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Cancer Research 67, 6333, July 1, 2007. doi: 10.1158/0008-5472.CAN-06-4344
© 2007 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Inhibition of Heat Shock Protein 27–Mediated Resistance to DNA Damaging Agents by a Novel PKC{delta}-V5 Heptapeptide

Eun-Ho Kim1,3, Hae-June Lee1, Dae-Hoon Lee4, Sangwoo Bae1, Jae-Won Soh5, Dooil Jeoung6, Joon Kim3, Chul-Koo Cho2, Yoon-Jin Lee1 and Yun-Sil Lee1

1 Laboratory of Radiation Effect and 2 Department of Radiation Oncology, Korea Institute of Radiological and Medical Sciences; 3 School of Life Sciences and Biotechnology, Korea University; 4 Seegene, Inc., Seoul, Korea; 5 Laboratory of Signal Transduction, Department of Chemistry, Inha University, Incheon, Korea; and 6 Division of Life Sciences, Kangwon National University College of Natural Sciences, Chuncheon, Korea

Requests for reprints: Yun-Sil Lee, Laboratory of Radiation Effect, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-Dong, Nowon-Ku, Seoul 139-706, Korea. Phone: 82-2-970-1325; Fax: 82-2-970-2402; E-mail: yslee{at}kcch.re.kr.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Heat shock protein 27 (HSP27), which is highly expressed in human lung and breast cancer tissues, induced resistance to cell death against various stimuli. Treatment of NCI-H1299 cells, which express a high level of HSP27, with small interference RNA specifically targeting HSP27 resulted in inhibition of their resistance to radiation or cisplatin, suggesting that HSP27 contributed to cellular resistance in these lung cancer cells. Furthermore, because HSP27 interacts directly with the COOH terminus of the protein kinase C{delta} (PKC{delta})-V5 region with ensuing inhibition of PKC{delta} activity and PKC{delta}-mediated cell death, we wished to determine amino acid residues in the V5 region that mediate its interaction with HSP27. Investigation with various deletion mutants of the region revealed that amino acid residues 668 to 674 of the V5 region mediate its interaction with HSP27. When NCI-H1299 cells were treated with biotin or with FITC-tagged heptapeptide of the residues 668 to 674 (E-F-Q-F-L-D-I), the cells exhibited dramatically increased cisplatin or radiation-induced cell death with the heptapeptide having efficient interaction with HSP27, which in turn restored the PKC{delta} activity that had been inhibited by HSP27. In vivo nude mice grafting data also suggested that NCI-H1299 cells were sensitized by this heptapeptide. The above data strongly show that the heptapeptide of the PKC{delta}-V5 region sensitized human cancer cells through its interaction with HSP27, thereby sequestering HSP27. The heptapeptide may provide a novel strategy for selective neutralization of HSP27. [Cancer Res 2007;67(13):6333–9]


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Apoptosis is responsible for the removal of unwanted or supernumerary cells during development as well as in adult homeostasis (1), and it is the predominant form of cell death triggered by cytotoxic drugs in tumor cells (2). Several members of the protein kinase C (PKC) family serve as substrates for caspases, and the PKC{delta} isozyme is associated with apoptosis. The activation and down-regulation of PKC{delta} have been found to be associated with inhibition of cell cycle progression (3) and tumor promotion (4), respectively, suggesting that PKC{delta} may have a negative effect on cell survival. In addition, the proteolytic activation of PKC{delta} is associated with apoptosis due to DNA damage induced by UV, ionizing radiation, cisplatin, etoposide, arabinoside, and doxorubicin (5–7). Moreover, several studies confirmed that the ectopic expression of a PKC{delta} catalytic fragment results in cell death (8, 9), and Mizuno et al. (8) showed that the kinase activity associated with the PKC{delta} catalytic fragment may be a key participant in the late stage of apoptosis.

Heat shock protein 27 (HSP27), a human form of small heat shock protein (in murine system, HSP25 is present and these two forms have homology of more than 90%), has been suggested to protect cells from apoptotic cell death triggered by hyperthermia, ionizing radiation, oxidative stress, Fas ligand, and cytotoxic drugs (10–12). Several mechanisms have been proposed to account for the HSP27-mediated apoptotic protection. For example, its specific interaction with cytochrome c in cytosol released from mitochondria prevents apoptosome formation (13, 14). The elimination of unfolded protein via the extra lysosomal, energy-dependent, ubiquitin-proteasome degradation pathway is another mechanism that contributes to protection of cells from stress stimuli. HSP27 binds to polyubiquitin chains as well as 26S proteasomes, and the ubiquitin-proteasome pathway is involved in the activation of transcription factor nuclear factor {kappa}B through the degradation of its main inhibitor I{kappa}B{alpha} (15). Moreover, phosphorylated HSP27 has been shown to bind an adaptor protein, Daxx, and then to inhibit Fas-mediated apoptosis (16). Interaction between HSP27 and Akt is also necessary for Akt activation, and this is followed by dissociation of phosphorylated HSP27 from Akt (17). We recently reported that the radioprotective effect of the related HSP25 involves delayed cell growth (18, 19) and HSP25-mediated MnSOD gene expression (20, 21). In addition, attenuation of oxidative stress–induced apoptosis by HSP25 was found to be due to inhibition of the PKC{delta}-mediated extracellular signal–regulated kinase 1/2 pathway and of PKC{delta}-mediated reactive oxygen species production (22). Furthermore, HSP25 was found to bind directly to the V5 region of PKC{delta} and inhibit the PKC{delta} activity, resulting in HSP25-mediated cytoprotection and apoptosis blockade (23).

