Parthenolide sensitizes ultraviolet (UV)-B-induced apoptosis via protein kinase C-dependent pathways

Yen-Kim Won, Choon-Nam Ong and Han-Ming Shen *

Department of Community, Occupational and Family Medicine, Yong Loo Lin School of Medicine, National University of Singapore, 16 Medical Drive, Singapore 117597, Republic of Singapore

* To whom the correspondence should be addressed. Tel: +65 6874 4998; Fax: +65 6779 1489; Email: cofshm{at}nus.edu.sg


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Parthenolide (PN) is the principal sesquiterpene lactone in feverfew (Tanacetum parthenium) with proven anti-inflammatory properties. We have previously reported that PN possesses strong anticancer activity in ultraviolet B (UVB)-induced skin cancer in SKH-1 hairless mice. In order to further understand the mechanism(s) involved in the anticancer activity of PN, we investigated the role of protein kinase C (PKC) in the sensitization activity of PN on UVB-induced apoptosis. Several subtypes of PKC have been reported to be involved in UVB-induced signaling cascade with both pro- and anti-apoptotic activities. Here we focused on two isoforms of PKC: novel PKC{delta} and atypical PKC{zeta}. In JB6 murine epidermal cells, UVB induces the membrane translocations of both PKCs, and PN pre-treatment enhances the membrane translocation of PKC{delta}, but inhibits the translocation of PKC{zeta}. Similar results were also detected when the activities of these PKCs were tested with the PKC kinase assay. Moreover, pre-treatment with a specific PKC{delta} inhibitor, rotterlin, completely diminishes the sensitization effect of PN on UVB-induced apoptosis. When cells were transiently transfected with dominant negative PKC{delta} or wild-type PKC{zeta}, the sensitization effect of PN on UVB-induced apoptosis was also drastically reduced. Further mechanistic study revealed that PKC{zeta}, but not PKC{delta}, is required for UVB-induced p38 MAPK activation and PN is likely to act through PKC{zeta} to suppress p38 activation in UVB-treated JB6 cells. In conclusion, we demonstrated that PN sensitizes UVB-induced apoptosis via PKC-dependent pathways.

Abbreviations: AP-1, activator protein-1; DAG, diacylglycerol; DN, dominant negative; MAPKs, mitogen activated protein kinases; NF-{kappa}B, nuclear transcription factor-kappa B; NF-AT, nuclear factor of activated T cells; PKC, protein kinase C; PN, parthenolide; UVB, ultraviolet B light


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Feverfew (Tanacetum parthenium) has been used as a herbal plant for centuries in Europe, with known anti-microbial and anti-inflammatory properties (1,2). Parthenolide (PN), a sesquiterpene lactone, is one of the principal bioactive components of this plant. It is believed that the bioactivity of PN is mediated via the highly electrophilic {alpha}-methylene-{gamma}-lactone ring and an epoxide residue which are capable of interacting rapidly with nucleophilic sites of biological molecules (3,4). PN is a potent inhibitor of DNA synthesis and cell proliferation in a number of cancer cell lines (57). In addition, PN has been reported to induce apoptosis via caspase activation and mitochondria dysfunction (8), disruption in intracellular thiols and calcium equilibrium (9), as well as activation of pro-apoptotic Bcl-2 family proteins (10). On the other hand, PN also has been shown to sensitize cells to apoptosis induced by various stimuli such as ultraviolet B (UVB), tumor necrosis factor-{alpha} (TNF-{alpha}) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), presumably through its action on activator protein-1 (AP-1) signaling pathway (1113). The in vivo anticancer activity of PN has been studied recently. For instance, we have demonstrated that PN possesses a strong chemopreventive property against UVB-induced skin cancer in SKH-1 hairless mice (12), whereas Sweeney et al. (14) revealed that PN in combination with docetaxel is capable of reducing metastasis and improving survival in the xenograft model of breast cancer.

