dominant-negative or cytokine response modifier A-overexpressing cells, and a possible role for DR4 and DR5 was investigated by antagonistic antibodies. Caspase activity and cleavage assays and treatment with the synthetic inhibitor zVAD-fmk were used to determine the involvement of caspases.

Results: Paclitaxel-treated cells displayed several features of apoptosis, including annexin V staining and poly(ADP-ribose) polymerase cleavage. The sequence of events suggested the involvement of a DR, as indicated by an early role for Fas-associated death domain and caspase-8, followed by cleavage of Bid and the disruption of mitochondria; nonetheless, we failed to demonstrate the involvement of DR4 and DR5. Interestingly, inhibition of either one of these routes only resulted in a 30% reduction of cell death that was in line with the observed small effect of caspase inhibition by zVAD-fmk on H460 cell survival.

Conclusion: Paclitaxel triggers cell death in H460 cells mainly via a currently unidentified caspase-independent mechanism in which the basic apoptotic machinery is merely coactivated. This finding is in sharp contrast with the largely caspase-dependent response elicited by DNA-damaging agents in these cells. We speculate on therapeutic implications.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemotherapy is an established treatment for advanced NSCLC.4 Anticancer agents exert at least part of their cell killing effects by inducing apoptosis (1) . The taxane paclitaxel is a widely used anticancer agent for the treatment of NSCLC. Paclitaxel stabilizes microtubules and, as a result, interferes with the dynamic changes that occur during the formation of the mitotic spindle, leading to mitotic arrest and subsequent induction of cell death (2) . Its action is different from other anticancer drugs, such as the DNA-damaging agents, and distinct mechanisms underlying the induction of apoptosis and drug resistance have been suggested (3) . Currently, the molecular mechanism by which paclitaxel-induced mitotic arrest leads to apoptosis is not clear, although evidence for the involvement of several signaling pathways has been shown, including the action of various protein kinases such as the mitogen-activated protein kinases, serine/threonine kinase-dependent phosphorylation of Bcl-2, and the p53 pathway (reviewed in Ref. 4 ). Phosphorylation of Bcl-2 appears to be a hallmark of cell death induced by paclitaxel, but the correlation between this event, mitotic arrest, and apoptosis remains controversial. Initial reports suggested that phosphorylation of Bcl-2 leads to inactivation of its antiapoptotic function (reviewed in Ref. 5 ); however, recent data demonstrated that Bcl-2 phosphorylation is a marker of mitotic arrest rather than a determinant of apoptosis (6 , 7) . p53 also seems to be dispensable for paclitaxel-induced apoptosis (8 , 9) , which is in agreement with the observation that clinical response rates to treatment with paclitaxel have been shown to be similar between patients with wild-type or mutant p53 cancers (10) . As noted by others, the variation in the concentration of paclitaxel used in studies aimed to analyze the mechanism underlying cell death triggered by this agent hampers the comparison and the interpretation of the results (11 , 12) .

Two major cellular apoptotic pathways have been identified that mediate apoptosis on exposure to different types of stimuli, such as anticancer drugs, ionizing radiation, and viruses (13) . One route, which is activated by the majority of anticancer drugs, involves the disruption of mitochondria that leads to the release of cytochrome c, which can trigger the activation of caspase-9 in a complex with Apaf-1 and the presence of ATP. Subsequently, additional caspases are activated that take care of the execution phase of the apoptotic process, causing the degradation of cellular proteins and the disassembly of the cell (14) . The Bcl-2 family of proteins play a key role in regulating mitochondria stability, and the antiapoptotic members Bcl-2 and Bcl-xL have been extensively characterized; the antiapoptotic function of Bcl-2 and Bcl-xL is opposed by apoptosis-promoting family members such as Bax (15) . The pro- and antiapoptotic Bcl-2 homologues can form heterodimers via the so-called Bcl-2 homology domain 3, thereby regulating each other’s activity and, thus, mitochondria integrity. The other apoptosis-mediating route is activated by the ligation of DRs, which belong to the tumor necrosis factor receptor gene superfamily and share a homologous cytoplasmatic sequence termed the death domain that enables engagement of the apoptotic machinery (16) . Among the best-characterized ligands that activate DRs are FasL and TRAIL that bind to FasR/CD95 or DR4 and DR5, respectively. On activation, the DRs recruit the adapter molecule FADD by the death domain that is also present on FADD, followed by the activation of caspase-8 (17 , 18) . The mitochondria route and the DR pathway are linked via the proapoptotic Bcl-2 family member Bid, which can be cleaved by active caspase-8 and subsequently stimulate the release of cytochrome c from mitochondria (19 , 20) .

In the present study we have examined the contribution of these apoptotic routes to the cell killing effect of physiologically relevant concentrations of paclitaxel in the NSCLC cell line H460. Interestingly, these pathways that form the basic apoptotic machinery in cells played only a minor role in triggering paclitaxel-induced cell death. Nevertheless, the sequence of events that started with partial activation of the DR pathway followed by disruption of mitochondria resembles that of the response induced by DR ligands. The response induced by paclitaxel is clearly different from that induced by DNA-damaging agents that is mediated primarily via mitochondria and caspases (21) . On the basis of these findings, we propose that paclitaxel kills H460 cells mainly via an as yet unknown caspase-independent mechanism, possibly in an uncharacterized connection with the DR pathway, and that the observed coactivation of caspases occurs rather as a bystander effect.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Drugs.
Drugs were provided as pure substances. Paclitaxel (Bristol-Myers Squibb, Woerden, The Netherlands) was diluted in ethanol. For each experiment, the stock solutions of the drugs were freshly diluted in culture medium to the indicated final concentration. All of the experiments were performed with IC80 values of paclitaxel, corresponding to clinically relevant concentrations: 50 nM for H460, 10 nM for SW1573, 120 nM for H322, 12 nM for GLC4, and 45 nM for Jurkat cells. The TRAIL/Apo2L protein, the anti-DR4(huTR1-M271)- and anti-DR5(huTRAILR2-M413)-specific mAbs were received from Genentech, Inc. under a joint MTA with Genentech, Inc. and Immunex Corp. The broad-spectrum caspase inhibitor zVAD-fmk was diluted to 20 mM in DMSO and added the cells at a final concentration of 50 µM 1 h before addition of paclitaxel.

Cell Lines and Transfectants.
The human NSCLC cell lines NCI-H460 (H460), SW1573, NCI-H322 (H322), the SCLC cell line GLC4, and Jurkat T-leukemia cells were used in the experiments. Cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FCS (Life Technologies, Inc., Breda, The Netherlands), 2 mM L-glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin, and grown at 37°C in a humidified atmosphere with 5% CO2. Cells from exponentially growing cultures were used in all of the experiments. Stable transfectants of H460 cells expressing Bcl-2, Bcl-xL, CrmA-WT, and FADD-DN were described previously (21) . These transfected cell lines were cultured in medium containing a final concentration of 1.5 µg/ml puromycin (Bcl-2, Bcl-xL, and CrmA-WT) or 200 µg/ml neomycin (FADD-DN), respectively. The cell lines were tested regularly for the absence of Mycoplasma infection.

