![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
CELLULAR AND MOLECULAR
Laboratory of Molecular and Cellular Pharmacology of Purinergic Transmission, Department of Pharmacological Sciences, School of Pharmacy, University of Milan, Milan, Italy
Received February 25, 2005; accepted May 4, 2005.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
and a short variant (caspase-9
), which, in other tumors, acts as a dominant negative of the long isoform. All analyzed clones showed a point mutation in the prodomain region that is known to interact with mitochondria-released factors. Thus, in these human astrocytoma cells, mitochondria-damaging agents induce a regulated form of mitochondrial-dependent necrotic cell death (oncosis). Resistance to apoptosis is due to an intrinsic defect of caspase-9, leading to inhibition of enzyme activation and/or impaired interaction with proteins released from depolarized mitochondria. These results may have implications for developing strategies aimed at overcoming tumor resistance to chemotherapy.
). The so-called "extrinsic pathway" is driven by the activation of extracellular death receptors, leading to the recruitment and activation of initiator caspase-8 and -10. Conversely, death at the mitochondrial level (the "intrinsic pathway") is initiated by a perturbation of the mitochondrial membrane potential, followed by release of cytochrome c in the cytosol that in turn contributes to the formation of a protein complex called apoptosome (Igney and Krammer, 2002). This complex eventually allows the activation of effector caspase-9. Both the intrinsic and the extrinsic pathway converge on downstream "executioner" caspases, mainly caspase-3, which are responsible for the cleavage of structural cytoplasmic and nuclear proteins, with consequent cell collapse and death.
Over the past few years, it has become clear that anticancer drugs are able to induce apoptosis and that this process is involved in the mediation of their cytotoxic effects. Furthermore, the induction of apoptosis by different classes of anticancer agents was demonstrated to converge into similar downstream mechanisms that are mainly represented by the two above-described biochemical pathways (Ferreira et al., 2002).
However, the success of chemotherapy is often hampered by the appearance of broad drug resistance, in several cases due to defective common apoptosis-inducing pathways. It is now clear that these defects are probably at the basis of the initial expansion of the population of neoplastic cells eventually originating the tumor (Igney and Krammer, 2002). Thus, it is very important to identify the biochemical defect(s) borne by a specific tumor to predict the possible outcome of a given drug treatment and to plan more focused and effective therapies aimed at bypassing the observed alterations. This is particularly important in the case of central nervous system tumors (e.g., gliomas, in particular those of astrocytic origin), which account for more than 40% of all brain tumors and are often characterized by a poor prognosis (Kleihues et al., 1995).
We have previously demonstrated that 2-chloro-2'-deoxyadenosine, a widely used chemotherapeutic drug for leukemia and lymphoma (known as cladribine; Bryson and Sorkin, 1993), is able to induce apoptotic cell death of human astrocytoma ADF cells through recruitment of an atypical caspase-2-dependent pathway (Ceruti et al., 2003). No activation of the mitochondria/caspase-9-dependent intrinsic pathway of apoptosis was detected in our experimental model, despite evidence demonstrating a key role played by this pathway in cladribine-induced cell death in other tumor cell lines (Marzo et al., 2001). Given the importance of the intrinsic pathway in the activation of apoptosis by chemotherapeutic agents (see above), we wondered whether insensitivity to this chemotherapeutic agent reflects a peculiar behavior of cladribine in these cells or is the result of a more generalized defect of the intrinsic pathway of apoptosis in astrocytoma cells. In the present study, we show that several mitochondria-damaging agents, including betulinic acid, a new, promising anticancer drug (Fulda and Debatin, 2000; Fulda et al., 2004), do not induce apoptosis in astrocytoma ADF cells, and we raise several hypotheses to explain this insensitivity that might have important implication in resistance to chemotherapy.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
) were maintained in culture in standard conditions (37°C, 95% humidity, 5% CO2) in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1% nonessential amino acids (all from Euroclone, Celbio, Italy), as described previously (Ceruti et al., 2003). Betulinic acid (BetA, 10–100 µg/ml; Alexis Biochemicals, Vinci-Biochem, Vinci, Italy), 2-deoxy-D-ribose (dRib, 10–50 mM; Sigma-Aldrich, Milan, Italy), and KCN (100–600 µM; Sigma-Aldrich) were added to cultures 24 h after plating cells on six-well plates (600,000 cells/well) and left in the culture medium for 24 to 96 h.
Cytofluorimetrical Studies
Evaluation of Apoptosis. The percentage of apoptotic cells in the total population (adhering + detached cells) was evaluated immediately at the end of the incubation period by means of propidium iodide (PI) staining of DNA followed by flow cytometric analysis, as described previously (Ceruti et al., 2000, 2003).
