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Cancer Research 68, 2795, April 15, 2008. doi: 10.1158/0008-5472.CAN-07-6244
© 2008 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Down-regulation of Caspase-2 by Rottlerin via Protein Kinase C-{delta}–Independent Pathway

Alakananda Basu, Brett Adkins and Chandreyi Basu

Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, Texas

Requests for reprints: Alakananda Basu, Department of Molecular Biology & Immunology, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107. Phone: 817-735-2487; Fax: 817-735-2118; E-mail: abasu{at}hsc.unt.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Protein kinase C-{delta} (PKC{delta}) plays an important role in DNA damage–induced apoptosis. We have previously shown that the PKC{delta} inhibitor rottlerin protects against cisplatin-induced apoptosis acting upstream of caspase-9. In the present study, we have investigated if rottlerin regulates caspase-2 activation. Knockdown of caspase-2 by siRNA inhibited processing of apical caspase-9 and caspase-8, whereas depletion of caspase-9 had little effect on caspase-2 processing. Rottlerin inhibited activation and processing of caspase-9 and caspase-8 and cleavage of poly(ADP)ribose polymerase. We made a novel observation that rottlerin induced down-regulation of caspase-2 but not of caspase-3, caspase-7, caspase-8, or caspase-9. Pharmacologic inhibitors of PKC, such as Gö 6983 and bisindolylmaleimide, or depletion of PKC{delta} by siRNA had no effect on the down-regulation of caspase-2 by rottlerin. The proteasome inhibitor MG132 reversed caspase-2 down-regulation by rottlerin, whereas calpain inhibitor had no effect. These results suggest that rottlerin induces down-regulation of caspase-2 via PKC{delta}-independent but ubiquitin proteasome–mediated pathway. Furthermore, down-regulation of caspase-2 by rottlerin can explain its antiapoptotic function during DNA damage–induced apoptosis. [Cancer Res 2008;68(8):2795–802]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Apoptosis is a genetically determined cell suicidal program that removes unwanted, redundant, and damaged cells and is required to maintain a balance between cell proliferation and cell death (1). Apoptosis is initiated by the activation of caspases, a family of interleukin-1β converting enzyme–like cysteine proteases that specifically cleave proteins after aspartate residues (2–4). Whereas caspase-8 and caspase-9 participate in the initiation phase of apoptosis, caspase-3, caspase-6, and caspase-7 are involved in the execution phase of apoptosis. Activation of these executioner caspases results in the cleavage of critical cellular proteins, including poly(ADP-ribose) polymerase (PARP), DNA-dependent protein kinase, lamin B, and protein kinase C-{delta} (PKC{delta}; refs. 3, 5). Caspase-2 is a unique caspase that can serve both as initiator and executioner caspase (6–10).

There are two major pathways of cell death. The extrinsic pathway is triggered by binding of ligand to members of tumor necrosis factor-{alpha} receptor superfamily, causing activation of procaspase-8 followed by activation of a caspase cascade (11). DNA-damaging agents are known to activate the intrinsic or mitochondrial pathway by inducing the release of mitochondrial cytochrome c, which allows Apaf-1 to interact with the initiator procaspase-9 to form an active apoptosome complex, resulting in activation of effector caspases, such as caspase-3 and caspase-7 (4). Recent studies from various laboratories have established a role for caspase-2 during genotoxic stress–induced apoptosis (7, 12–25).