Cell Death Measurement.
Cells were plated at a density of 0.5 x 106 cells in six-well plates (Costar, Cambridge, MA) 24 h before treatment. Cells were incubated for 8 to 96 h with paclitaxel. For apoptosis experiments with TRAIL/Apo2L and paclitaxel, drugs were added in the presence or absence of antagonistic anti-DR4- and anti-DR5-specific mAbs. The analysis of apoptosis was performed as described previously (21) . Briefly, the extent of cell death was determined by PI staining of hypodiploid DNA or by annexin V-FITC and 7-AAD double staining. For the PI staining, 3 x 105 cells were resuspended in Nicoletti buffer (50 µg/ml propidium iodide (Sigma Chemicals, St. Louis, MO), 0.1% sodium citrate, 0.1% Triton X-100 and 1 mg/ml RNA-seA (Roche, Basel, Switzerland) in PBS) as described before (21) and analyzed by FACScan (Becton Dickinson, Mountainview, CA). The fraction of cells with sub-G1 DNA content was assessed using the Lysis program (Becton Dickinson). Annexin V staining was performed according to the manufacturer’s protocol (Nexins Research, Kattendijke, the Netherlands). After incubation with annexin V, 10 µl of 7-AAD (PharMingen, San Diego, CA) was added, and analysis was performed on FACScalibur using CELLQuest software (Becton Dickinson). The percentage of specific apoptosis was calculated by subtracting the percentage of spontaneous apoptosis of the relevant controls from the total percentage of apoptosis. For morphological studies, apoptosis was evaluated either by fluorescent microscopic analysis of fragmented nuclei stained with Hoechst 33342 or by light microscopy of cytospin preparations stained with May-Grünwald Giemsa.

Detection of Expression of DR4 and DR5.
Cells were stained with DR4- and DR5-specific mAbs for 45 min at 4°C. Isotype-matched nonbinding antibodies (DAKO, Santa Barbara, CA) were used to control for nonspecific binding. The equivalent to 1 µg of protein of DR4 and DR5 antibodies or the respective control antibody was used in each sample. After the incubation with the primary antibody, cells were washed twice with cold PBS and incubated with FITC secondary antibody at the dilution 1:50 at 4°C in the dark for 30 min. Two new steps of washing with cold PBS were performed before the cells were analyzed by FACScalibur using CELLQuest software. The same instrument settings were used for all of the experiments, and 5000 events were analyzed. MFI ratio was defined as the MFI of gated live cells stained with anti-Fas:MFI of cells stained with isotype-matched antibody.

Electrophoresis and Western Blotting.
Western blot analysis was performed essentially as described previously (22) . In brief, from each sample, 25 µg of protein/lane were separated on 8–15% SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes (Amersham, Braunschweig, Germany). Subsequently, membranes were incubated overnight at 4°C in a solution containing PBS supplemented with 5% nonfat dry milk. For immunodetection, the following antibodies were used: anti-Bcl-2 mAb (Dako); anticytochrome c mAb and anticaspase-3 polyclonal antibody (both from PharMingen, San Diego, CA); anticaspase-8 mAb (Immunotech, Marseille, France); anti-Bcl-xL polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit polyclonal anti-PARP (Boehringer Mannheim, Germany); and rabbit polyclonal anti-Bid (provided by Dr. Xiadong Wang). After a 2-h incubation with the primary antibody in dilutions that ranged from 1:500 to 1:2000, membranes were washed in tris-buffered saline [10 mM Tris-HCl (pH 8.0), 0.15 M NaCl, and 0.05% Tween 20] followed by horseradish peroxidase-conjugated goat-antimouse or goat-antirabbit antibody. Enhanced chemiluminescence (Amersham) was used for detection, and protein expression was quantified by densitometry of autoradiographs (Model GS-690 imaging densitometer; Bio-Rad, Richmond, CA). Protein loading equivalence was corrected in relation to the expression of ß-actin.

Preparation of Cytosol for Measurement of Cytochrome c.
Cytosolic extracts were prepared essentially as described before (21) . In brief, cells were pelleted by centrifugation after a washing step with ice-cold PBS. Subsequently, cell pellets were resuspended in 2 volumes of buffer A [50 mM Tris, 1 mM EGTA, 5 mM ß-mercaptoethanol, 0.2% BSA, 10 mM KH2PO4 (pH 7.6), and 0.4 M sucrose], incubated for 20 min on ice, and disrupted by 20 passages through a 26-gauge needle. Cell extracts were clarified of mitochondria by centrifugation at 20,000 x g for 30 min at 4°C. The harvested cytosolic extracts were stored at -80°C and thawed on ice before use.

Fluorimetric Assay for Caspase Activity.
Caspase-8- and caspase-3-like enzyme activity was assayed in cells using caspase-8 and caspase-3 activity kits (Clontech Laboratories Inc., Palo Alto, CA) according to the manufacturer’s instructions. Relative percentage of activity, as measured by DEVD-AFC and IETD-AFC cleavage, respectively, was determined by comparing the levels of treated cells with untreated controls.

Statistics.
Quantitative experiments were analyzed by the Student’s t test. All of the Ps resulted from the use of two-sided tests and were considered significant when <0.05.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Paclitaxel Induces Cell Death in H460 Cells.
The NSCLC cell line H460 has been extensively characterized previously in our laboratory in regard to its sensitivity to drug- and Fas-induced apoptosis (21 , 22) . For all of the experiments, IC80 concentrations corresponding to clinically relevant concentrations were used. H460 cells were incubated with 50 nM of paclitaxel for 16, 24, and 48 h, stained with PI, and analyzed by FACS. Cell cycle distribution at these time points showed accumulation of the cells in the G2-M phases (Fig. 1A)Citation . Approximately 7, 17, and 30%, respectively, of the cells showed a sub-G1 DNA content, indicative of apoptosis (Fig. 1, A and B)Citation . These results were confirmed by annexin V-FITC 7-AAD double staining assays, showing the induction of apoptosis already after 8 h of treatment with paclitaxel (Fig. 1B)Citation . Morphological features of cells treated with paclitaxel included rounding up, loss of contact with neighboring cells, and detachment from plate. Cytospin preparations demonstrated membrane blebbing, shrinkage, and nuclear condensation, whereas Hoechst 33342 confirmed nuclear condensation and fragmentation (data not shown), consistent with previous reports (12 , 23) .



View larger version (23K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Paclitaxel induces apoptosis in H460 cells. A, H460 cells were treated with IC80 concentrations of paclitaxel for sequential duration (16, 24, and 48 h), and PI-labeled cells were analyzed by flow cytometry, demonstrating accumulation of the cells in the G2-M phase and the development of a sub-G1-population. Representative figures of multiple experiments are shown. B, H460 cells were exposed to IC80 concentrations of paclitaxel, and apoptosis was assessed by PI and annexin V-FITC 7-AAD double staining at the indicated time points. The results are represented as the mean of at least three independent experiments; bars, ± SD. C, Western blot analysis showing cleavage of PARP on treatment with paclitaxel at the indicated hours after treatment.

 
As a marker of activation of caspases after treatment, the cleavage of the nuclear protein PARP was determined by monitoring the occurrence of the Mr 89,000 cleaved product by Western blotting. PARP cleavage became evident at 24 h after treatment (Fig. 1C)Citation , and increased cleavage was observed after 48 h. These results indicate that paclitaxel-induced cell death has several characteristics of apoptotic death.