Analysis of Mitochondrial Membrane Potential. Changes in mitochondrial membrane potential (
m) induced in the total cell population by different pharmacological treatments were analyzed by means of the fluorescent dye 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1; Molecular Probes, Società Italiana Chimici, Rome, Italy), as described previously (Ceruti et al., 1997, 2003). J-Aggregate fluorescence was recorded by flow cytometry in the fluorescence channel 2 (FL2) and monomer fluorescence in the fluorescence channel 1 (FL1). Necrotic fragments were electronically gated out on the basis of morphological characteristics on the forward light scatter (FSC) versus the side light scatter dot plot.
Analysis of Phosphatidylserine Externalization and Membrane Integrity. Phosphatidylserine (PS) exposure on the outer leaflet of the plasma membrane was evaluated by staining ADF cells with Annexin V-FITC (apoptosis detection kit; Vinci-Biochem), following the manufacturer's instruction. Annexin V binding to PS was evaluated in the FL1. Cells were counterstained with an isotonic PI solution (20 µg/ml in phosphate-buffered saline) to evaluate cell membrane integrity. In fact, under these experimental conditions intact cell membranes exclude PI, which can only permeate cells undergoing necrotic cell death (Scholz et al., 2005). PI positivity was detected in the FL2. On the same experimental samples, cell swelling was evaluated by analyzing cell size distribution as a function of the FSC values.
Detection of Lactate Dehydrogenase Activity in Culture Supernatants. Lactate dehydrogenase (LDH) release into the cell culture supernatant occurs upon damage of the plasma membrane, and it is therefore considered a typical hallmark of necrosis. LDH activity was measured by means of a spectrophotometric assay kit (cytotoxicity detection kit-LDH; Roche, Milan, Italy), following the manufacturer's instructions. At the end of the incubation with the tested agents, culture supernatants from both control and drug-treated ADF cells were collected and centrifuged to discard floating cells. Determination of LDH activity was then carried out in a 96-well plate in a total volume of 200 µl (100 µl of supernatant in each sample and 100 µl of reaction mixture). After a 10- to 20-min incubation at 25°C, LDH enzyme activity was measured in a spectrophotometer at 490 nm. To assess maximal LDH activity, cells were exposed for 18 h to 1% Triton X-100 and processed as described above. To detect spontaneous LDH activity due to the presence of 10% FCS (blank), parallel samples were run with medium alone. Each condition was run in triplicate.
Detection of Caspase Activity
Caspase activity was measured by means of a spectrophotometric assay kit (CaspACE assay system colorimetric; Promega, Milan, Italy), following the manufacturer's instructions with some minor modifications, as described previously (Ceruti et al., 2003). The determination of caspase activity was carried out in a 96-well plate in a total volume of 100 µl, with a total amount of 70 µg of protein in each sample, and in the presence of tetrapeptide substrates conjugated to paranitroaniline (i.e., N-acetyl-Asp-Glu-Val-Asp-paranitroaniline, N-acetyl-Leu-Glu-His-Asp-paranitroaniline, and N-acetyl-Ile-Glu-Thr-Asp-paranitroaniline in caspase-3, -9, and -8, respectively, final concentration 200 µM). Extracts were incubated for 4 h at 37°C and released paranitroaniline was then measured in a spectrophotometer at 405 nm. Each condition was run in triplicate, and for each experimental condition at least three independent experiments were performed.
Western Blotting Analysis
Whole-cell lysates were prepared and analyzed by immunoblotting, as described previously (Ceruti et al., 2003). For caspase-3 detection, 50 µg of proteins in each lane was size fractionated by SDS-polyacrylamide gel electrophoresis in 11% acrylamide gel, whereas for gp-170 (Mdr-1), 100 µg of proteins was subjected to electrophoretic separation in 7.5% acrylamide gel. After electroblotting onto nitrocellulose membrane, an overnight incubation with primary antibodies was performed. In particular, mouse monoclonal anti-caspase-3 antibody (1:750 in 3% nonfat dry milk in TBS; Alexis Biochemicals), followed by goat anti-mouse secondary antibody conjugated to horseradish peroxidase (1:2000 in 3% nonfat dry milk in TBS, 1 h at room temperature; Sigma Aldrich), was used to detect caspase-3 activation. A goat polyclonal anti-Mdr-1 antibody (1:100 in 5% nonfat dry milk in TBS; Santa Cruz Biotechnology, Inc., Santa Cruz, CA; kindly provided by Dr. Paola Perego, Istituto Nazionale dei Tumori, Milan, Italy), followed by monkey anti-goat secondary antibody conjugated to horseradish peroxidase (1:4000 in 5% nonfat dry milk in TBS, 1 h at room temperature; Sigma-Aldrich) was used to detect gp-170 expression. In this case, whole-cell lysates from LoVo/DX cells, known to express Mdr-1 (Soranzo et al., 1990) were processed in parallel as positive control. Afterward, to check for correct protein loading in the gel, the same filters were probed using a rabbit anti-actin polyclonal antibody (1:500 in 3% nonfat dry milk in TBS; Sigma-Aldrich) followed by a goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:3000 in 3% nonfat dry milk in TBS,1 h at room temperature; Sigma-Aldrich). Detection of proteins was performed by enhanced chemiluminescence (Amersham Biosciences, Inc., Milan, Italy) and autoradiography.