Overexpression of HSP27 may predict poor response to chemotherapy and radiotherapy and, hence, poor prognosis in general. In addition to its putative involvement in drug resistance and radioresistance, HSP27 has also been shown to be related to cell growth and motility (24, 25). High HSP27 levels may be associated with aggressive growth and infiltration and with poor prognosis (26–28).

In the present study, we showed that amino acid residues 668 to 674 of the V5 region of PKC{delta} was necessary for HSP27 binding. Based on this information, we prepared a novel heptapeptide containing the region required for HSP27 binding and showed that the heptapeptide attenuated HSP27-mediated resistance and neutralized endogenous HSP27 in human lung cancer cells, which frequently express high level of HSP27.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. Human non–small cell lung cancer cell lines, NCI-H23, NCI-H358, NCI-H460, NCI-H596, and NCI-H1299, were grown in RPMI 1640 supplemented with 10% FBS, glutamine, HEPES, and antibiotics at 37°C in a 5% CO2 humidified incubator. Murine fibroblast L929 cells were cultured in DMEM (Life Technologies, Inc.) supplemented with 10% FBS, glutamine, HEPES, and antibiotics at 37°C in a 5% CO2 humidified incubator.

Chemicals and reagents. Anti-HSP27, anti–ß-actin, anti-PKC{delta}, and anti–glutathione S-transferase (GST) were purchased from Santa Cruz Biotechnology. Anti–cytochrome c (PharMingen) was also used. Cisplatin was purchased from Calbiochem. Anti-biotin, streptavidin-linked agarose beads, cisplatin, and Taxol were from Sigma Chemical Co.

Plasmids. Wild-type PKC{delta} (GenBank accession no. AY545076), the catalytic domain (amino acids 334–674, CAT), and the dominant-negative catalytic domain (amino acids 334–674, CAT-KR) were cloned into pcDNA3 that contains a COOH-terminal hemagglutinin (HA) tag (23). To construct the PKC{delta}-deletion mutants, each DNA fragment was amplified using mutagenic primers containing an EcoRI site by PCR. The amplified PKC{delta} mutants were also cloned into pcDNA3 that contains a GST tag. Wild-type mouse HSP25 (GenBank accession no. XM124655) was cloned into pcDNA4HisMaxC (Invitrogen), which contains an NH2-terminal His tag (23).

Irradiation. Cells were plated in 60-mm dishes and incubated at 37°C under humidified 5% CO2 in culture medium until 70% to 80% confluent. Cells were then exposed to {gamma}-rays with 137Cs {gamma}-ray source (Atomic Energy of Canada, Ltd.) with a dose rate of 3.81 Gy/min.

Cell transfection. Predesigned small interference RNA (siRNA) for human HSP27 (5'-GUUCAAAGCAACCACCUGUtt-3') was purchased from Ambion, Inc., and negative control siRNA (5'-UAGCGACUAAACACAUCAA-3') was purchased from Dharmacon, Inc. Cells were transfected with peptides (Peptron) or siRNAs by using Lipofectamine 2000 (Invitrogen) and with plasmids by Lipofectamine Plus reagent and Lipofectamine reagent (Invitrogen) according to the manufacturer's guidelines.

Colony-forming assay. Clonogenicity was examined by the colony-forming assay as previously described (29). Cells were seeded into 60-mm dishes at a density to produce ~500 colonies per dish in the control and were incubated for 7 to 14 days. Colonies were fixed with a mixture of 75% methanol and 25% acetic acid and stained with 0.4% trypan blue. The number of colonies consisting of 50 or more cells was scored.

MTT assay. After cisplatin treatment, the cells were assayed for their growth activity using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) test. The MTT test was done as previously described (29).

Flow cytometry. Cells were cultured, harvested at the indicated times, stained with propidium iodide (1 µg/mL) according to the manufacturer's protocol, and then analyzed using a FACScan flow cytometer (Becton Dickinson).

Immunoblotting. For PAGE and Western blotting, cells were solubilized with lysis buffer [120 mmol/L NaCl, 40 mmol/L Tris (pH 8.0), 0.1% NP40]. Immunoblotting was done as previously described (29).