Mitochondria Play a Minor Role in Paclitaxel-induced Cell Death.
The role of mitochondria in mediating cell death in H460 cells exposed to paclitaxel was investigated by analyzing both mitochondria stability as determined by the release of cytochrome c and by studying the involvement of different Bcl-2 family members in this process. As shown in Fig. 2ACitation , the release of cytochrome c by Western blotting in cytosolic extracts derived from paclitaxel-treated H460 cells could not be detected earlier than 48 h after treatment. In addition, paclitaxel treatment resulted in phosphorylation of Bcl-2, characteristic of microtubule-interacting agents, most clearly visible at 16 and 24 h after exposure to the drug as indicated by the occurrence of Bcl-2-specific bands with lower mobility in the gel (Fig. 2B)Citation . Bcl-xL phosphorylation was not detected, and the level of expression remained unchanged (Fig. 2B)Citation . To additionally examine the role of antiapoptotic Bcl-2 and Bcl-xL, we exploited previously generated and characterized H460 cells stably overexpressing these proteins in which the apoptosis triggering effect of DNA damaging agents such as cisplatin and topotecan was strongly reduced (21) . Interestingly, the overexpression of these proteins resulted in only a partial reduction of apoptotic cells by 25% for Bcl-2 (P = 0.15) and 30% for Bcl-xL (P = 0.17) after exposure to paclitaxel (Fig. 2C)Citation . Moreover, in time course experiments, the partial inhibitory effect of Bcl-2 and Bcl-xL on apoptosis occurred no earlier than after an exposure time of 48 h, which is in line with the observed cytochrome c release at the same time post-treatment in the parental cells (Fig. 2A)Citation . Overexpression of Bcl-2 was effective in preventing the release of cytochrome c, additionally indicating the functional activity of Bcl-2 in these cells (Fig. 2D)Citation . Thus, these results show that the role of mitochondria in mediating the cell killing effect of paclitaxel is limited, indicating that alternative mechanisms of cell death must be involved.



View larger version (30K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. The role of mitochondria in paclitaxel-induced apoptosis is minor and occurs only at later stages. A, cytochrome c (Cyt-c) release from mitochondria was determined in cytosolic extracts at 16, 24, and 48 h after treatment with IC80 concentrations of paclitaxel by Western blotting. B, Bcl-2 and Bcl-xL expression in paclitaxel-treated H460 cells. Slower migrating Bcl-2-specific bands represent phosphorylated Bcl-2 variants. C, level of paclitaxel-induced apoptosis in H460 cells stably overexpressing Bcl-2, Bcl-xL, or the empty vector. After 16, 24, and 48 h of exposure, the percentage of apoptotic cells was analyzed by PI staining. The results are represented as the mean of at least three independent experiments; bars, ± SD. D, cytochrome c release assays in H460 cells stably overexpressing Bcl-2. Cytosolic extracts from cells harvested at the indicated time points were subjected to Western blotting with anticytochrome c mAb.

 
The Early Phase of Paclitaxel-induced Cell Death Resembles a DR-mediated Pathway.
The findings described above suggest that mitochondria play only a minor and secondary role in mediating the toxic effect of paclitaxel in H460 cells. Involvement of DRs in mediating chemotherapy-induced apoptosis has been described previously (24) , and we demonstrated recently the existence of alternative drug-induced routes in H460 cells that trigger DR-independent activation of caspase-8 via an as yet unidentified mechanism (21) . Therefore, we examined the possible contribution of the DR pathway to paclitaxel-induced cytotoxicity in H460 cells stably overexpressing FADD-DN that is able to interfere with the function of normal FADD as an adaptor molecule between DRs and caspase-8. This cell line was generated and characterized earlier (21) . In H460 cells overexpressing FADD-DN, cell death induced by paclitaxel at 16 h after treatment was reduced from 10% to 3.5% (P = 0.01). At later time points the apoptosis-protecting effect decreased, with a reduction from 17% to 9% after 24 h (P = 0.02) and a lack of apoptosis inhibition at 48 h (Fig. 3A)Citation . Next we used the CrmA derived from cowpox virus, which is a selective inhibitor of caspase-1 and -8 that functions downstream from FADD. Transfectants stably overexpressing CrmA (see Ref. 21 ) showed 2.3% of death cells at 16 h after treatment versus 7% in cells expressing the empty vector (P = 0.03). After 24 h these numbers were 7.7% versus 14%, respectively (P = 0.03). In line with the results obtained with FADD-DN, CrmA failed to protect at 48 h after exposure to paclitaxel (Fig. 3B)Citation .



View larger version (29K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Paclitaxel-induced mode of cell death resembles a death-receptor-mediated pathway. A, H460 cells transfected with FADD-DN or the empty vector were treated with paclitaxel and analyzed for apoptosis by PI staining. B, H460 cells transfected with the caspase-8 inhibitor CrmA or the empty vector were exposed to paclitaxel. After 16, 24, and 48 h, the percentage of apoptotic cells was determined by PI staining. All of the results depicted represent a mean of at least three independent experiments; bars, ± SD. C, Western blot analysis showing the cleavage of Bid on treatment with paclitaxel. ß-actin mAb was used as a control for the amount of protein loaded.

 
The results obtained thus far indicate that paclitaxel induces cell death in two phases, an early phase involving the action of FADD and a later phase in which mitochondria contribute to cell death. These two phases resemble the way in which DR ligands trigger apoptosis, involving the initial activation of caspase-8 and at a later step the activation of the Bcl-2-related protein Bid that is cleaved from a precursor by active caspase-8 and subsequently induces mitochondria instability and the release of cytochrome c. In H460 cells exposed to paclitaxel, cleavage of Bid was first detected at 32 h after treatment and increased to near complete cleavage at 48 h (Fig. 3C)Citation . The timing of this event parallels the involvement of FADD and caspase-8 in apoptosis induced by paclitaxel at early time points (16 h and 24 h), and the subsequent release of cytochrome c, which does not occur before 48 h of treatment.

The DRs DR4 and DR5 Do Not Mediate Paclitaxel-triggered Apoptosis.
We have shown previously that paclitaxel-induced cytotoxicity is not mediated by the Fas system (21) . However, other DRs can also act via FADD and may play a role in transducing the cytotoxic effect of paclitaxel. These include DR4 and DR5, which are membrane receptors for TRAIL/Apo2L (25, 26, 27) . The involvement of DRs in mediating chemotherapy-induced apoptosis has been proposed to occur via the up-regulation of receptor and/or ligand expression (24) . Assessment of DR4 and DR5 levels on H460 cells, as evaluated by FACS analysis using specific mAbs against the receptors, showed that DR4 is abundantly expressed, whereas levels of DR5 are lower (Fig. 4A)Citation . Apoptosis in H460 cells was readily induced by exposure to TRAIL/Apo2L, as depicted in Fig. 4BCitation ; 100 ng/ml TRAIL/Apo2L caused a time-dependent increase in the level of apoptotic cells with an onset at 4 h after treatment. These results indicate the functionality of this pathway in H460 cells. Thus, we investigated the possibility that the early phase of paclitaxel-induced cytotoxicity could be mediated by the TRAIL-receptor pathway. Antagonistic DR4 and/or DR5 mAbs were used to study the cell killing effect of paclitaxel in the absence of a functional DR4/DR5 pathway. As a control for the antagonistic effects, first the potential of anti-DR4 and -DR5 were tested in blocking TRAIL/Apo2L-induced apoptosis, which appeared to be almost complete in case of anti-DR4 (Fig. 4C)Citation , whereas anti-DR5 had hardly any effect (data not shown). These results are in agreement with the relative low levels of DR5 expression on these cells. We then additionally studied the potential role of DR4 in mediating the toxic effects of paclitaxel. Antagonistic DR4 antibodies were added 1 h before the addition of paclitaxel, and apoptosis was determined after 16 h, the time point at which the protection provided by FADD-DN was most significant. In contrast to the nearly complete blockade of TRAIL/Apo2L-induced apoptosis, anti-DR4 failed to protect against paclitaxel-induced cell death (Fig. 4C)Citation . This result was not affected by varying the concentration of the antibody (10 or 20 µg/ml) or exposure time to paclitaxel (data not shown). In addition, the level of expression of these DRs during paclitaxel-induced apoptosis was examined. Table 1Citation shows that paclitaxel did not induce an increase in the protein levels of DR4 and DR5 when compared with the levels of untreated controls. Taken together, these observations indicate that the early phase of paclitaxel-induced cell death in H460 cells involves FADD; however, we failed to identify a DR that is responsible for transducing the signal via FADD.