|
). For RT-PCR analysis, 1 µg of RNA was treated with RQ1 RNase-free DNase (Promega, Milan, Italy), and RNA was then reverse-transcribed with Superscript II RNA H- reverse transcriptase (200 U/sample; Invitrogen, Milan, Italy). cDNAs were amplified in each PCR assay with Platinum TaqDNA polymerase (1.25 U/sample; Invitrogen) in a 25-µl reaction mixture containing 20 pmol of 5' and 3' primers in a standard PCR buffer (50 mM KCl, 1.5 mM MgCl2, and 20 mM Tris-HCl, pH 8.4). Amplification were performed in a Gene-Amp 9700 thermal cycler (Applied Biosystems, Milan, Italy) for 35 to 40 cycles (typically, 94°C/45 s, 45 s at the annealing temperature of 68°C, 56°C and 67°C for caspase-3, caspase-9, and caspase-8, respectively, followed by 45 s at 72°C) after an initial denaturation at 95°C for 2 min.
The following forward (Fw) and reverse (Rw) oligonucleotide primers were used: caspase-3, Fw 5'-TTCAGAGGGGATCGTTGTAGAAGTC-3' and Rw 5'-CAAGCTTGTCGGCATACTGTTTCAG-3'; caspase-9, Fw 5'-ATGGACGAAGCGGATCGGCGGCTCC-3' and Rw 5'-GCACCACTGGGGTAAGGTTTTCTAG-3'; caspase,-8 Fw 5'-CACTAGAAAGGAGGAGATGGAAAG-3' and Rw 5'-CTATCCTGTTCTCTTGGAGAGTCC-3'; and -actin, Fw 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3' and Rw 5'-CTAGAAGCATTGCGGTGGACGATGGAGGG-3'. Ten microliter aliquots of the PCR products were finally size separated by electrophoresis in 1% agarose gels.
Cloning of Human Caspase-9 and Caspase-9
Amplification of caspase-9 and caspase-9 mRNAs was performed with specific oligonucleotide primers (Fw 5'-CCATGGACGAAGCGGATC-3' and Rw 5'-GCCCTGGCCTTATGATGTTTT-3') for 35 cycles (initial denaturation at 95°C for 2 min, 95°C/60 s, 60 s at the annealing temperature of 58.8°C, followed by 70 s at 72°C). The PCR products were cloned into the pcDNA3.1 expression vector using the pcDNA3.1/V5-His TOPO TA expression kit (Invitrogen). Positive colonies were identified with a double enzymatic digestion, the first with HindIII and EcoRV (Roche) and the second with PvuII (Promega). The construct was verified by sequencing using the Applied Biosystems Terminator cycle sequencing kit.
|
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
; Fulda and Debatin, 2000). Hence, we exposed human astrocytoma ADF cells to increasing concentrations of the drug (10–100 µg/ml) for various times (24–72 h; Fig. 1A). No significant induction of apoptosis was detected at any of the times tested, with only a slight, although significant increase in the percentage of necrosis obtained after a 72-h incubation with the drug. Not even prolongation of the incubation time to a total of 96 h (Fig. 1B) was able to activate apoptotic cell death, but it did further increase the necrotic signs of toxicity. Despite the lack of nuclear signs of apoptosis, BetA was able to induce a highly significant depolarization of mitochondria. This is clearly shown by graphs in Fig. 2A, which were obtained after staining both control and BetA-treated cultures with the mitochondria-specific dye JC-1. A high fluorescence emission of the dye in both the yellow (FL2; y-axis) and the green (FL1; x-axis) channels of the flow cytometer is representative of healthy and polarized mitochondria. Depolarization induces a significant drop in the yellow emission, accompanied by a contemporary increase of the green emission, due to the prevalence of the monomeric form of the dye with respect to J-aggregates. Depolarization is time-dependent, starting from a 48-h incubation time (Fig. 2B), and it can be achieved with any of the drug concentrations tested (10–50 µg/ml).
We next wondered whether the inability to activate the mitochondrial pathway of apoptosis represented a drug-related effect or whether it was a more general behavior of our cell line, mirroring some defects in the intrinsic pathway of death. Hence, we exposed ADF cells to dRib and KCN, two well known "mitochondriotropic" molecules, acting on different molecular targets inside the organelle (Kletsas et al., 1998; Shou et al., 2000). Also with dRib, a very low percentage of apoptotic and necrotic death was detected, despite the use of elevated concentrations of the drug (10–50 mM) and the long incubation periods (24–72 h; Fig. 3A). A 96-h incubation with the highest concentration tested was able to induce necrosis in only the 27% of the total cell population (Fig. 3B), despite concentration- and time-dependent depolarization of mitochondria (Fig. 4, A and B).