The electromagnetic spectrum of ultraviolet (UV) can be grouped into UVA (320–400 nm), UVB (280–320 nm) and UVC (200–280 nm). UVB exposure is the main etiological factor for non-melanoma skin cancer in human (15). The mechanism(s) of UVB-induced skin cancer have not been fully understood. Several transcriptional factors including AP-1, nuclear transcription factor-kappa B (NF-{kappa}B), nuclear factor of activated T cells (NF-AT), and signal transducers and activators of transcription have been linked to the tumor-promoting ability of UVB (1619).

Protein kinase C (PKC) is a group of serine/threonine kinases that regulate many cellular functions such as proliferation, differentiation, transformation, survival and apoptosis (20). PKC can be classified into three groups based on the co-factors required for activation: (i) the Ca2+ and diacylglycerol (DAG)-dependent classical or conventional PKC that consists of isotypes {alpha}, ß1, ß2 and {gamma}; (ii) the DAG-dependent, Ca2+-independent novel PKC that consist of {delta}, {eta}, {varepsilon} and {theta}; and (iii) the DAG- and Ca2+-independent atypical PKC that consist of {iota}/{lambda} and {zeta}. The consequences of PKC activation by UVB is rather cell-type specific and could lead to inhibition on cell proliferation or even induction of apoptosis. Among all, PKC{delta} seems to be the main PKC subtype with pro-apoptotic functions in response to various extracellular stimuli including UVB (21,22), whereas PKC{zeta} has been shown to be anti-apoptotic in response to UV (23,24).

Our previous findings (12) showed that the suppression of mitogen activated protein kinase (MAPK) and AP-1 signaling cascade by PN contributes to its sensitization effect on UVB-induced apoptosis in JB6 cells. It is also known that certain subtypes of PKC regulate the MAPK-AP-1 pathway in UVB-treated cells (21,25). Hence, the main objective of this study is to explore the involvement of PKC to further understand the underlying mechanism(s) of the chemopreventive property of PN. Our data demonstrated here that PN selectively inhibits UVB-induced PKC{zeta} activation and subsequent p38 MAPK activation, while further enhancing UVB-induced PKC{delta} activity; both contribute to the sensitization effect of PN on UVB-induced apoptosis in murine epidermal JB6 cells.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals and reagents
PN (97% pure) was purchased from Biomol (Plymouth Meeting, PA). Anti-HA, PKC-{delta} and {zeta} polyclonal antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). Secondary antibodies (horseradish peroxidase conjugated goat anti-rabbit IgG and goat anti-mouse IgG) and enhanced chemiluminescence substrate were from Pierce (Rockford, IL). {gamma}-p32 ATP was obtained from Perkin–Elmer (Boston, MA). PKC inhibitors GF109203X and rotterlin were purchased from Calbiochem (San Diego, CA). Other common chemicals were from Sigma–Aldrich (St Louis, MO).

Cell culture and UVB exposure
JB6 murine epidermal cells were cultured in minimum essential medium (MEM) supplemented with 5% fetal bovine serum (FBS) and 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in 5% CO2/air atmosphere as described previously (12). Cells were seeded in 60 mm cultural dishes and starved with MEM containing 0.5% FBS for 24 h after reaching 80% confluence. After pre-treatment with various reagents at designated conditions, cells were washed with PBS once and then exposed to UVB in fresh PBS. UVB was delivered through a bank of FS24 lamps (Light Sources, Orange, CT) with spectral irradiance of 280–400 nm, 80% of which was in the UVB region (280–320 nm) with a peak at ~313 nm. The emitted UVB dose was quantified using a phototherapy radiometer (International Light, Newburyport, MA) equipped with IL SED 240 detector. Cells were returned to the incubator with the addition of the previous culture medium until the time of collection.

Transient transfection
Wild-type and dominant negative (DN) PKC{delta} and PKC{zeta} plasmids were kindly provided by Dr J.W.Soh from Inha University, Incheon, Korea. DN-p38{alpha} and DN-p38ß2 plasmids were gifts from Dr J.Han (Scripps Research Institute, La Jolla, CA, USA). Cells were co-transfected with designated PKC plasmids and a transfection marker pDsRed (Clontech, Palo Alto, CA) using LipofectAMINE reagent (Invitrogen, Carlsbad, CA) according to manufacturer's protocol. Cells were subjected to various treatments 24 h after transfection.