View larger version (22K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. The TRAIL receptors DR4 and DR5 are not involved in mediating paclitaxel-induced apoptosis in H460 cells. A, the level of constitutional expression of DR4 and DR5 in H460 cells was analyzed by flow cytometry with specific antibodies (····). Isotype-matched antibodies were used to control for unspecific binding. Data were statistically analyzed for histogram plots using CellQuest software as described (see "Materials and Methods"). B, representative time course experiment demonstrating the susceptibility of H460 cells to TRAIL/Apo2L-induced apoptosis (100 ng/ml). At the indicated time points, the percentage of apoptotic cells was analyzed by PI staining and compared with untreated cells. C, effect of antagonizing DR4 mAb on TRAIL/Apo2L- and paclitaxel-induced apoptosis. H460 cells were incubated with anti-DR4 1 h before the addition of TRAIL/Apo2L or paclitaxel, and apoptotic cells were monitored after 16 h by PI staining assays. A representative experiment out of three independent experiments is shown.

 

View this table:
[in this window]
[in a new window]

  		Table 1 Relative expression of DR4 and DR5 on paclitaxel-treated H460 cells

DR4 and DR5 levels were determined at the indicated time points after paclitaxel treatment, and are depicted relative to the untreated controls (MFI). The values represent the mean of three or more independent experiments ± SD.

 
zVAD-fmk Blocks Caspase Activation but Does Not Prevent Paclitaxel-induced Cell Death.
Our results indicate that the two major apoptotic pathways only play a minor role in paclitaxel-induced cell death. To additionally substantiate these findings, we studied the contribution of caspases to the cell-killing effects of paclitaxel in more detail. Exposure of H460 cells to 50 nM of paclitaxel for 24 h and 48 h resulted in an increase of caspase-8-like and especially caspase-3-like protease activity as compared with untreated control cells (Fig. 5A)Citation and was accompanied by cleavage of the proforms of caspase-8 as well as caspase-3 to their active products (Fig. 5B)Citation . Caspase-9 was not studied here, because we showed previously that this caspase is not activated by chemotherapeutic drugs in H460 cells (21) . The processing of cleaved forms of caspase-8 and -3 could be blocked by pretreatment of the cells with 50 µM of the broad-spectrum caspase inhibitor zVAD-fmk (Fig. 5B)Citation . Likewise, pretreatment with zVAD-fmk was effective in preventing PARP cleavage (Fig. 5C)Citation .



View larger version (51K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Paclitaxel-induced cell death in H460 occurs independent of caspase activation. A, caspase-8- and -3-like protease activity on exposure to 50 nM paclitaxel for the indicated hours, as measured by DEVD-AFC and IETD-AFC cleavage, respectively. The changes in activity are shown relative to control cell activity, which is set as 1. B, cleavage of caspase-8 and -3 in H460 cells was detected by Western blotting after culturing the cells in the presence (+) or absence (-) of IC80 concentrations of paclitaxel for 24 h and 48 h after (+) or without (-) pretreatment of 50 µM of zVAD-fmk. Cleaved products of caspase-8 (top panel) and -3 (bottom panel) are indicated. C, cleavage of PARP, detected under similar conditions as the caspase cleavage assays described under B. D, H460 cells were exposed to IC80 concentrations of paclitaxel and compared with cells pretreated with 50 µM of zVAD-fmk. Apoptosis was assessed by PI staining at the indicated time points. E, H460 cells stably overexpressing Bcl-2 or the empty vector control were exposed to IC80 concentrations of paclitaxel for 48 h in the presence or absence of 50 µM of zVAD-fmk. Apoptosis was assessed by PI staining. F, the experiments described under D were extended to the NSCLC cell lines SW1573 and H322 as well as SCLC cell line GLC4 and compared with Jurkat cells. The exposure time to paclitaxel was 48 h. All results are represented as the mean of at least three independent experiments; bars, ± SD.

 
Next, we evaluated the effect of caspase inhibition on paclitaxel-induced cell death. In line with the observations pointing at a minor role for DR and mitochondria pathways, pretreatment with zVAD-fmk hardly affected paclitaxel-induced cell death (Fig. 5D)Citation . At 16 h and 24 h after treatment, cells exposed to paclitaxel had a similar percentage of apoptotic cells as cells pretreated with zVAD-fmk, whereas after 48 h, 72 h, and 96 h, zVAD-fmk reduced the percentage of apoptosis only from 34.5% to 30.5%, from 38% to 32%, and from 33% to 28.5%, respectively. Morphologically, cells pretreated with zVAD-fmk could not be distinguished from cells exposed to paclitaxel alone, because they still displayed apoptotic features such as nuclear condensation and apoptotic bodies (data not shown).

In addition, to study the effect of combined inhibition of the mitochondrial and caspase pathways on paclitaxel cytotoxicity, H460 cells stably overexpressing Bcl-2 were treated with zVAD-fmk. Paclitaxel-triggered cell death was somewhat reduced on zVAD-fmk treatment, from 26% to 18% at 48 h after treatment, whereas the empty vector control cells showed a minor reduction from 32% to 27% (Fig. 5E)Citation , indicating that simultaneous inhibition of the mitochondria pathway and caspases results in a slightly increased but still incomplete protection against paclitaxel-induced cytotoxicity. Finally, we investigated whether the lack of effect of zVAD-fmk on paclitaxel-induced cell death would also apply to other lung cancer cell lines. The NSCLC cell lines SW1573 and H322, and the SCLC cell line GLC4 were exposed to their respective IC80 concentrations of paclitaxel for 48 h and compared with cells pretreated with zVAD-fmk. Jurkat T-leukemia cells were used as control cells in these experiments, because leukemic and lymphoid cells are known to be more apoptosis-prone than most solid tumors (28) . As shown in Fig. 5FCitation , zVAD-fmk showed a similar inability to prevent paclitaxel-induced apoptosis in SW1573 and H322 as in H460 cells. In contrast, in Jurkat cells, zVAD-fmk provided a 75% protection, whereas in GLC4 a reduction from 24% to 16.5% was observed. Thus, we conclude that our findings of a caspase-independent mechanism of paclitaxel-induced cell death can likely be extrapolated to other NSCLC cell lines.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The molecular mechanisms involved in the mediation of cell death induced by the potent anticancer agent paclitaxel are only partially understood. In this study the role of mitochondria- and DR-dependent apoptosis was examined in the NSCLC cell line H460 on treatment with a clinically relevant concentration of paclitaxel. These two distinct routes were found to contribute at two different stages. In an early phase, at 16 to 24 h after treatment, FADD partially mediated paclitaxel-induced cytotoxicity, whereas at a later stage (48 h) the mitochondrial pathway enhanced the triggering of cell death. However, our results indicate that these apoptotic routes do not play an essential role in mediating paclitaxel-induced cell death as demonstrated by the finding that inhibition of these pathways by stable overexpression of FADD DN or Bcl-2 resulted only in a temporary protection until 24 h after exposure and in a 30% reduction in the fraction of death cells after 48 h, respectively. In line with this, the broad-spectrum caspase inhibitor zVAD-fmk did not substantially rescue the cells from cell death, indicating the existence of an alternative, caspase-independent mechanism that is instrumental for the cell killing effect of paclitaxel.