|
|
Similar results were also obtained by exposing cultures to the mitochondrial poison KCN (Fig. 5). Concentrations up to 400 µM were completely ineffective in inducing death of ADF cells (Fig. 5, A and B). A higher concentration (600 µM) was instead able to kill almost all of the cells, mainly by inducing necrosis, through massive mitochondria depolarization (Fig. 5C), accompanied by widespread cytotoxicity.
|
|
No Activation of Caspases by BetA and dRib in Human Astrocytoma ADF Cells. Resistance to apoptotic stimuli may also arise from defects in the expression of caspases (Philchenkov et al., 2004; Reed, 2004). Thus, we decided to verify whether, in our experimental model, lack of apoptosis by BetA and dRib may arise from a defective expression and/or activation of the most important "initiator" caspases (i.e., caspase-8 and caspase-9 for the intrinsic and extrinsic pathways, respectively) and of "executioner" caspase-3 (Igney and Krammer, 2002). As a first step, we looked at the presence of caspase-8 in the human astrocytoma ADF cells. RT-PCR analysis with specific oligonucleotide primers for this caspase revealed the presence of an amplification product at the expected molecular weight of 411 bp (Fig. 7A). However, despite the presence of caspase-8 mRNA, neither BetA nor dRib used at the same concentrations that induced cell death (see above) were able to activate this caspase (Fig. 7B). To check for the liability of the colorimetric assay, a positive control was used in parallel (see legend to Fig. 7).
|
; Debatin, 2004). We have already shown a significant release of cytochrome c from the mitochondrial to the cytosolic cellular compartment after exposing ADF cells to BetA, dRib, and KCN (Ceruti et al., 2003), suggesting that no alterations in the mechanism(s) of protein release from depolarized mitochondria are detectable in our experimental model. We thus checked the expression of caspase-9 and caspase-3 by RT-PCR in our experimental model. Amplification of cDNA with specific oligonucleotide primers for these caspases revealed two products (Fig. 8A) at the expected molecular weights of 264 and 330 bp for caspase-3 and caspase-9, respectively. Actin was used as an internal control for the amplification reaction. Translation of RNAs to proteins was checked by Western blotting analysis (see lower part of Fig. 8C for caspase-3; data not shown for caspase-9).
|
Nevertheless, no activation of either caspase-9 (Fig. 8B) or caspase-3 (Fig. 8C) was detectable in ADF cells after exposure to both BetA and dRib at any of the times tested, in agreement with the lack of apoptotic cell death (Figs. 1 and 3). Also for these caspases, positive controls were run in parallel, leading to a statistically significant caspase-9 and -3 activation (see Discussion). For effector caspase-3, Western blotting analysis was also performed. Low molecular weight active fragments, which are known to be produced upon enzyme activation, were not detected after either dRib or BetA exposure, but only in the lane corresponding to the positive control (Fig. 8C). Lack of caspase activation was also observed in the presence of 100 to 600 µM KCN (data not shown).
Expression of a Caspase-9 Short Isoform in Human ADF Cells. Two splicing variants of caspase-9 have been described previously (Seol and Billiar, 1999; Srinivasula et al., 1999; Angelastro et al., 2001; Waltereit and Weller, 2002): one long isoform corresponding to the full-length protein (called caspase-9) and one short variant (identified as caspase-9) missing most of the large subunit (indicated with "B" in Fig. 9A) containing the catalytic site of the enzyme (see Fig. 9A for a schematic representation of the two variants). To check for the presence of these two splice variants, we designed oligonucleotide primers external to the caspase coding sequence (see Materials and Methods). Two RT-PCR products were identified in ADF cells (Fig. 9B) with the expected molecular weight of 1250 and 800 base pairs, corresponding to caspase-9 and -9, respectively.
|
and -9 obtained from ADF cells are 99% identical to that reported for the human caspase-9 (GenBank accession nos. NM_001229
[GenBank]
and NM_032996
[GenBank]
for caspase-9 and -9, respectively), except for a single C-to-T nucleotide substitution in position 83 of the coding sequence, which was found in each of the 11 clones analyzed. Figure 9C shows the deduced amino acid sequence for ADF caspase-9 and its alignment with the previously reported human caspase-9 isoform. The portion of the protein outside the boxes, containing the part that is missing in the short isoform, represents caspase-9. The reported polymorphism leads to a single amino acid substitution at position 28 (A
V), which belongs to the prodomain portion of the protein (Allan et al., 2003). Therefore, we conclude that not only do ADF cells express the short isoform of caspase-9 but also they have a point mutation in the gene sequence encoding for the enzyme that may contribute to their resistance to various apoptotic stimuli (see Discussion).
Finally, to rule out the possibility that resistance to induction of apoptosis may be due to expression, in ADF cells, of the glycoprotein p-170 (gp-170 or Mdr-1) known to act as a pump extruding drugs out of cells (thus reducing their intracellular concentrations and efficacy as chemotherapeutic agents; Moran et al., 1997), we also looked at the presence of this protein in the human astrocytoma cells used here. Western blot analysis with an antibody that specifically recognized the gp-170 protein did not reveal any immunoreactive band in lysates from ADF cells, whereas a specific band with the expected molecular weight of 170 was detected in lysates from LoVo/DX cells (Fig. 10), which have been reported to express the multidrug resistance protein to significant levels (Soranzo et al., 1990).