PKC{delta} is a substrate for caspase-3, and it is believed that proteolytic activation of PKC{delta} is necessary for DNA damage–induced apoptosis (26–29). We have previously shown that rottlerin, a pharmacologic inhibitor of PKC{delta}, inhibits activation of caspase-3 and caspase-9 induced by the DNA-damaging anticancer agent cisplatin (30, 31), suggesting that PKC{delta} acts at an early stage of DNA damage–induced apoptosis that precedes activation of caspase-3 and caspase-9. Several studies showed that caspase-2 is activated early in response to DNA damage acting upstream of mitochondria, suggesting that it functions as an apical caspase (7, 13, 21, 32, 33). Because rottlerin inhibits cisplatin-induced processing of caspase-9, we wanted to know if it acts upstream of caspase-2. We made a novel observation that rottlerin-induced down-regulation of caspase-2 via ubiquitin proteasome–mediated pathway. Furthermore, the ability of rottlerin to induce caspase-2 down-regulation was independent of its ability to inhibit PKC{delta}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials. Rottlerin, Gö 6983, bisindolylmaleimide (BIM) II, MG132, ALLN, PSI, calpeptin, and protease inhibitor cocktail were obtained from Calbiochem. Cisplatin was from Sigma. siRNA SMARTpool against caspase-2, PKC{delta}, and nontargeting SMARTpool siRNA were obtained from Dharmacon. Monoclonal antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and polyclonal antibody to PKC{delta} were from Santa Cruz Biotechnology, Inc. Monoclonal antibody to caspase-2, PARP, and polyclonal antibody to caspase-9 were purchased from BD PharMingen. Mouse monoclonal antibody to caspase-2 was obtained from Imgenex Corp. and rat monoclonal caspase-2 antibody from Alexis. Polyclonal antibody to caspase-2 and caspase substrates were obtained from BioVision. Polyclonal antibody to caspase-3 and monoclonal antibody to caspase-8 were obtained from BioSource/Invitrogen. Horseradish peroxidase–conjugated goat anti-mouse and donkey anti-rabbit antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. Polyvinylidene difluoride (PVDF) membrane was from Millipore, and enhanced chemiluminescence detection kit was from Amersham. Lipofectamine 2000 transfection reagent was obtained from Invitrogen.

Cell culture. HeLa cells were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 2 mmol/L glutamine. Ovarian cancer 2008 cells were maintained in RPMI 1640 supplemented with 5% heat-inactivated FBS and 2 mmol/L glutamine. Cells were kept in a humidified incubator at 37°C with 95% air and 5% CO2.

Knockdown of caspase-2 and PKC{delta}. Control nontargeting siRNA SMART pool or siRNA SMART pool targeted against caspase-2 or PKC{delta} were introduced into HeLa or 2008 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Briefly, cells were seeded 1 d before transfection. Forty-eight hours after siRNA transfection, cells were treated as indicated in the text and processed for Western blot analysis as described before (34, 35).

Immunoblot analysis. Equivalent amounts of total cellular extracts were separated by SDS-PAGE and transferred electrophoretically to PVDF membrane. Immunoblot analyses were performed as described previously (30).

Caspase assay. Cells were treated with rottlerin and cisplatin as indicated in the text. Caspase activity of cell extracts treated with or without cisplatin was determined at 37°C using 40 µmol/L caspase substrate z-VDVAD-AFC, z-IETD-AFC, or z-LEHD-AFC in 50 mmol/L HEPES (pH 7.4), 100 mmol/L NaCl, 10 mmol/L DTT, 1 mmol/L EDTA, 0.1% CHAPS, and 5% glycerol. The release of 7-amino-4-trifluoromethylcoumarin (AFC) was measured at excitation wavelength 400 nm and emission wavelength 505 nm using a SPECTRAMAX 340 microplate reader (Molecular Devices) and SOFTmax PRO software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Rottlerin induces down-regulation of caspase-2. We have previously shown that rottlerin acts upstream of caspase-9 during cisplatin-induced apoptosis (31). Because caspase-2 has been shown to act as an apical caspase during DNA damage–induced apoptosis, we wanted to know if rottlerin acts upstream of caspase-2. Figure 1 shows that caspase-2 siRNA effectively depleted caspase-2 in HeLa cells and ovarian cancer 2008 cells but it did not decrease the levels of other caspases, such as caspase-8, caspase-9, (Fig. 1A), caspase-3, and caspase-7 (data not shown). Because proteolytic processing of caspase-2 is not essential for its activation (36), we also determined caspase activity using a fluorogenic substrate of caspase-2. Both constitutive and cisplatin-induced caspase-2 activity was attenuated in cells transfected with caspase-2 siRNA compared with cells transfected with control siRNA (Fig. 1B). Depletion of caspase-2 was associated with inhibition of cisplatin-induced proteolytic processing of caspase-8 and caspase-9, and cleavage of PARP and PKC{delta} in both HeLa and 2008 cells (Fig. 1A). The effect of caspase-2 knockdown on the inhibition of caspase activation and cleavage of PARP or PKC{delta} was more pronounced in HeLa cells compared with 2008 cells, presumably because reduction of caspase-2 level by siRNA was less in 2008 cells compared with HeLa cells. These results suggest that depletion of caspase-2 inhibits cisplatin-induced apoptosis.