Detection of apoptotic cell death
Following the designated treatments, apoptotic cell death was quantified using DNA content analysis (sub-G1 cells) as described previously (26). In some experiments, cells were first transiently transfected with various PKC plasmids and pDsRed. Apoptotic cell death was then determined using DNA content analysis coupled with flow cytometry after 4',6-diamidino-2-phenylindole (DAPI) staining. Briefly, cells were washed with phosphate buffered saline (PBS), fixed first with 0.5% para-formaldehyde and then with 70% ethanol. After staining with DAPI, 20 000 cells from each group were analyzed by flow cytometry using Becton Dickinson FACSVantage SE system (Franklin Lakes, NJ) (27). Only those transfected cells with expression of the red fluorescence protein were then gated for analyzing the percentage of sub-G1 cells using WinMDI 2.7 software (Scripps Institute, La Jolla, CA).

PKC translocation assay
PKC translocation assay was performed based on a published method (21) with modifications. In brief, 15 min after UVB irradiation cells were washed once with ice-cold PBS and then sonicated in homogenization buffer A [20 mM Tris–HCl (pH 8.0), 10 mM EGTA, 2 mM EDTA, 2 mM DTT, 1 mM PMSF and protease inhibitor cocktail] for 10 s on ice. The lysate was then centrifuged at 100 000 g for 1 h at 4°C. The supernatant was collected as the cytosolic fraction. The pellet was then resuspended in homogenization buffer B (with 1% Triton X-100 in buffer A) and sonicated for another 10 s on ice. The suspension was centrifuged at 15 000 g for 15 min at 4°C. The supernatant was collected as the membrane fraction. Using 8% SDS–polyacrylamide gel 30 µg of proteins were separated in Mini-Protein II system (Bio-Rad, Hercules, CA). Following electrophoresis the protein was transferred to a PVDF membrane (Millipore, Bedford, MA) and subsequently hybridized with anti-PKC{delta} and anti-PKC{zeta} antibodies. The blots were detected using the enhanced chemiluminescence method (Pierce). The blots were scanned using Kodak Image Station (New Haven, CT) and the densitometric measurements of the bands were performed using Kodak 1D 3.5 software.

PKC kinase assay
PKC kinase assay was performed according to a published method (28) with modifications. Briefly, cells were harvested 30 min after UVB irradiation in PKC lysis buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 0.1% Tween-20, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, 0.1 mM PMSF, 1 mM NaF, 0.1 mM Na3VO4, 10 mM ß-glycerophosphate and protease inhibitor cocktail]. The cell lysate was then centrifuged at 12 000 g for 15 min at 4°C and the supernatant was collected as cellular protein. Four hundred micrograms of protein were immunoprecipitated with anti-PKC antibodies (4 µg) overnight and followed by incubation with protein G-sepharose for 1 h. The immunoprecipitates were washed five times with ice-cold kinase buffer [50 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM DTT, 2.5 mM EGTA, 1 mM NaF, 0.1 mM Na3VO4 and 10 mM ß-glycerophosphate]. The kinase assay was initiated by adding 30 µl of kinase buffer containing 10 µg of GST-MARCKS substrate, 0.5 µCi of [{gamma}-32P]ATP and protease inhibitor cocktail. The reaction were performed for 30 min at 30°C and was terminated by adding 3x sampler buffer. All reaction mixes were then boiled for 5 min before being separated on 10% SDS–polyacrylamide gel in Mini-Protein II system (Bio-Rad). Gels were then dried and exposed to an X-ray film (Kodak) at room temperature.