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
), which can vary greatly depending upon the histotype, and the degree of malignity. Thus, important needs in the pharmacological approach to cancer are represented by 1) understanding whether apoptotic pathways can be successfully recruited by chemotherapeutic agents in specific tumors, 2) determining whether alterations of the "classical" cell death pathways are present, and 3) assessing the possibility to activate alternative and effective ways of killing cancer cells.
Our data clearly show that human ADF astrocytoma cells are resistant to agents known to interfere with cell survival by altering mitochondrial functions. Among the agents tested here, BetA is a natural product derived from the bark of the white birch tree (Eiznhamer and Xu, 2004) and represents a promising new drug for the treatment of various neoplasms. BetA has been demonstrated to potently inhibit the growth of neuroectodermal tumors, such as neuroblastoma, medulloblastoma, and Ewing sarcoma cell lines (Fulda and Debatin, 2000) as well as of several human carcinoma (Zuco et al., 2002). Although its intracellular molecular target has never been described, it is generally claimed that, in neuroectodermal tumors, BetA directly affects mitochondria (Fulda and Debatin, 2000), causing a drop in mitochondrial membrane potential, cytochrome c release, and caspase-9 activation (Fulda et al., 1998; Costantini et al., 2000), leading to the typical signs of apoptotic cell death. For glial tumors, conflicting results have been published. In fact, although this drug was originally claimed to also induce apoptosis in glioblastoma cell lines (Fulda and Debatin, 2000), very high concentrations of BetA (as high as 50 µg/ml) were needed to detect signs of apoptosis in quite a few malignant glioma cell lines (Wick et al., 1999), and only the concomitant exposure to BetA and TRAIL was effective in inducing death (Fulda et al., 2004). The present results represent the first in-depth evaluation of -induced effects on a human astrocytoma cell line and might have important clinical implications.
dRib has never been used as an anticancer agent, but it is an extensively used in vitro tool to induce mitochondriadependent cell death in experimental systems, through glutathione depletion and consequent induction of oxidative stress (Ceruti et al., 1997; Kletsas et al., 1998; Ikeda et al., 2002). On the other hand, KCN represents a well known mitochondrial poison that uncouples the respiratory chain from ATP production, leading to a dramatic energetic defect and cell death, mainly necrotic (Li et al., 2002; Prabhakaran et al., 2004; Fig. 5). Despite very different types of mitochondrial interference, these three agents induced very similar effects on human astrocytoma cells.
In our experimental model, mitochondria-damaging agents are still able to activate the very first steps of the intrinsic pathway of apoptosis (i.e., disruption of mitochondrial membrane potential and release of cytochrome c in the cytoplasm; see Figs. 2, 4, and 5 and Ceruti et al., 2003). Nevertheless, despite the appearance of these dramatic biochemical events, caspase-9 activation is defective (Fig. 8). Thus, the prolonged energetic imbalance induced by these agents leads, in turn, to necrotic death (although of modest entity and after very long exposure periods; Figs. 1, 3, and 5). The nature of cell death upon exposure to these agents has been also confirmed by other parameters of necrosis, including LDH release, cellular swelling, and flow cytometry positivity to isotonic PI (Fig. 6, A and B). Interestingly, we also found that, despite lack of significant apoptosis, drug-treated cells show externalization of PS, which is generally taken as a marker of apoptotic death. This confirms recent data from other authors showing that early exposure of PS at the outer face of plasma membrane is not unequivocally associated to induction of apoptosis but also occurs during oncosis (early primary necrosis; Krysko et al., 2004), probably depending upon the loss of aminophospholipid translocase activity, due to the rapid decrease in ATP levels. Moreover, the pathway of death we observed in our experimental model is remarkably similar to what recently reported for arsenic trioxide in several tumoral cell lines. Also in this case, cells underwent potent mitochondrial depolarization, and a regulated form of mitochondrial-dependent necrotic cell death, with no involvement of caspases (Scholz et al., 2005).