Figure 1
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  		Figure 1. Knockdown of caspase-2 inhibits cisplatin-induced activation of caspases (cas). A, HeLa cells or ovarian cancer 2008 cells were transfected with either control nontargeting or caspase-2 siRNA as described under Materials and Methods. After 48 h, cells were treated with indicated concentrations of cisplatin for 2 h and then incubated in drug-free medium for 24 h. Western blot analyses were performed with total cell lysates using indicated antibodies. Arrows, processed forms of caspases, PARP, and PKC{delta}. GAPDH was used to control for loading differences. B, caspase-2 activity was determined using VDVAD-AFC as the substrate as described under Materials and Methods. C, HeLa cells were transfected with either control nontargeting, caspase-2, or caspase-9 siRNA. After 48 h, cells were treated with 25 µmol/L cisplatin for the indicated time period, and Western blot analyses were performed with indicated antibodies. Tubulin was used to control for equal loading. Results are representative of at least two separate experiments.

 
Because caspase-9 is believed to be the initiator caspase during DNA damage–induced apoptosis, we compared how depletion of caspase-2 or caspase-9 affects cisplatin-induced processing of each other in HeLa cells (Fig. 1C). Although knockdown of caspase-2 decreased processing of caspase-8 and caspase-9, depletion of caspase-9 did not decrease the processing of these caspases as determined by the amount of cleaved caspases. Knockdown of either caspase-2 or caspase-9 decreased cisplatin-induced cleavage of PARP, although caspase-2 knockdown was more effective. These results suggest that caspase-2 acts upstream of caspase-9 during cisplatin-induced apoptosis.

We then examined the effect of rottlerin on cisplatin-induced caspase activation. We monitored processing of caspases by Western blot analysis (Fig. 2 ). Because activation of caspase-2 and caspase-9 may not require proteolytic processing (36), we analyzed samples from the same experiment for caspase activity using fluorogenic peptide substrates (Table 1 ). Pretreatment of 2008 cells with rottlerin inhibited cisplatin-induced activation of caspase-2, caspase-8, and caspase-9 (Table 1) and processing of caspase-8 and caspase-9 (Fig. 2). Interestingly, rottlerin induced down-regulation of caspase-2. Figure 3 shows that treatment of HeLa cells with rottlerin for 7 hours or longer induced down-regulation of caspase-2 but not of other caspases, such as caspase-3, caspase-7, and caspase-9. We have previously shown that cisplatin or rottlerin had no effect on caspase-2 using a caspase-2 antibody purchased from Transduction Laboratories (30). This antibody was later discontinued by the company. We therefore used several antibodies that recognize different epitopes to monitor down-regulation of caspase-2. These antibodies also showed decrease in caspase-2 in cells treated with caspase-2 siRNA (Fig. 1A and C; data not shown). As shown in Fig. 3, both mouse monoclonal antibody to caspase-2 obtained from BD PharMingen and rat monoclonal antibody from Alexis showed similar time course of caspase-2 down-regulation. Thus, the effect of rottlerin on caspase-2 down-regulation was not due to epitope masking.


Figure 2
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  		Figure 2. Effect of rottlerin on cisplatin-induced caspase activation. 2008 cells were pretreated with 10 µmol/L rottlerin for 45 min and then treated with the indicated concentrations of cisplatin for 20 h. Total cell lysates were processed either for caspase activity assay using fluorogenic substrates (Table 1) or for Western blot analysis. Arrows, processed forms. GAPDH was used to control for loading differences. Results are representative of two separate experiments.

 

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  		Table 1. Effect of rottlerin on cisplatin-induced caspase activation

 

Figure 3
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  		Figure 3. Time course of caspase-2 down-regulation by rottlerin. HeLa cells were treated with 10 µmol/L rottlerin for the indicated time period. Western blot analyses were performed with total cell lysates using the indicated antibodies. Two different caspase-2 antibodies were used (monoclonal caspase-2 antibody from BD PharMingen and rat monoclonal caspase-2 antibody from Alexis). GAPDH was used to control for loading differences. Results are representative of at least two separate experiments.