Statistical analysis
All numeric data are presented as mean ± standard deviations (SD) from at least three independent experiments and analyzed using one-way ANOVA with Student-Newman–Keul as post hoc comparison. A P-value < 0.05 is considered as statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
PN sensitizes UVB-induced apoptosis via PKC-dependent pathways
Our previous findings demonstrated that PN is capable of sensitizing JB6 cells to UVB-induced apoptosis via the MAPK-AP-1 signaling pathway (12). It is known that UVB activates PKC, and PKC regulates the MAPK-AP-1 signaling pathway (29). Here we first investigated if PKC plays a role in PN-sensitized UVB-induced apoptosis by using 2 PKC inhibitors. As shown in Figure 1A, pre-treatment with GF109203X, a pan-PKC inhibitor also sensitizes cells to UVB-induced apoptosis, although to a lesser extent than PN. In contrast, pre-treatment with a specific PKC{delta} inhibitor, rotterlin, is capable of completely protecting cells from apoptosis induced by UVB alone or PN plus UVB (Figure 1B). Data from this study thus suggested that PKC{delta} is critical in both UVB and PN-UVB-induced apoptosis, consistent with some of the earlier reports that PKC{delta} possesses mainly pro-apoptotic functions in response to various extracellular stimuli (21,22).



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Fig. 1. Involvement of PKC in cell death induced by PN-UVB. (A) Pre-treatment with a pan-specific PKC inhibitor (GF109203X); (B) pre-treatment with a specific PKC{delta} inhibitor (rotterlin). JB6 cells were pre-treated with 5 µM PN for 2 h, 20 µM GF109203X or 1 µM rotterlin for 1 h and then subjected to 50 mJ/cm2 of UVB. In some groups, cells were first pre-treated with GF109203X or rotterlin for 1 h, followed by PN and UVB. Apoptosis was quantified with DNA content/sub-G1 analysis 24 h after UVB irradiation. Data were presented in mean ± SD from three independent experiments. An asterisk indicates statistically significant comparison with the untreated control group (P < 0.05).

 
PN selectively regulates different isoforms of PKC in UVB-induced activations
UVB is known to activate certain subtypes of PKC such as PKC{delta} and PKC{zeta} (21,23). One of the critical events of PKC activation is the translocation from the cytosol to membrane (30). In this study, we first measured UVB-induced PKC activation by determining PKC membrane translocation. Figure 2 provides convincing evidence that UVB-induced membrane translocations of PKC{delta} and PKC{zeta} 15 min post-irradiation in JB6 cells. Similar changes were also observed in other PKC isoforms such as PKC{eta} and PKC{lambda} (data not shown). When cells were pre-treated with 5 µM of PN, the UVB-induced translocation of PKC{zeta} was significantly inhibited (Figure 2B) while the translocation of PKC{delta} was further enhanced (Figure 2A). A consistent pattern of changes was also detected when the activation of PKC was measured using the in vitro PKC kinase assay: PN inhibited the UVB-induced activation of PKC{zeta} (Figure 3B) while further enhancing that of PKC{delta} (Figure 3A).



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Fig. 2. PKC activation in cells treated with PN and UVB measured by PKC membrane translocation. (A) PN enhanced UVB-induced PKC{delta} membrane translocation; (B) PN inhibited UVB-induced PKC{zeta} membrane translocation. Cells were pre-treated with 5 µM PN for 2 h, 20 µM GF109203X or 1 µM rotterlin for 1 h and then subjected to 50 mJ/cm2 of UVB. Cells were harvested 15 min after UVB irradiation. Using 8% SDS–polyacrylamide gels 30 µg of cytosolic or membrane proteins were separated and blotted with respective anti-PKC antibodies. The blots were scanned and the densitometric measurements of the bands were performed.

 


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Fig. 3. PKC activation in cells treated with PN and UVB measured by PKC kinase assay. (A) PN enhanced UVB-induced PKC{delta} kinase activity; (B) PN inhibited UVB-induced PKC{zeta} kinase activity. Cells were treated as described in Figure 2 and harvested 30 min after UVB irradiation. Cell lysate was immunoprecipitated with anti-PKCs antibodies and then subjected to PKC kinase assay as described in Materials and methods. Data were quantified as described in Figure 2.