Treatment with paclitaxel elicited several events in H460 cells that have been found to be characteristic for this drug, such as G2-M arrest and the phosphorylation of Bcl-2. Recent studies have demonstrated that Bcl-2 phosphorylation is a marker of mitotic arrest rather than a determinant of apoptosis (6 , 7) . Therefore, phosphorylation of Bcl-2 at early time points in H460 cells cannot be considered as a key event in mediating cell death, consistent with the lack of effect observed with the overexpression of Bcl-2 and Bcl-xL. Our finding that the antiapoptotic Bcl-2 family members had only a small protecting effect at later stages of apoptosis in H460 cells is in contrast with reports that have indicated stronger suppression of apoptosis by Bcl-2 and Bcl-xL (29, 30, 31, 32, 33) . The use of higher concentrations of paclitaxel than in the current study as well as unknown cell line specific factors may account for this discrepancy.

The biphasic response to paclitaxel treatment that we found in H460 cells followed the sequence of a DR pathway: an early involvement of FADD (16–24 h), cleavage of Bid (32–48 h), and at later stages (48 h) an enhancing effect involving mitochondria. The ability of DRs to mediate chemotherapy-induced cytotoxicity, which has been proposed to occur via the up-regulation of receptor and/or ligand expression in treated cells (24) , is currently debated. Although several investigators have shown that the Fas/FasL system contributes to chemotherapy-induced apoptosis (reviewed in Ref. 34 ), genetic studies have demonstrated that the apoptotic response to treatment with cytotoxic drugs is largely intact in FADD knockout mice, arguing against the involvement of DRs (35 , 36) . In addition, the activation of caspase-8 in colon cancer cells and in B-lymphoma cells by paclitaxel was found to be independent of Fas/FasL (37 , 38) . We established previously that paclitaxel-induced cytotoxicity is not mediated by the Fas system (22) , and in the current study we assessed a possible role for DR4 and DR5, which are membrane receptors for TRAIL/Apo2L. Although H460 cells abundantly express DR4, which effectively mediates TRAIL-induced apoptosis in these cells, antagonistic antibodies did not prevent paclitaxel-triggered cell death. These experiments exclude a role of the TRAIL/Apo2L system in transducing the cytotoxic effect of paclitaxel.

Altogether, the contribution of these caspase-dependent routes to the cytotoxic effects triggered by paclitaxel was not essential. Although overexpression of CrmA rescued H460 cells from early paclitaxel-induced cell death, this protection was lost after 48 h of treatment. These findings are corroborated by the absence of substantial protection against paclitaxel-induced apoptosis provided by zVAD-fmk, implying the existence of caspase-independent routes. Moreover, a double antiapoptotic block in H460 cells, provided by overexpression of Bcl-2 in combination with pretreatment with zVAD-fmk, resulted in only a slightly increased and still incomplete protection against paclitaxel-induced cytotoxicity. Therefore, the activation of caspases on paclitaxel exposure, as established by caspase-activity assays and cleavage of proforms of caspase-8 and -3, is not instrumental for the execution of cell death and can be considered a bystander effect.

The observation of induction of caspase-independent pathways by paclitaxel is not unprecedented. Okano and Rustgi (12) reported recently that physiologically relevant concentrations of paclitaxel caused cell death via both caspase-dependent and -independent pathways in human esophageal squamous cancer cells. Other studies in H460 and H520 NSCLC cells found a more pronounced protective effect of zVAD-fmk against paclitaxel-induced apoptosis, although the authors mentioned that zVAD-fmk did not reverse paclitaxel-induced inhibition of cell growth but resulted in homeostasis (23) . The main cause for the difference with our findings probably is the use of a 10-fold higher concentration of paclitaxel than the one we applied in the current study. Activation of other effector caspases on paclitaxel-treatment, such as caspase-7, which has been reported previously (23) , was not directly evaluated in our study.

Whether apoptosis contributes to drug-induced cell death is a current controversy in the field (39) . Evidence has accumulated that apoptosis is not the main mediator of cell killing after anticancer therapy in solid tumors, in contrast to lymphomas and leukemias, although the specific triggering of apoptosis in tumor cells by molecular intervention is a promising novel anticancer approach that is currently being developed and tested in the clinic (40) . However, evolving nomenclature as well as the use of different assays to measure cell death has led to substantial increase of confusion in the field. ZVAD-fmk has been widely used to assess caspase-independent cell death but can also inhibit other noncaspase cysteine proteases (41) . Chemotherapeutic drugs often cause so-called "slow-cell death" without caspase activation, in which cells die in a protractive and inefficient manner but which may nevertheless be associated with apoptotic features (28) . In a recent review, four patterns of cell death mode are described with apoptosis and necrosis on opposite far ends. Apoptosis-like programmed cell death and necrosis-like programmed cell death are used to define the many cases of caspase-independent and specialized caspase-independent signaling pathways, respectively (42) . This knowledge substantially weakens the paradigmatic antithesis of apoptosis and necrosis (43) . The choice between apoptosis and slow cell death is cell-type dependent, and the availability of classical apoptotic pathways is usually more pronounced in lymphoid and leukemia cells than in solid tumors (28) . This is in line with our finding that in contrast to NSCLC cells, zVAD-fmk did rescue Jurkat cells from paclitaxel-induced apoptosis, again emphasizing the role of cell-specific factors in death responses. Because DR and mitochondria-independent pathways can be triggered in Jurkat cells as well, which has been shown recently for the sesquiterpene lactone helenalin (44) , stimulus-specific factors are also involved in determining the relative importance of different death mechanisms. It is likely that in many cell types multiple pathways leading to cell death are latently present (45) . Participation of other, noncaspase proteases, such as calpains and cathepsins, in chemotherapy-induced apoptosis has been reported (46, 47, 48, 49) . Whether similar mechanisms could account for paclitaxel-induced apoptosis in NSCLC cells remains to be demonstrated. Additional studies to delineate the signaling pathways triggered by paclitaxel in NSCLC cells leading to cell death are currently being carried out in our laboratory.

The results of our study support the notion that the mechanism underlying the induction of apoptosis by microtubule-interacting agents such as paclitaxel differs in some respects from that of other anticancer drugs (3) and could involve proapoptotic factors interacting with microtubules, such as Bim. However, Bim activation is abrogated by Bcl-2 overexpression (50) , which makes its involvement in paclitaxel-induced cell death unlikely. In particular, our findings provide a molecular basis for the distinct cytotoxic activities of paclitaxel in comparison with DNA-damaging agents, including cisplatin, gemcitabine, and topotecan. As depicted in Fig. 6Citation , apoptosis triggered by DNA-damaging agents is largely dependent on caspase-8 and the mitochondrial pathway (21) . In contrast, activation of caspases and mitochondria seems to be a mere bystander effect on paclitaxel treatment. In H460 cells, paclitaxel induces cell death via an as yet uncharacterized caspase-independent mechanism, which might be connected with FADD, whereas apoptosis induced by DNA-damaging agents does not involve FADD or DRs. These differences seem to be specific for NSCLC cells and could not be demonstrated for Jurkat cells, which suggests that in particular tumor types it is interesting to study the application of cell death mechanisms in relation to the response to chemotherapy and prognosis.