 
The effect of rottlerin on caspase-2 down-regulation is independent of PKC{delta} inhibition. Because rottlerin is an inhibitor of PKC{delta}, we compared the effects of rottlerin with other inhibitors of PKC on caspase-2 down-regulation. Whereas rottlerin is believed to be a specific inhibitor of PKC{delta} (37), BIM, and Gö 6983 inhibit all PKC isozymes albeit with different potencies (38). cPKCs are most sensitive and can be inhibited at nanomolar concentrations, whereas nPKCs can be inhibited at submicromolar concentrations in in vitro kinase assays. As shown in Fig. 4A , rottlerin induced down-regulation of caspase-2 as well as proteolytic processing of PKC{delta} in both HeLa and 2008 cells. Rottlerin alone caused a slight increase in proteolytic cleavage of PKC{delta} and PARP. This is consistent with our previous report that rottlerin by itself could cause a small increase in cytochrome c release (31). The PKC inhibitors Gö 6983 or BIM caused a modest decrease in the proteolytic activation of PKC{delta} as determined by the levels of catalytic fragment (CF) of PKC{delta}. These inhibitors, however, had little effect on caspase-2 down-regulation in HeLa cells.


Figure 4
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  		Figure 4. Effect of PKC{delta} on caspase-2 down-regulation. A, HeLa and 2008 cells were treated with 10 µmol/L rottlerin (rot), 1 µmol/L BIM, or 1 µmol/L Gö 6983 (Gö) for 30 min and then treated with 5 µmol/L cisplatin for 15 h. Cells were then processed for Western blot analysis. GAPDH was used to control for loading differences. Arrows, processed form of PARP. B, HeLa and 2008 cells were transfected with control (con) or PKC{delta} siRNA as described in the Materials and Methods. Cells were treated with or without 10 µmol/L rottlerin for 18 h, and then total cell lysates were processed for Western blot analysis. C, cells transfected with control or PKC{delta} siRNA were treated with (solid bar) or without (hatched bar) rottlerin as described above. Densitometric quantification of caspase-2 levels from three separate experiments corrected for loading is shown. D, HeLa cells transfected with control or PKC{delta} siRNA were treated with indicated concentrations of cisplatin for 15 h. Western blot analyses were performed with indicated antibodies. Arrows, processed forms of caspases and PARP. Results are representative of at least two separate experiments.

 
Because rottlerin is a pharmacologic inhibitor and it may act on additional targets besides PKC{delta}, we also examined if depletion of PKC{delta} by siRNA leads to down-regulation of caspase-2. siRNA against PKC{delta} caused efficient depletion of PKC{delta} in both HeLa and 2008 (Fig. 4B) cells but did not affect the levels of other PKC isozymes (data not shown). Knockdown of PKC{delta} failed to induce down-regulation of caspase-2 in these cells. In fact, depletion of PKC{delta} seems to increase caspase-2 level slightly. Furthermore, rottlerin induced down-regulation of caspase-2 in PKC{delta}-depleted cells (Fig. 4B and C). These results suggest that the effect of rottlerin on caspase-2 down-regulation was independent of PKC{delta}.

Because PKC{delta} functions as a proapoptotic protein and rottlerin is believed to inhibit PKC{delta}, we examined the consequence of PKC{delta} knockdown on the activation of caspases and cleavage of PARP in HeLa cells. Figure 4D shows that cisplatin caused a concentration-dependent increase in the processing of caspase-2, caspase-8, caspase-9, and caspase-3, and cleavage of PARP. PKC{delta} siRNA effectively depleted PKC{delta} in HeLa cells. Although cisplatin caused a concentration-dependent increase in the levels of catalytic fragment of PKC{delta} in control siRNA–transfected cells, we were unable to detect PKC{delta}-CF in cells transfected with PKC{delta} siRNA. Depletion of PKC{delta} by siRNA, however, attenuated but did not prevent processing of caspases and cleavage of PARP. PKC{delta} knockdown had little effect on the processing of caspases in 2008 cells (data not shown). Thus, the antiapoptotic effect of rottlerin may be independent of PKC{delta}.