 
In order to further understand the differential roles of PKC isoforms on the sensitization effect of PN on UVB-induced apoptosis, cells were transiently transfected with wild-type or DN forms of PKC{delta} or PKC{zeta} plasmids, together with pDsRed as the transfection marker, followed by PN-UVB treatment. When the morphological changes of apoptotic cell death were examined under an inverted fluorescence microscope, it was found that the DN-PKC{delta} transfected cells became rather resistant to PN-UVB-induced apoptosis whereas the wild-type PKC{delta} transfected cells underwent massive apoptosis upon PN-UVB treatment (Figure 4). On the contrary, overexpression of wild-type PKC{zeta} offered significant protection against PN and UVB-induced apoptosis (Figure 4). Such findings are basically consistent with the effect of PKC inhibitors as shown earlier (Figure 1A and B).



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Fig. 4. Impact of PKC overexpressions on apoptosis induced by PN-UVB. JB6 cells were transiently transfected with pcDNA, wild-type (wt) or DN forms of PKC{delta} or PKC{zeta} and a transfection marker pDsRed as described in Materials and methods. Twenty-four hours after transfection, cells were pre-treated with 5 µM of PN for 2 h and then subjected to 50 mJ/cm2 of UVB. The morphological changes of apoptotic cell death were examined under an inverted fluorescence microscope 24 h after UVB irradiation. Cells with successful transfection were marked in red.

 
In order to obtain more quantitative data, we used another approach by analyzing DNA content/sub-G1 cells among those transfected cells (Figure 5A). Being consistent with the morphological changes, cells transfected with DN-PKC{delta} or wild-type PKC{zeta} were resistant to PN and UVB-induced apoptotic cell death (Figure 5B and C). In contrast, overexpression of wild-type PKC{delta} or DN-PKC{zeta} significantly enhanced cell death induced by PN and UVB treatment (Figure 5B and C).



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Fig. 5. Effect of PKC expression on apoptosis induced by PN-UVB. (A) Apoptotic cell death was determined with DNA content/sub-G1 analysis coupled with flow cytometry after DAPI staining. JB6 cells were first transiently transfected with various PKC plasmids and pDsRed as a transfection marker, followed by being treated with PN and UVB. In a total of 20 000 cells from each group analyzed using flow cytometry, only those transfected cells with expression of the red fluorescence protein were then selected for further analysis for percentage of sub-G1 cells. (B) Effect of wild-type (wt)- and DN-PKC{delta} expression on PN-UVB-induced apoptosis in JB6 cells. (C) Effect of wild-type (wt)- and DN-PKC {zeta} expression on PN-UVB-induced apoptosis in JB6 cells. In both (B) and (C), JB6 cells were first transfected for 24 h, followed by treatment with PN and UVB. Cells were collected for DNA content analysis 24 h after UVB irradiation. Data were presented in mean ± SD from four independent experiments. An asterisk indicates statistically significant comparison with the group transfected with pcDNA only (P < 0.05).

 
PKC{zeta} acts upstream of p38 MAPK but not JNK
Our previous findings illustrated that the MAPK-AP-1 pathway is one of the molecular targets of PN (12). Certain PKC isoforms have been shown to regulate the MAPK signaling cascade. For example, PKC{delta} has been reported to affect the UVB-induced phosphorylations of Erk 1/2 and JNK (21) while PKC{zeta} primarily targets Erk 1/2 (25). Since PKCs are known to respond differently depending on cell type and stimuli, we set out to identify the effect of PKC{delta} and {zeta} on MAPK activation in our experimental system. Here we first confirmed our previous findings that PN pre-treatment is capable of blocking UVB-induced p38 activation, similar to the effect of a specific p38 inhibitor SB203580 (Figure 6A). Interestingly, pre-treatment with a pan-specific PKC inhibitor, GF109203X, inhibited the UVB-induced p38 activation to a certain extent, while the PKC{delta} specific inhibitor, rotterlin, has no effect on p38 activation (Figure 6A). Next we examined the relationship between PKC and p38 activation using genetic approaches. When cells were transfected with wild-type or DN-PKC{delta}, neither UVB-induced p38 activation nor the inhibitory effect of PN was affected as compared with the pcDNA-transfected control (Figure 6B). Intriguingly, overexpression of wild-type PKC{zeta} abolished the inhibitory effect of PN on UVB-induced p38 phosphorylation, while the overexpression of DN-PKC{delta} even completely blocked UVB-induced p38 activation (Figure 6C). Therefore, these data clearly suggest that it is PKC{zeta}, but not PKC{delta}, that is responsible for UVB-induced p38 activation.