Insensitivity of ADF cells to the agents tested here is not due to a defect in the expression of caspase-9, since both the long and the short forms of this caspase were detected. Even though we cannot rule out that other genetic and/or biochemical alterations may account for ADF cells resistance to chemotherapy (e.g., loss of the apoptotic protease activating factor-1 protein, subcellular distribution of caspases, or overexpression of antiapoptotic proteins), expression of the short caspase-9 isoform (caspase-9) in these cells might contribute to their insensitivity to chemotherapy. In fact, it has been demonstrated that this form of caspase-9 can behave as a dominant negative of the caspase-9 long isoform (caspase-9) and inhibit its activation in response to various proapoptotic stimuli (Srinivasula et al., 1999). Both neuronal and non-neuronal cells transfected with caspase-9 were shown to be resistant to death evoked by trophic factor deprivation or DNA damage (Angelastro et al., 2001). Overexpression of caspase-9 inhibited tumor necrosis factor-- and Bax-mediated apoptosis in MCF-7 carcinoma cells (Seol and Billiar, 1999). Moreover, it has been already demonstrated that other astrocytoma cell lines express very high level of both and caspase-9 isoforms with respect to normal astrocytes (Waltereit and Weller, 2002). In these cells, overexpression of both isoforms by exogenous transfection did not modulate their sensitivity to the apoptosis induced by either CD95L plus cycloheximide or teniposide. Nevertheless, it should be emphasized that these apoptotic stimuli do not directly target mitochondria; thus, despite the overexpression of the pro- and antiapoptotic caspase-9 isoforms, the final degree of apoptosis could likely be maintained unchanged by recruitment of other pathways (e.g., the caspase-8- and/or the caspase-2-dependent pathways). This is not the case of the agents used in the present study, which directly act on mitochondria and whose apoptotic activity can be more heavily influenced by alterations of the intrinsic death pathway. Thus, our results seem to confirm the low sensitivity of tumor of glial origin to BetA, suggesting that expression of caspase-9 may represent one of the reasons for this resistance, and may also be used to predict the success of a given chemotherapy. Indeed, a very interesting study have been recently published showing that 13 of 21 glioblastoma cell lines established directly from patients were sensitive to BetA-induced cell death (Jeremias et al., 2004). Although this represents a very high percentage of success (around 62%), the reason for the remaining cell lines to be insensitive to this pharmacological treatment is currently unknown.
We also detected an A-to-V mutation at position 28 of the caspase-9 protein expressed by ADF cells, which may as well influence caspase-9 activity. This polymorphism has already been reported (GenBank single nucleotide polymorphism ID rs1052571), but no information regarding the possible biological significance of this mutation is currently available. The mutated amino acid is localized in the region of the protein corresponding to the enzyme prodomain (Allan et al., 2003), which is responsible for the interactions with apoptotic protease activating factor-1 in the apoptosome complex (Shiozaki et al., 2002). It is tempting to speculate that the introduction of a more lipophilic and ramified amino acid, such as valin, in this region of the protein may somehow modify the ternary structure of the protein, leading to a defective apoptosome formation.
In previous studies, we have been able to activate the apoptotic program, and therefore induce cell death, in ADF astrocytoma cells only by using agents able to bypass the caspase-9-dependent pathway of death. In fact, we have demonstrated that 2CdA is a very effective apoptotic stimulus for this cell line only due to the fact that it is able to recruit a caspase-2-dependent/mitochondria-independent pathway of death (Ceruti et al., 2003). Indeed, ADF cells show resistance to another typical chemotherapeutic agent, etoposide, and this resistance can be overcome by the concomitant exposure to pharmacological agents that can promote the activation of the caspase-2-dependent pathway of apoptosis (S. Ceruti, A. Mazzola, and M. P. Abbracchio, manuscript in preparation). In both pharmacological paradigms, caspase-9 activation can be achieved at very late time points (see positive control in Fig. 8; Ceruti et al., 2003), only as a consequence of the direct intervention of the other caspases that can proteolytically cleave and activate the enzyme, as a positive feedback loop aimed at amplifying the death process.
Thus, since induction of apoptosis represents a common event for different classes of anticancer agents converging into common downstream mechanisms, these observations confirm that disruption and/or alteration of only one of such mechanisms can led to broad drug resistance. For this reason, the setting of therapeutic strategies aimed at overcoming resistance and at recruiting the pathways that are still active in a given tumor represents one of the most important goals in cancer chemotherapy.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: BetA, betulinic acid; dRib, 2-deoxy-D-ribose; PI, propidium iodide; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimida-zolcarbocyanine iodide; FL2, fluorescence channel 2; FL1, fluorescence channel 1; FSC, forward light scatter; PS, phosphatidylserine; FITC, fluorescein isothiocyanate; LDH, lactate dehydrogenase; Mdr-1, multidrug resistance protein-1; TBS, Tris-buffered saline; RT-PCR, reverse transcriptase-polymerase chain reaction; PCR, polymerase chain reaction; Fw, forward; Rw, reverse; ANOVA, analysis of variance; bp, base pair(s); KCN, potassium cyanide.
Address correspondence to: Prof. Maria P. Abbracchio, Department of Pharmacological Sciences, Via Balzaretti, 9, University of Milan, 20133 Milan, Italy. E-mail: mariapia.abbracchio{at}unimi.it
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Allan LA, Morrice N, Brady S, Magee G, Pathak S, and Clarke PR (2003) Inhibition of caspase-9 through phosphorylation at Thr 125 by ERK MAPK. Nat Cell Biol 5: 647-654.[CrossRef][Medline]
Angelastro JM, Moon NY, Liu DX, Yang AS, Greene LA, and Franker T (2001) Characterization of a novel isoform of caspase-9 that inhibits apoptosis. J Biol Chem 276: 12190-12200.