Rottlerin induces caspase-2 down-regulation via the proteasome-mediated pathway. We then examined the mechanism by which rottlerin induces down-regulation of caspase-2. Rottlerin induced down-regulation of caspase-2 in both HeLa (Fig. 5A ) and 2008 (Fig. 5B) cells but it did not induce down-regulation of caspase-9, caspase-3, caspase-8, or caspase-7. We compared the ability of several protease inhibitors in preventing caspase-2 down-regulation by rottlerin in HeLa cells (Fig. 5A). Two different proteasome inhibitors, MG132 and PSI, inhibited rottlerin-induced down-regulation of caspase-2 in HeLa cells. ALLN, an inhibitor of the proteasome, cathepsins, and calpains, also inhibited caspase-2 down-regulation by rottlerin. In contrast, the calpain inhibitor calpeptin failed to prevent rottlerin-induced down-regulation of caspase-2. Figure 5B shows that pretreatment of 2008 cells with MG132 before rottlerin treatment also blocked rottlerin-induced down-regulation of caspase-2. These results suggest that rottlerin triggers caspase-2 down-regulation via the proteasome-mediated pathway.


Figure 5
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  		Figure 5. Effect of proteasome inhibitor on caspase-2 down-regulation. A, HeLa cells were pretreated with 10 µmol/L MG132, 50 µmol/L calpeptin (cal), 10 µmol/L ALLN, or 10 µmol/L PSI for 1 h and then treated with 10 µmol/L rottlerin for 18 h. Western blot analyses were performed with indicated antibodies. B, 2008 cells were pretreated with 10 µmol/L MG132 for 1 h and then treated with 10 µmol/L rottlerin for 18 h. Total cell lysates were processed for Western blot analysis with indicated antibodies. GAPDH was used to control for loading differences. Results are representative of at least three experiments.

 
Because MG132 inhibited caspase-2 down-regulation by rottlerin, we examined if MG132 could reverse the effect of rottlerin in protecting cells against cisplatin-induced apoptosis. Figure 6A shows that pretreatment of HeLa cells with MG132 before cisplatin treatment enhanced caspase-2 processing and cleavage of PARP, whereas pretreatment with rottlerin induced caspase-2 down-regulation and inhibited PARP cleavage. Cisplatin-induced processing of caspase-3 was not inhibited by MG132 but was apparently enhanced. The increase in caspase-3 processing by MG132 was associated with an increase in proteolytic processing of PKC{delta} (Fig. 6B). Although pretreatment of cells with MG132 before rottlerin treatment inhibited caspase-2 down-regulation, it failed to enhance cisplatin-induced caspase activation or PARP cleavage (Fig. 6A).


Figure 6
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  		Figure 6. Effect of MG132 and rottlerin on caspase activation. A, HeLa cells were pretreated with or without MG132 for 30 min before treatment with rottlerin for another 30 min. Cells were then treated with or without the indicated concentrations of cisplatin for 14 h. Cells were processed for Western blot analysis with the indicated antibodies. Tubulin was used to control for loading differences. Arrows, processed forms of caspases and PARP. B, HeLa cells were treated with MG132, rottlerin, and cisplatin as described above and processed for Western blot analysis. C, caspase-2 activity was determined using VDVAD-AFC as the substrate as described under Materials and Methods. Columns, mean of four separate measurements; bars, SE.

 
We also examined the effect of MG132 on caspase activity using a fluorometric assay. Consistent with the Western blot analysis (Fig. 6A), MG132 enhanced cisplatin-induced activation of caspase-2, but rottlerin inhibited caspase-2 activation even when cells were pretreated with MG132 (Fig. 6C). Thus, rottlerin may also inhibit caspase-2 activation and processing.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we made a novel observation that rottlerin induces down-regulation of caspase-2, and this may explain the ability of rottlerin to protect against DNA damage–induced apoptosis. Down-regulation of caspase-2 by rottlerin was triggered by the proteasome-mediated pathway. Furthermore, the effect of rottlerin on caspase-2 down-regulation was independent of its ability to inhibit PKC{delta}.

Rottlerin was originally identified as a specific inhibitor of PKC{delta} (37). Since the demonstration that PKC{delta} is a substrate for caspase-3, there have been numerous studies that used rottlerin to link PKC{delta} with DNA damage–induced apoptosis (39). We have previously shown that rottlerin inhibited cisplatin-induced caspase activation and cell death, suggesting that PKC{delta} not only acts as a substrate for caspase-3 but also regulates caspase activation (30). Rottlerin not only inhibited activation of caspase-3 but it also inhibited processing of caspase-9 (31). We proposed that rottlerin acts at an early stage before caspase-9 activation to regulate cisplatin-induced cell death (31).