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Fig. 6. Effect of PKC on p38 activation in UVB-treated cells. (A) PN and pan-specific PKC inhibitor GF109203X, but not the specific PKC{delta} inhibitor rotterlin, inhibited UVB-induced p38 phosphorylation. Cells were pre-treated with 5 µM PN for 2 h, 20 µM GF109203X or 1 µM rotterlin for 1 h and then subjected to 50 mJ/cm2 of UVB. (B) Overexpressions of wild-type (wt)- or DN forms of PKC{delta} did not affect p38 activation induced by either UVB alone or PN plus UVB. (C) Overexpression of DN-PKC{zeta} blocked UVB-induced p38 activation and overexpression of wt-PKC{zeta} reversed the inhibitory effect of PN on UVB-induced p38 activation. JB6 cells were first transiently transfected with pcDNA, wt- or DN forms of PKC{delta} or PKC{zeta} for 24 h, followed by treatment with PN and UVB. Cells were harvested 30 min after UVB irradiation. Thirty µg of cellular proteins were separated on 10% SDS–polyacrylamide gels and the subsequent membranes were hybridized with anti-p-p38, p38 and HA antibodies. The western blot data were collected and quantified as described in Figure 2.

 
Since PN is able to inhibit the phosphorylation of JNK induced by UVB as shown previously (12), here we also examined whether PKC has any functional role in UVB-induced JNK activation. As shown in Figure 7A, the two PKC inhibitors had no effect on UVB-induced JNK activation. Furthermore, overexpressions of the two DN forms of PKC{delta} and PKC{zeta} had no effect on either UVB-induced JNK activation or the inhibitory effect of PN (Figure 7B). Unlike the previous findings (21,25), the total levels of MAPKs are not affected by the overexpressions of either the wild-type or DN forms of PKC{delta} and PKC{zeta}. It is thus believed that UVB-induced JNK activation is independent of PKC activation.



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Fig. 7. No evident effect of PKC on JNK activation in UVB-treated cells. (A) PKC inhibitor GF109203X and rotterlin has no evident effect on UVB-induced JNK activation. JB6 cells were treated as described in Figure 6A. (B) Overexpressions of DN forms of PKC{delta} and PKC{zeta} failed to affect JNK activation induced by UVB alone or PN plus UVB. JB6 cells were transiently transfected with pcDNA, DN forms of PKC{delta} or PKC{zeta} for 24 h, followed by treatment with PN and UVB. Cells were harvested 30 min after UVB irradiation. The phosphorylated JNK was determined by western blot and quantified as described in Figure 2.

 
In order to further confirm the functional linkage between PKC{zeta} and p38 activation in protection of apoptosis induced by PN and UVB, JB6 cells were transiently transfected with the wild-type PKC{zeta} together with DN-p38{alpha} and DN-p38ß2 plasmids. As shown in Figure 8A, overexpression of DN-p38{alpha} and DN-p38ß2 completely inhibited UVB-induced p38 activation. More importantly, while the overexpression of wild-type PKC{zeta} offered significant protection against apoptosis induced by PN-UVB, the co-transfection of the DN forms of p38 protein abolished the protective effect of wild-type PKC{zeta} and greatly sensitized cells to PN-UVB-induced apoptosis (Figure 8B). Such observations thus provide strong evidence that the anti-apoptotic function of PKC{zeta} is achieved via p38 activation and PN is likely to act through PKC{zeta} to suppress p38 activation, and then enhance apoptosis in UVB-treated JB6 cells.