Bryson HM and Sorkin EM (1993) Cladribine. A review of its pharmacodynamic and pharmacokinetic properties and therapeutic potential in haematological malignancies. Drugs 46: 872-894.[Medline]
Ceruti S, Barbieri D, Veronese E, Cattabeni F, Cossarizza A, Giammarioli AM, Malorni W, Franceschi C, and Abbracchio MP (1997) Different pathways of apoptosis revealed by 2-chloro-adenosine and deoxy-D-ribose in mammalian astroglial cells. J Neurosci Res 47: 372-383.[CrossRef][Medline]
Ceruti S, Beltrami E, Matarrese P, Mazzola A, Cattabeni F, Malorni W, and Abbracchio MP (2003) A key role of caspase-2 and caspase-3 in the apoptosis induced by 2-chloro-2'-deoxy-adenosine (cladribine) and 2-chloro-adenosine in human astrocytoma cells. Mol Pharmacol 63: 1437-1447.
Ceruti S, Franceschi C, Barbieri D, Malorni W, Camurri A, Giammarioli AM, Ambrosini A, Racagni G, Cattabeni F, and Abbracchio MP (2000) Apoptosis induced by 2-chloro-adenosine and 2-chloro-deoxy-adenosine in human astrocytoma cell line: differential mechanisms and possible clinical relevance. J Neurosci Res 60: 388-400.[CrossRef][Medline]
Costantini P, Jacotot E, Decaudin D, and Kroemer G (2000) Mitochondrion as a novel target of anticancer chemotherapy. J Natl Cancer Inst 92: 1042-1053.
Debatin KM (2004) Apoptosis pathways in cancer and cancer therapy. Cancer Immunol Immunother 53: 153-159.[CrossRef][Medline]
Earnshaw WC, Martins LM, and Kaufmann SH (1999) Mammalian caspases: structures, activation, substrates and functions during apoptosis. Annu Rev Biochem 68: 383-424.[CrossRef][Medline]
Eiznhamer DA and Xu ZQ (2004) Betulinic acid: a promising anticancer candidate. Drugs 7: 359-373.
Ferreira CG, Epping M, Kruyt FAE, and Giaccone G (2002) Apoptosis: target of cancer therapy. Clin Cancer Res 8: 2024-2034.
Fulda S and Debatin KM (2000) Betulinic acid induced apoptosis through a direct effect on mitochondria. Med Pediatr Oncol 35: 616-618.[CrossRef][Medline]
Fulda S, Jeremias I, and Debatin KM (2004) Cooperation of betulinic acid and TRAIL to induce apoptosis in tumor cells. Oncogene 23: 7611-7620.[CrossRef][Medline]
Fulda S, Jeremias I, Steiner HH, Pietsch T, and Debatin KM (1999) Betulinic acid: a new cytotoxic agent against malignant brain-tumour cells. Int J Cancer 82: 435-441.[CrossRef][Medline]
Fulda S, Scaffidi C, Susin SA, Krammer PH, Kroemer G, Peter E, and Debatin KM (1998) Activation of mitochondria and release of mitochondrial apoptogenic factors by betulinic acid. J Biol Chem 18: 33942-33948.
Fumagalli M, Trincavelli L, Lecca D, Martini C, Ciana P, and Abbracchio MP (2004) Cloning, pharmacological characterization and distribution of the rat G-protein-coupled P2Y(13) receptor. Biochem Pharmacol 68: 113-124.[CrossRef][Medline]
Igney FH and Krammer PH (2002) Death and anti-death: tumour resistance to apoptosis. Nat Rev 2: 277-288.
Ikeda R, Furukawa T, Kitazono M, Ishitsuka K, Okumura H, Tani A, Sumizawa T, Araguchi M, Komatsu M, Uchimiya H, et al. (2002) Molecular basis for the inhibition of hypoxia-induced apoptosis by 2-deoxy-D-ribose. Biochem Biophys Res Commun 291: 806-812.[CrossRef][Medline]
Jeremias I, Steiner HH, Benner A, Debatin M, and Herold-Mende C (2004) Cell death induction by betulinic acid, ceramide and TRAIL in primary glioblastoma multiforme cells. Acta Neurochir 146: 721-729.
Kleihues P, Soylemezoglu F, Schauble B, Scheithauer BW, and Burger P (1995) Histopathology, classification and grading of gliomas. Glia 15: 211-221.[CrossRef][Medline]
Kletsas D, Barbieri D, Stathakos D, Botti B, Bergamini S, Tomasi A, Monti D, Malorni W, and Franceschi C (1998) The highly reducing sugar 2-deoxy-D-ribose induces apoptosis in human fibroblasts by reduced glutathione depletion and cytoskeletal disruption. Biochem Biophys Res Commun 243: 416-425.[CrossRef][Medline]
Krysko O, de Ridder L, and Cornelissen M (2004) Phosphatidylserine exposure during early primary necrosis (oncosis) in JB6 cells as evidenced by immunogold labelling technique. Apoptosis 9: 495-500.[CrossRef][Medline]
Li L, Prabhakaran K, Shou Y, Borowitz JL, and Isom GE (2002) Oxidative stress and cyclooxygenase-2 induction mediate cyanide-induced apoptosis of cortical cells. Toxicol Appl Pharmacol 15: 55-63.