Caspase-9 is believed to be the apical caspase during DNA damage–induced apoptosis, although recent studies suggest that caspase-2 may act upstream of caspase-9 (7, 13, 21, 24, 32, 33). Our results show that knockdown of caspase-2 inhibited the processing of caspase-9 and caspase-8. In contrast, knockdown of caspase-9 failed to inhibit processing of caspase-2 and caspase-8. These results suggest that caspase-2 may indeed act upstream of caspase-9 during cisplatin-induced apoptosis. Although rottlerin inhibited activation and processing of apical caspase-8 and caspase-9, we made an unexpected observation that rottlerin induced down-regulation of caspase-2. Rottlerin, however, did not influence the levels of other caspases, such as caspase-3, caspase-7, caspase-8, and caspase-9. It is conceivable that down-regulation of caspase-2 by rottlerin may inhibit activation and processing of caspase-9 and downstream caspases.

Because PKC{delta} plays an important role in DNA damage–induced apoptosis, it is conceivable that rottlerin induces caspase-2 down-regulation via PKC{delta}-dependent pathway. However, several studies reported that rottlerin acts on additional targets besides PKC{delta} (40–46). For example, rottlerin acts as a mitochondrial uncoupler independent of its effects on PKC{delta} (40, 43, 46). We therefore compared the effects of rottlerin with other pharmacologic inhibitors of PKC. Rottlerin inhibits PKC isozymes with IC50 values for PKC{delta} (3–6 µmol/L), cPKCs (30–40 µmol/L), and PKC{varepsilon}, PKC{eta}, and PKC{zeta} (80–100 µmol/L; ref. 37). BIM inhibits all PKC isozymes with rank order of potency {alpha} > βI > {varepsilon} > {delta} > {zeta} (38). Although cPKCs are inhibited at nanomolar concentrations, nPKCs are inhibited at submicromolar concentrations. One of the caveats with the use of these broad specificity PKC inhibitors is that they inhibit multiple PKC isozymes, which may have opposite effects on apoptosis. For example, we have shown that inhibition of PKC{alpha} by cPKC-specific inhibitor Gö 6976 or knockdown of PKC{alpha} by siRNA enhanced cisplatin-induced apoptosis (47). Thus, depending on the concentrations of these inhibitors, they may increase or decrease activation of caspases and proteolytic cleavage of PKC{delta} and PARP. We have found that although 1 µmol/L BIM or Gö 6983 attenuated proteolytic cleavage of PKC{delta}, they did not induce caspase-2 down-regulation. Furthermore, silencing of PKC{delta} by siRNA failed to induce caspase-2 down-regulation. These results suggest that the effect of rottlerin on caspase-2 down-regulation was PKC{delta} independent.

PKC{delta} may be activated by cofactors or by proteolytic activation. During apoptosis, cleavage of PKC{delta} at the hinge region by caspase-3 removes the autoinhibitory regulatory domain leading to its proteolytic activation (39). Because down-regulation of caspase-2 prevents activation of downstream caspases, including caspase-3, it also decreases proteolytic activation of PKC{delta}, which is cleaved by caspase-3. To determine if inhibition of PKC{delta} processing by caspase-2 knockdown was responsible for the antiapoptotic effect of rottlerin, we examined the effect of PKC{delta} knockdown on caspase activation and cisplatin-induced apoptosis. Silencing of PKC{delta} had modest effect on the processing of caspase-2 and caspase-9, and cleavage of PARP in HeLa cells, but it had little effect on the processing of caspases in 2008 cells (data not shown). Because rottlerin effectively inhibited processing of these caspases in both cell types, these results suggest that rottlerin acts on additional targets besides PKC{delta}.

To understand how rottlerin induces caspase-2 down-regulation, we compared the effects of several protease inhibitors, including calpain inhibitor calpeptin and proteasome inhibitors MG132 and PSI. Only proteasome inhibitors were able to prevent rottlerin-mediated down-regulation of caspase-2, suggesting that rottlerin triggers caspase-2 down-regulation via proteasome-mediated pathway. Because MG132 prevents caspase-2 down-regulation by rottlerin, we expected that pretreatment with MG132 would reverse the protective effect of rottlerin on cisplatin-induced cell death. However, MG132 had no effect on rottlerin-mediated protection against cisplatin-induced cell death. One possibility is that caspase-2 is not important for cisplatin-induced cell death, and thus, inhibition of caspase-2 down-regulation has no effect on cisplatin-induced cell death. This is unlikely because knockdown of caspase-2 by siRNA inhibited cisplatin-induced cell death. Rottlerin also prevented activation of caspase-2 even when caspase-2 down-regulation was prevented by MG132. Thus, rottlerin not only induces caspase-2 down-regulation but it also inhibits caspase-2 activity.