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Fig. 8. Functional relationship between PKC{zeta} and p38 in UVB-induced apoptosis. (A) Overexpressions of DN-p38{alpha} and DN-p38ß2 completely blocked the UVB-induced p38 phosphorylation. JB6 cells were transiently transfected with DN-p38{alpha} and DN-p38ß2 for 24 h, followed by UVB exposure (50 mJ/cm2). Cells were harvested 30 min after UVB irradiation. Thirty µg of cellular proteins were separated on 10% SDS–polyacrylamide gels and the subsequent membranes were hybridized with anti-p-p38, p38 and Flag antibodies. (B) Overexpressions of DN-p38{alpha} and DN-p38ß2 completely abolished the protective effect of wilt-type (wt)-PKC{zeta} on apoptosis induced by PN and UVB. Cells were transfected with wt-PKC{delta}, with or without DN-p38{alpha} and DN-p38ß2 for 24 h, followed by treatment with PN and UVB. Cells were collected for DNA content analysis for sub-G1 cells 24 h after UVB irradiation. Data were presented in mean ± SD from three independent experiments. An asterisk indicates statistically significant comparison with the group transfected with pcDNA only (P < 0.05).

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Even though the underlying mechanism(s) of the carcinogenic ability of UVB have not been fully understood, a number of molecular targets have been identified to be involved in UVB carcinogenesis, which include AP-1, NF-{kappa}B, NF-AT and STATs (1619). We have previously shown that PN possesses a strong chemopreventive property against UVB-induced skin cancer via its potent inhibitory effect on the MAPK-AP-1 signaling pathway (12). Here we further demonstrated that PN selectively regulates the activities of PKC: promotion of the pro-apoptotic PKC{delta} and suppression of the pro-survival PKC{zeta}. Moreover, we also provided evidence that PN acts on PKC{zeta} upstream of p38 to sensitize UVB-induced apoptotic cell death. Thus, we reveal a new mechanism involved in the anticancer function of PN.

PKC can be classified into 11 isoforms based on the co-factors required for activation. UVB is known to activate certain PKC isoforms such as PKC{delta}, {varepsilon}, {zeta}, {lambda}/{iota} and {eta}, leading to apoptosis or cell survival (21,23,25,31). It has been reported that UVB induces phospholipase A2 activation and arachidonic acid release, and activates PKC (32). Furthermore, UVB irradiation of cultured cells is also known to elevate levels of DAG (33) as well as to generate reactive oxygen radicals that may activate PKC (34). Among all the UVB-activated PCK isoforms, PKC{delta} seems to be the main subtype involved in apoptotic signaling induced by various stimuli including UVB (22). It is generally believed that activated PKC{delta} decreases mitochondrial membrane potential, resulting in cytochrome c release, caspase activation and apoptosis (22). On the other hand, PKC{zeta} has been shown to promote cell survival by either stimulating the nucleotide excision repair activity (35) or phosphorylating Rel A and subsequently activating the NF-{kappa}B survival pathway (36).

One important observation of this study is the differential effect by PN on different PKC isoforms in cells treated with UVB: PN selectively enhances the pro-apoptotic PKC{delta} and suppresses the anti-apoptotic PKC{zeta} (Figures 2 and 3). Indeed, selective effects on different PKC isoforms have been reported previously. Sodium butyrate has been shown to upregulate PKC{varepsilon} while downregulating PKCß during erythroid differentiation (37). UCN-01, a staurosporine analog, has been illustrated to have selective effect on different PKC isoforms used in vitro kinase assay (38). In addition, a diazene carbonyl derivative diamide, which oxidizes thiols to disulfides through addition/displacement reactions at the diazene bond, stimulates the pro-apoptotic PKC{delta} while inactivating the oncogenic PKC{varepsilon} (39).