View larger version (35K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 6. Activation of distinct cell death pathways by DNA-damaging agents and paclitaxel in H460 cells. Whereas DNA-damaging agents clearly trigger apoptosis in H460 cells, paclitaxel induces cell death mainly via caspase-independent pathways. Activation of DR and mitochondria (mito) routes is not instrumental and should be considered a bystander effect.

 
The triggering of distinct apoptotic pathways by paclitaxel in NSCLC cells might have therapeutic implications, because our results were obtained with clinically relevant drug concentrations. The absence of an essential role for mitochondria and caspases in paclitaxel-induced apoptosis in H460 cells could have clinical consequences. Bcl-2 is abnormally expressed in 15–25% of NSCLCs (51 , 52) , and on the basis of the current study, this would not be relevant for the response to treatment with paclitaxel. Likewise, it can be concluded from our study that the expression of inhibitors of apoptosis on NSCLC tumors would not be predictive of response, which adds up to previous evidence that the relation between expression of inhibitors of apoptosis and chemosensitivity in NSCLC may be more complex than anticipated by in vitro data (53 , 54) . Finally, paclitaxel is used in several combination regimens, and the combination of paclitaxel and cisplatin or gemcitabine was found to be sequence dependent (55 , 56) . The current study may open up new ways to study the effect of combination therapy in relation to the apoptotic mechanism activated.


   ACKNOWLEDGMENTS
 
We thank Dr. David Lynch (Immunex Corp., Seattle, WA) for TRAIL/Apo2L and anti-DR4- and anti-DR5-specific mAbs and Dr. Xiadong Wang (University of Texas Southwestern Medical Center, Dallas, TX) for the Bid antibody. We also thank Prof. Paul van der Valk (Department of Pathology, University Hospital Vrije Universiteit, the Netherlands) for helpful discussions.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 C. H. is supported by a Dutch Cancer Society Grant. C. G. F. is supported by a personal grant from Capes (Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), Brasília, Brazil. Back

2 Both authors contributed equally to this work. Back

3 To whom requests for reprints should be addressed, at University Hospital Vrije Universiteit, Department of Medical Oncology, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. Phone: 31-20-444-4321; Fax: 31-20-444-4079; E-mail: g.giaccone{at}vumc.nl Back

4 The abbreviations used are: NSCLC, non-small cell lung cancer; DR, death receptor; TRAIL, tumor necrosis factor-related apoptosis inducing ligand; FADD, Fas-associated death domain; FADD-DN, dominant-negative Fas-associated death domain; FasL, Fas ligand; mAb, monoclonal antibody; CrmA, cytokine response modifier A; PARP, poly(ADP-ribose) polymerase; SCLC, small cell lung cancer; PI, propidium iodide; 7-AAD, 7-amino-actinomycin D; MFI, mean fluorescence intensity. Back