Malorni W, Rainaldi G, Rivabene R, and Santini MT (1994) Different susceptibilities to cell death induced by t-butylhydroperoxide could depend upon cell histotype-associated growth features. Cell Biol Toxicol 10: 207-218.[CrossRef][Medline]
Marzo I, Perez-Galan P, Giraldo P, Rubio-Felix D, Anel A, and Naval J (2001) Cladribine induces apoptosis in human leukemia cells by caspase-dependent and -independent pathway acting on mitochondria. Biochem J 359: 537-546.[CrossRef][Medline]
Moran E, Clearly I, Larkin AM, Nic Amhlaoibh R, Masterson A, Scheper RJ, Izquierdo MA, Center M, O'Sullivan F, and Clynes M (1997) Co-expression of MDR-associated markers, including P-170, MRP and LRP and cytoskeletal proteins, in three resistant variants of the human ovarian carcinoma cell line, OAW42. Eur J Cancer 33: 652-660.
Philchenkov A, Zavelevich M, Kroczak TJ, and Los M (2004) Caspases and cancer: mechanisms of inactivation and new treatment modalities. Exp Oncol 26: 82-97.[Medline]
Prabhakaran K, Li L, Borowitz JL, and Isom GE (2004) Caspase inhibition switches the mode of cell death induced by cyanide by enhancing reactive oxygen species generation and PARP-1 activation. Toxicol Appl Pharmacol 195: 194-202.[CrossRef][Medline]
Reed JC (2004) Apoptosis mechanisms: implications for cancer drug discovery. Oncology 18: 11-20.
Scholz C, Wieder T, Stärck L, Essmann F, Schulze-Osthoff K, Dörken B, and Daniel PT (2005) Arsenic trioxide triggers a regulated form of caspase-independent necrotic cell death via the mitochondrial death pathway. Oncogene 24: 1904-1913.[CrossRef][Medline]
Seol DW and Billiar TR (1999) A caspase-9 variant missing the catalytic site is an endogenous inhibitor of apoptosis. J Biol Chem 274: 2072-2076.
Shiozaki EN, Chai J, and Shi Y (2002) Oligomerization of caspase-9 induced by Apaf-1 CARD. Proc Natl Acad Sci USA 99: 4197-4202.
Shou Y, Gunassekar PG, Borowitz JL, and Isom GE (2000) Cyanide-induced apoptosis involves oxidative-stress-activated NF-
B in cortical neurons. Toxicol Appl Pharmacol 164: 196-205.[CrossRef][Medline]
Soranzo C, Perego P, and Zunino F (1990) Effect of tumor necrosis factor on human tumor cell lines sensitive and resistant to cytotoxic drugs and its interaction with chemotherapeutic agents. Anticancer Drugs 1: 157-163.[Medline]
Srinivasula SM, Ahmad M, Guo Y, Zhan Y, Lazebnik Y, Fernandes-Alnemri T, and Alnemri ES (1999) Identification of an endogenous dominant-negative short isoform of caspase-9 that can regulate apoptosis. Cancer Res 59: 999-1002.
Waltereit R and Weller M (2002) The role of caspases 9 and 9-short (9S) in death ligand- and drug-induced apoptosis in human astrocytoma cells. Mol Brain Res 106: 42-49.[Medline]
Wick W, Grimmel C, Wagenknecht B, Dichgans J, and Weller M (1999) Betulinic acid-induced apoptosis in glioma cells: a sequential requirement for new protein synthesis, formation of reactive oxygen species and caspase processing. J Pharmacol Exp Ther 289: 1306-1312.
Zuco V, Supino R, Righetti SC, Cleris L, Marchesi E, Gambacorti-Passerini C, and Formelli F (2002) Selective cytotoxicity of betulinic acid an tumor cell lines, but not on normal cells. Cancer Lett 175: 17-25.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  G. J. Arismendi-Morillo and A. V. Castellano-Ramirez Ultrastructural mitochondrial pathology in human astrocytic tumors: potentials implications pro-therapeutics strategies J. Electron Microsc. (Tokyo), January 1, 2008; 57(1): 33 - 39. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Ceruti, A. Mazzola, and M. P. Abbracchio Proteasome Inhibitors Potentiate Etoposide-Induced Cell Death in Human Astrocytoma Cells Bearing a Mutated p53 Isoform J. Pharmacol. Exp. Ther., December 1, 2006; 319(3): 1424 - 1434. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||