The selective regulation on PKC{delta} and PKC{zeta} can also be appreciated by the difference in their molecular structures. Although both PKCs contain an N-terminal regulatory domain and a C-terminal catalytic kinase domain, PKC{delta} contains two cysteine-rich C1 subdomains (40) whereas PKC{zeta} has only one zinc finger binding region (41). DAG is known to bind to one of the cysteine-rich C1 subdomains and activate PKC{delta}. In contrast, PKC{zeta} is regulated by other lipid co-factors such as phosphatidylinositol (PI) 3,4,5-P3 and ceramide (42). The exact mechanisms responsible for the differential regulation of PN on UVB-induced PKC activation are currently not known. One possible explanation is related to the involvement of the caspase cascade in PKC activation. For instance, PKC{delta} is cleaved by caspase-3 during apoptosis to a more catalytically active fragment (43). In contrast, caspase-3-dependent cleavage of PKC{zeta} generates a fragment that corresponds to its catalytic domain and is enzymatically inactive (24). Since PN is a potent activator of caspase-3 (8,9), it is possible that both PKC{delta} and PKC{zeta} were cleaved by caspase-3 in response to PN-UVB treatment. As a result, the catalytically active PKC{delta} and the inactive fragment of PKC{zeta} are generated, leading to more profound cell death induced by PN-UVB.

Currently, there is still controversy with regard to the exact role of JNK and p38 in UV-induced apoptosis, with both pro- and anti-apoptotic activities being reported (4446). Our previous findings indicated that PN sensitizes UVB-induced apoptosis by inhibiting both JNK and p38, leading to blockade in the pro-survival AP-1 pathway (12). The link between MAPK and PKC has been reported previously. Chen et al. (21) showed that PKC{delta} and PKC{varepsilon} mediate UVB-induced signal transduction and apoptosis through the activation of ERK and JNK. Furthermore, inhibition of PKC{lambda}/{iota} with a dominant negative mutant suppressed UVB-induced ERK and the subsequent AP-1 activation (47). On the other hand, the antisense oligonucleotide of PKC{zeta} has been shown to inhibit UVB-induced AP-1 activation (25). In this study, we provide convincing evidence suggesting that PKC{zeta} acts upstream of p38, but not JNK, to protect cell death induced by PN-UVB, based on experimental data using both pharmacological and genetic approaches. For instance, overexpression of wide-type PKC{zeta} reverses the inhibitory effect of PN on UVB-induced p38 phosphorylation (Figure 6C), and transfections with wild-type PKC{zeta} together with DN-p38{alpha} and DN-p38ß2 plasmids completely abolishing the protective effect of wild-type PKC{zeta} and greatly sensitizes cells to PN-UVB-induced apoptosis (Figure 8B). Therefore, it is clear that the sensitization activity of PN on UVB-induced apoptosis is probably achieved through its inhibitory effect on the PKC{zeta} and p38 signaling pathway.

In summary, we show for the first time that PN sensitizes JB6 cells to UVB-induced apoptosis through selective regulation on the pro-apoptotic PKC{delta} and the pro-survival PKC{zeta} functions. Furthermore, our data also suggest that the UVB-induced p38 MAPK activation is regulated via a PKC{zeta}-dependent mechanism. These findings may shed new light in understanding the anticancer activity of PN.


    Acknowledgments
 
The authors thank Dr J.W.Soh for providing PKC plasmids, Dr J.Han for giving the DN-p38{alpha} and DN-p38ß2 plasmids, Drs W.Duan and Y.M.Zhu for their help on PKC kinase assay. We also thank S.Y.Zhang, Q.Huang, R.X.Shi, M.Zhao and Y.B.Ong for their technical assistance. Y.K.W. is supported by a research scholarship from the National University of Singapore. This work is supported by a research grant from NUS Academic Research Fund.

Conflict of Interest Statement: None declared.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 17, 2005; revised July 10, 2005; accepted July 22, 2005.





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