It has been shown that the proteolytic fragment of PKC{delta} generated by caspase-3–mediated cleavage is necessary for DNA damage–induced apoptosis (26–29). It has also been reported that PKC{delta} is a substrate for caspase-2 because recombinant PKC{delta} was cleaved by recombinant caspase-2 in vitro (19). The authors suggested that caspase-2 is primarily responsible for doxorubicin-induced cleavage of PKC{delta} because 5 µmol/L caspase-3 inhibitor zDEVD partially blocked doxorubicin-induced PKC{delta} cleavage, whereas 10 µmol/L caspase-2 inhibitor zVDVAD completely blocked it. Thus, inhibition of caspase-2 may inhibit proteolysis of PKC{delta}. We, however, failed to detect any cleavage of PKC{delta} in MCF-7 cells (48) that lack functional caspase-3 (49), although these cells express caspase-2 (20). Furthermore, we have shown that caspase-2 acts upstream of caspase-3. Thus, inhibition of caspase-2 by zVDVAD is expected to inhibit caspase-3 and, thus, cleavage of PKC{delta}. Regardless of whether PKC{delta} is cleaved by caspase-3 or caspase-2, rottlerin can still inhibit apoptosis by directly inhibiting the catalytic fragment of PKC{delta} generated by these caspases. Consequently, even when caspase-2 down-regulation is inhibited by proteasome inhibitors, rottlerin can still prevent cisplatin-induced apoptosis. Thus, one possibility is that rottlerin not only acts upstream of caspase-2 to regulate its level, but it also acts downstream of caspase-2 and caspase-3 to directly inhibit PKC{delta} catalytic fragment.

There are, however, controversies whether rottlerin inhibits PKC{delta} or not. Gschwendt et al. (37) have shown that rottlerin directly inhibits PKC{delta} catalytic activity, whereas Davies et al. (50) failed to show inhibition of PKC{delta} activity by rottlerin in vitro. Our results show that knockdown of PKC{delta}, which inhibited cisplatin-induced generation of PKC{delta} catalytic fragment, had modest effect on the processing of caspases, suggesting that rottlerin may act on additional targets besides PKC{delta}. Several studies have shown that mitochondrial uncoupling effect of rottlerin could contribute to apoptosis in a PKC{delta}-independent manner (41, 46). These studies, however, do not explain how rottlerin exerts its antiapoptotic function during DNA damage–induced apoptosis. We have found that rottlerin inhibited processing of caspase-2 as well as caspase-9, caspase-3, and caspase-7 even when caspase-2 down-regulation was inhibited by MG132, suggesting that rottlerin acts upstream of caspase-9 in the presence of the proteasome inhibitor.

The mechanism of caspase-2 activation remains elusive. Caspase-2 can function both as an apical as well as an effector caspase. It has been reported that caspase-2 is processed by caspase-3, and this processing could result in an amplification loop (20). Because caspase-3 is a substrate for PKC{delta} and phosphorylation of caspase-3 by PKC{delta} results in its activation (51), inhibition of PKC{delta} by rottlerin may inhibit caspase-3 and further processing of caspase-2. Blockage of caspase-2 processing may in turn result in inhibition of caspase-3 and processing of PKC{delta}, forming a negative feedback loop. Thus, proteolytic activation of PKC{delta} may be dependent on the generation of the processed form of caspase-2.

In summary, we have shown that caspase-2 functions as an apical caspase during DNA damage–induced apoptosis, and rottlerin acts upstream of caspase-2 via PKC{delta}-independent pathway. We made a novel observation that down-regulation of caspase-2 by rottlerin rather than inhibition of PKC{delta} activity can explain the antiapoptotic function of rottlerin.


   Acknowledgments
 
Grant support: CA85682 and CA71727 (A. Basu) from the NIH/National Cancer Institute.

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.

We thank Dr. Usha Sivaprasad, Shalini Persaud, and Soumya Krishnamurthy for critical reading of the manuscript, and Jiyoung Lee and Rohini Dhar for their assistance with some experiments.

Received 11/14/07. Revised 2/ 7/08. Accepted 2/19/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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