Received 8/27/01; revised 10/24/01; accepted 11/ 5/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Fisher D. E. Apoptosis in cancer therapy: crossing the threshold. Cell, 78: 539-542, 1994.[CrossRef][Medline]
  2. Jordan M. A., Toso R. J., Thrower D., Wilson L. Mechanism of mitotic block and inhibition of cell proliferation by taxol at low concentrations. Proc. Natl. Acad. Sci. USA, 90: 9552-9556, 1993.[Abstract/Free Full Text]
  3. O’Connor P. M., Jackman J., Bae I., Myers T. G., Fan S., Mutoh M., Scudiero D. A., Monks A., Sausville E. A., Weinstein J. N., Friend S., Fornace A. J. J., Kohn K. W. Characterization of the p53 tumor suppressor pathway in cell lines of the National Cancer Institute anticancer drug screen and correlations with the growth-inhibitory potency of 123 anticancer agents. Cancer Res., 57: 4285-4300, 1997.[Abstract/Free Full Text]
  4. Wang L. G., Liu X. M., Kreis W., Budman D. R. The effect of antimicrotubule agents on signal transduction pathways of apoptosis: a review. Cancer Chemother. Pharmacol, 44: 355-361, 1999.[CrossRef][Medline]
  5. Wang T. H., Wang H. S., Soong Y. K. Paclitaxel-induced cell death: where the cell cycle and apoptosis come together. Cancer (Phila.), 88: 2619-2628, 2000.[CrossRef][Medline]
  6. Fan M., Du L., Stone A. A., Gilbert K. M., Chambers T. C. Modulation of mitogen-activated protein kinases and phosphorylation of Bcl-2 by vinblastine represent persistent forms of normal fluctuations at G2–M1. Cancer Res., 60: 6403-6407, 2000.[Abstract/Free Full Text]
  7. Ling Y. H., Tornos C., Perez-Soler R. Phosphorylation of Bcl-2 is a marker of M phase events and not a determinant of apoptosis. J. Biol. Chem., 273: 18984-18991, 1998.[Abstract/Free Full Text]
  8. Bacus S. S., Gudkov A. V., Lowe M., Lyass L., Yung Y., Komarov A. P., Keyomarsi K., Yarden Y., Seger R. Taxol-induced apoptosis depends on MAP kinase pathways (ERK and p38) and is independent of p53. Oncogene, 20: 147-155, 2001.[CrossRef][Medline]
  9. Das G. C., Holiday D., Gallardo R., Haas C. Taxol-induced cell cycle arrest and apoptosis: dose-response relationship in lung cancer cells of different wild-type p53 status and under isogenic condition. Cancer Lett., 165: 147-153, 2001.[CrossRef][Medline]
  10. Safran H., King T., Choy H., Gollerkeri A., Kwakwa H., Lopez F., Cole B., Myers J., Tarpey J., Rosmarin A. p53 mutations do not predict response to paclitaxel/radiation for nonsmall cell lung carcinoma. Cancer (Phila.), 78: 1203-1210, 1996.[CrossRef][Medline]
  11. Blagosklonny M. V., Fojo T. Molecular effects of paclitaxel: myths and reality (a critical review). Int. J. Cancer, 83: 151-156, 1999.[Medline]
  12. Okano J., Rustgi A. K. Paclitaxel induces prolonged activation of the Ras/MEK/ERK pathway independently of activating the programmed cell death machinery. J. Biol. Chem., 276: 19555-19564, 2001.[Abstract/Free Full Text]
  13. Sun X. M., MacFarlane M., Zhuang J., Wolf B. B., Green D. R., Cohen G. M. Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis. J. Biol. Chem., 274: 5053-5060, 1999.[Abstract/Free Full Text]
  14. Earnshaw W. C., Martins L. M., Kaufmann S. H. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu. Rev. Biochem., 68: 383-424, 1999.[CrossRef][Medline]
  15. Green D. R. Apoptotic pathways: paper wraps stone blunts scissors. Cell, 102: 1-4, 2000.[CrossRef][Medline]
  16. Ashkenazi A., Dixit V. M. Apoptosis control by death and decoy receptors. Curr. Opin. Cell Biol., 11: 255-260, 1999.[CrossRef][Medline]
  17. Boldin M. P., Varfolomeev E. E., Pancer Z., Mett I. L., Camonis J. H., Wallach D. A novel protein that interacts with the death domain of Fas/APO1 contains a sequence motif related to the death domain. J. Biol. Chem., 270: 7795-7798, 1995.[Abstract/Free Full Text]
  18. Peter M. E. The TRAIL Discussion: it is FADD and caspase-8!. Cell Death Differ., 7: 759-760, 2000.[CrossRef][Medline]
  19. Li H., Zhu H., Xu C. J., Yuan J. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell, 94: 491-501, 1998.[CrossRef][Medline]
  20. Luo X., Budihardjo I., Zou H., Slaughter C., Wang X. Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell, 94: 481-490, 1998.[CrossRef][Medline]
  21. Ferreira C. G., Span S. W., Peters G. J., Kruyt F., Giaccone G. Chemotherapy triggers apoptosis in a caspase-8-dependent and mitochondria-controlled manner in non-small cell lung cancer cells. Cancer Res., 60: 7133-7141, 2000.[Abstract/Free Full Text]
  22. Ferreira C. G., Tolis C., Span S. W., Peters G. J., van Lopik T., Kummer A. J., Pinedo H. M., Giaccone G. Drug-induced apoptosis in lung cancer cells is not mediated by the Fas/FasL (CD95/APO1) signaling pathway. Clin. Cancer Res., 6: 203-212, 2000.[Abstract/Free Full Text]
  23. Ling Y., Zhong Y., Perez-Soler R. Disruption of cell adhesion and caspase-mediated proteolysis of ß- and {gamma}-catenins and APC protein in paclitaxel-induced apoptosis. Mol. Pharmacol., 59: 593-603, 2001.[Abstract/Free Full Text]
  24. Friesen C., Fulda S., Debatin K. M. Cytotoxic drugs and the CD95 pathway. Leukemia (Baltimore), 13: 1854-1858, 1999.[CrossRef][Medline]
  25. Ashkenazi A., Pai R. C., Fong S., Leung S., Lawrence D. A., Marsters S. A., Blackie C., Chang L., McMurtrey A. E., Hebert A., DeForge L., Koumenis I. L., Lewis D., Harris L., Bussiere J., Koeppen H., Shahrokh Z., Schwall R. H. Safety and antitumor activity of recombinant soluble Apo2 ligand. J. Clin. Investig., 104: 155-162, 1999.[Medline]
  26. Pitti R. M., Marsters S. A., Ruppert S., Donahue C. J., Moore A., Ashkenazi A. Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family. J. Biol. Chem., 271: 12687-12690, 1996.[Abstract/Free Full Text]
  27. Wiley S. R., Schooley K., Smolak P. J., Din W. S., Huang C. P., Nicholl J. K., Sutherland G. R., Smith T. D., Rauch C., Smith C. A. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity, 3: 673-682, 1995.[CrossRef][Medline]
  28. Blagosklonny M. V. Cell death beyond apoptosis. Leukemia (Baltimore), 14: 1502-1508, 2000.[CrossRef][Medline]
  29. Gazitt Y., Fey V., Thomas C., Alvarez R. Bcl-2 overexpression is associated with resistance to dexamethasone, but not melphalan, in multiple myeloma cells. Int. J. Oncol., 13: 397-405, 1998.[Medline]
  30. Huang Y., Ibrado A. M., Reed J. C., Bullock G., Ray S., Tang C., Bhalla K. Co-expression of several molecular mechanisms of multidrug resistance and their significance for paclitaxel cytotoxicity in human AML HL-60 cells. Leukemia (Baltimore), 11: 253-257, 1997.[CrossRef][Medline]
  31. Ibrado A. M., Liu L., Bhalla K. Bcl-xL overexpression inhibits progression of molecular events leading to paclitaxel-induced apoptosis of human acute myeloid leukemia HL-60 cells. Cancer Res., 57: 1109-1115, 1997.[Abstract/Free Full Text]
  32. Liu J. R., Fletcher B., Page C., Hu C., Nunez G., Baker V. Bcl-xL is expressed in ovarian carcinoma and modulates chemotherapy-induced apoptosis. Gynecol. Oncol., 70: 398-403, 1998.[CrossRef][Medline]
  33. Tang C., Willingham M. C., Reed J. C., Miyashita T., Ray S., Ponnathpur V., Huang Y., Mahoney M. E., Bullock G., Bhalla K. High levels of p26BCL-2 oncoprotein retard taxol-induced apoptosis in human pre-B leukemia cells. Leukemia (Baltimore), 8: 1960-1969, 1994.[Medline]
  34. Fulda S., Strauss G., Meyer E., Debatin K. M. Functional CD95 ligand and CD95 death-inducing signaling complex in activation-induced cell death and doxorubicin-induced apoptosis in leukemic T cells. Blood, 95: 301-308, 2000.[Abstract/Free Full Text]
  35. Varfolomeev E. E., Schuchmann M., Luria V., Chiannilkulchai N., Beckmann J. S., Mett I. L., Rebrikov D., Brodianski V. M., Kemper O. C., Kollet O., Lapidot T., Soffer D., Sobe T., Avraham K. B., Goncharov T., Holtmann H., Lonai P., Wallach D. Targeted disruption of the mouse Caspase 8 gene ablates cell death induction by the TNF receptors, Fas/Apo1, and DR3 and is lethal prenatally. Immunity, 9: 267-276, 1998.[CrossRef][Medline]
  36. Yeh W. C., Pompa J. L., McCurrach M. E., Shu H. B., Elia A. J., Shahinian A., Ng M., Wakeham A., Khoo W., Mitchell K., El-Deiry W. S., Lowe S. W., Goeddel D. V., Mak T. W. FADD: essential for embryo development and signaling from some, but not all, inducers of apoptosis. Science (Wash. DC), 279: 1954-1958, 1998.[Abstract/Free Full Text]
  37. Goncalves A., Braguer D., Carles G., Andre N., Prevot C., Briand C. Caspase-8 activation independent of CD95/CD95-L interaction during paclitaxel-induced apoptosis in human colon cancer cells (HT29–D4). Biochem. Pharmacol., 60: 1579-1584, 2000.[CrossRef][Medline]
  38. Wieder T., Essmann F., Prokop A., Schmelz K., Schulze-Osthoff K., Beyaert R., Dorken B., Daniel P. T. Activation of caspase-8 in drug-induced apoptosis of B-lymphoid cells is independent of CD95/Fas receptor-ligand interaction and occurs downstream of caspase-3. Blood, 97: 1378-1387, 2001.[Abstract/Free Full Text]
  39. Schmitt C. A., Lowe S. W. Apoptosis is critical for drug response in vivo. Drug Resistance Updates, 4: 132-134, 2001.[CrossRef][Medline]
  40. Brown J. M., Wouters B. G. Apoptosis: mediator of mode of cell killing by anticancer agents?. Drug Resistance Updates, 4: 135-136, 2001.[CrossRef][Medline]
  41. Borner C., Monney L. Apoptosis without caspases: an inefficient molecular guillotine?. Cell Death Differ., 6: 497-507, 1999.[CrossRef][Medline]
  42. Leist M., Jaattela M. Four deaths and a funeral: from caspases to alternative mechanisms. Nat. Rev. Mol. Cell Biol., 2: 589-598, 2001.[CrossRef][Medline]
  43. Hirsch T., Marchetti P., Susin S. A., Dallaporta B., Zamzami N., Marzo I., Geuskens M., Kroemer G. The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death. Oncogene, 15: 1573-1581, 1997.[CrossRef][Medline]
  44. Dirsch V. M., Stuppner H., Vollmar A. M. Helenalin triggers a CD95 death receptor-independent apoptosis that is not affected by overexpression of Bcl-x(L) or Bcl-2. Cancer Res., 61: 5817-5823, 2001.[Abstract/Free Full Text]
  45. Fiers W., Beyaert R., Declercq W., Vandenabeele P. More than one way to die: apoptosis, necrosis and reactive oxygen damage. Oncogene, 18: 7719-7730, 1999.[CrossRef][Medline]
  46. Roberts L. R., Adjei P. N., Gores G. J. Cathepsins as effector proteases in hepatocyte apoptosis. Cell. Biochem. Biophys., 30: 71-88, 1999.[Medline]
  47. Wood D. E., Thomas A., Devi L. A., Berman Y., Beavis R. C., Reed J. C., Newcomb E. W. Bax cleavage is mediated by calpain during drug-induced apoptosis. Oncogene, 17: 1069-1078, 1998.[CrossRef][Medline]
  48. Wood D. E., Newcomb E. W. Caspase-dependent activation of calpain during drug-induced apoptosis. J. Biol. Chem., 274: 8309-8315, 1999.[Abstract/Free Full Text]
  49. Wu G. S., Saftig P., Peters C., El-Deiry W. S. Potential role for cathepsin D in p53-dependent tumor suppression and chemosensitivity. Oncogene, 16: 2177-2183, 1998.[CrossRef][Medline]
  50. Puthalakath H., Huang D. C. S., O’Reilly L. A., King S. M., Strasser A. The proapoptotic activity of the Bcl-2 family member Bim is regulated by interaction with the dynein motor complex. Mol. Cell, 3: 287-296, 1999.[CrossRef][Medline]
  51. Apolinario R. M., van d. V., de Jong J. S., Deville W., van Ark-Otte J., Dingemans A. M., van Mourik J. C., Postmus P. E., Pinedo H. M., Giaccone G. Prognostic value of the expression of p53, bcl-2, and bax oncoproteins, and neovascularization in patients with radically resected non-small-cell lung cancer. J. Clin. Oncol., 15: 2456-2466, 1997.[Abstract/Free Full Text]
  52. Pezzella F., Turley H., Kuzu I., Tungekar M. F., Dunnill M. S., Pierce C. B., Harris A., Gatter K. C., Mason D. Y. bcl-2 protein in non-small-cell lung carcinoma. N. Engl. J. Med., 329: 690-694, 1993.[Abstract/Free Full Text]
  53. Ferreira C. G., van d. V., Span S. W., Ludwig I., Smit E. F., Kruyt F. A., Pinedo H. M., van Tinteren H., Giaccone G. Expression of X-linked inhibitor of apoptosis as a novel prognostic marker in radically resected non-small cell lung cancer patients. Clin. Cancer Res., 7: 2468-2474, 2001.[Abstract/Free Full Text]
  54. Ferreira C. G., van d. V., Span S. W., Jonker J. M., Postmus P. E., Kruyt F. A., Giaccone G. Assessment of IAP (inhibitor of apoptosis) proteins as predictors of response to chemotherapy in advanced non-small-cell lung cancer patients. Ann. Oncol., 12: 799-805, 2001.[Abstract/Free Full Text]
  55. Kroep J. R., Giaccone G., Tolis C., Voorn D. A., Loves W. J., Groeningen C. J., Pinedo H. M., Peters G. J. Sequence dependent effect of paclitaxel on gemcitabine metabolism in relation to cell cycle and cytotoxicity in non-small-cell lung cancer cell lines. Br. J. Cancer, 83: 1069-1076, 2000.[CrossRef][Medline]
  56. Rowinsky E. K., Citardi M. J., Noe D. A., Donehower R. C. Sequence-dependent cytotoxic effects due to combinations of cisplatin and the antimicrotubule agents taxol and vincristine. J. Cancer Res. Clin. Oncol, 119: 727-733, 1993.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Clin. Cancer Res.Home page
M. S. Choe, Z. Chen, C. M. Klass, X. Zhang, and D. M. Shin
Enhancement of Docetaxel-Induced Cytotoxicity by Blocking Epidermal Growth Factor Receptor and Cyclooxygenase-2 Pathways in Squamous Cell Carcinoma of the Head and Neck
Clin. Cancer Res., May 15, 2007; 13(10): 3015 - 3023.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
U. Vijapurkar, W. Wang, and R. Herbst
Potentiation of Kinesin Spindle Protein Inhibitor-Induced Cell Death by Modulation of Mitochondrial and Death Receptor Apoptotic Pathways
Cancer Res., January 1, 2007; 67(1): 237 - 245.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
L. E. Broker, F. A.E. Kruyt, and G. Giaccone
Cell Death Independent of Caspases: A Review
Clin. Cancer Res., May 1, 2005; 11(9): 3155 - 3162.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S.-J. Park, C.-H. Wu, J. D. Gordon, X. Zhong, A. Emami, and A. R. Safa
Taxol Induces Caspase-10-dependent Apoptosis
J. Biol. Chem., December 3, 2004; 279(49): 51057 - 51067.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
Y. Zou, H. Fu, S. Ghosh, D. Farquhar, and J. Klostergaard
Antitumor Activity of Hydrophilic Paclitaxel Copolymer Prodrug Using Locoregional Delivery in Human Orthotopic Non-Small Cell Lung Cancer Xenograft Models
Clin. Cancer Res., November 1, 2004; 10(21): 7382 - 7391.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
L. E. Broker, C. Huisman, S. W. Span, J. A. Rodriguez, F. A. E. Kruyt, and G. Giaccone
Cathepsin B Mediates Caspase-Independent Cell Death Induced by Microtubule Stabilizing Agents in Non-Small Cell Lung Cancer Cells
Cancer Res., January 1, 2004; 64(1): 27 - 30.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
D. J. Waxman and P. S. Schwartz
Harnessing Apoptosis for Improved Anticancer Gene Therapy
Cancer Res., December 15, 2003; 63(24): 8563 - 8572.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
A. Masuda, K. Maeno, T. Nakagawa, H. Saito, and T. Takahashi
Association between Mitotic Spindle Checkpoint Impairment and Susceptibility to the Induction of Apoptosis by Anti-Microtubule Agents in Human Lung Cancers
Am. J. Pathol., September 1, 2003; 163(3): 1109 - 1116.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
L. Yang, T. Mashima, S. Sato, M. Mochizuki, H. Sakamoto, T. Yamori, T. Oh-hara, and T. Tsuruo
Predominant Suppression of Apoptosome by Inhibitor of Apoptosis Protein in Non-Small Cell Lung Cancer H460 Cells: Therapeutic Effect of a Novel Polyarginine-conjugated Smac Peptide
Cancer Res., February 15, 2003; 63(4): 831 - 837.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
T. M. LaVallee, X. H. Zhan, M. S. Johnson, C. J. Herbstritt, G. Swartz, M. S. Williams, W. A. Hembrough, S. J. Green, and V. S. Pribluda
2-Methoxyestradiol Up-Regulates Death Receptor 5 and Induces Apoptosis through Activation of the Extrinsic Pathway
Cancer Res., January 15, 2003; 63(2): 468 - 475.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
L. E. Broker, C. Huisman, C. G. Ferreira, J. A. Rodriguez, F. A. E. Kruyt, and G. Giaccone
Late Activation of Apoptotic Pathways Plays a Negligible Role in Mediating the Cytotoxic Effects of Discodermolide and Epothilone B in Non-Small Cell Lung Cancer Cells
Cancer Res., July 15, 2002; 62(14): 4081 - 4088.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Huisman, C.
Right arrow Articles by Giaccone, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Huisman, C.
Right arrow Articles by Giaccone, G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online