Immunoprecipitation. Cells (1 x 107) were lysed in immunoprecipitation buffer [50 mmol/L HEPES (pH 7.6), 150 mmol/L NaCl, 5 mmol/L EDTA, 0.1% NP40]. After centrifugation (10 min at 15,000 x g) to remove particulate material, the supernatant was incubated with antibodies (1:100) with constant agitation at 4°C. The immunocomplexes were precipitated with protein A-Sepharose (Sigma) and analyzed by immunoblotting.

PKC assay. Cellular proteins were extracted with PKC extraction buffer [50 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 0.1% Tween 20, 1 mmol/L EDTA, 2.5 mmol/L EGTA and 10% glycerol], containing protease inhibitors (10 µg/mL each of aprotinin and leupeptin and 0.1 mmol/L phenylmethylsufonyl fluoride) and phosphatase inhibitors (1 mmol/L NaF, 0.1 mmol/L Na3VO4, and 10 mmol/L ß-glycerophosphate). One hundred micrograms of protein of cell extracts were immunoprecipitated with 3 µg of anti-PKC{delta} antibody and 30 µL of protein G-Sepharose by incubation for 3 h at 4°C. The immunoprecipitates were washed twice each with PKC extraction buffer and PKC reaction buffer [50 mmol/L HEPES (pH 7.5), 10 mmol/L MgCl2, 1 mmol/L DTT, 2.5 mmol/L EGTA, 1 mmol/L NaF, 0.1 mmol/L Na3VO4 and 10 mmol/L ß-glycerophosphate] and then resuspended in 20 µL of PKC reaction buffer. The kinase assay was initiated by adding 40 µL of PKC reaction buffer, containing 10 µg of myelin basic protein as a substrate (Upstate) and 5 µCi of [{gamma}-32P]ATP. The reactions were carried out at 30°C for 30 min and terminated by adding SDS sample buffer; the mixtures were then boiled for 5 min. The reaction products were analyzed by SDS-PAGE and autoradiography.

Immunofluorescence analysis. Cells transfected with FITC-peptides were analyzed with a confocal laser-scanning microscope (Leica Microsystems).

Immunohistochemistry. Tissue microarray of lung tissues was used for the immunostaining of HSP27. Tissue microarray of human lung cancers was purchased from US Biomax, Inc. Tumor slides were deparaffinized and rehydrated using xylene and alcohol; for immunoperoxidase labeling, endogenous peroxidase was blocked with 0.3% H2O2 in absolute methanol for 15 min at room temperature. Primary anti-HSP27 antibody was reacted with the tissue for 2 h in a humid chamber at room temperature and washed with PBS for 10 min, and the sections were incubated for 20 min at room temperature with secondary antibody. After additional incubation with streptavidin-HRP for 10 min, immunoreactive sites were visualized using 3,3'-diaminobenzidine for 5 min. The sections were counterstained with Harris' hematoxylin, dehydrated, and mounted with coverslips.

Histidine pull-down assay. After transfection with histidine-tagged DNA for 48 h, cells were harvested and whole-cell extracts were prepared as described above. Cell extracts were mixed and incubated with nickel-nitrilotriacetic acid-agarose beads for 30 min at 4°C in the presence of 10 mmol/L imidazole. After washing the resin with buffer containing 10 mmol/L imidazole, proteins were recovered by suspending in Laemmli sample buffer and were then subjected to SDS-PAGE and Western blot analysis with anti-GST antibodies.

Biotinylated peptide binding assay. Biotin-labeled peptides (Peptron) were transfected into cells by using Lipofectamine 2000 (Invitrogen). After incubation for 24 h, cells were harvested and whole-cell extracts were prepared as described above. Cell extracts were mixed with 5 µL of streptavidin-linked agarose beads (1:1 suspension) and incubated for 30 min at 4°C. After washing the resin with lysis buffer, proteins were recovered by suspending in Laemmli sample buffer and were then subjected to SDS-PAGE and Western blot analysis with anti-HSP27 antibodies.

Tumor xenografts in nude mice. A single cell suspension (3 x 106 cells) with a viability of 95% was s.c. injected into the hind legs of 5-week-old BALB/c athymic nude mice (Charles River Japan). The volume injected was 50 µL per mouse to avoid leakage, and a different site was used for each injection. When the tumor reached a minimal volume of 150 to 250 mm3, radiation (12 Gy) with local regional application was started. Each group consisted of three mice, and tumor volumes were determined according to the formula (L x l2) / 2 by measuring tumor length (L) and width (l) with a caliper. All animal work was carried out in accordance with our institute policies.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
HSP27 is highly expressed in human lung and breast cancer tissues. To examine the HSP27 expression in normal and cancerous tissues, especially in lung cancer, and whether HSP27 can be used as a cancer marker, immunocytochemical analysis was done using a tissue array that consisted of 45 lung tissues including squamous cell carcinoma, large-cell carcinoma, and adenocarcinoma. As seen in Fig. 1 , HSP27 was found to be overexpressed in cancer tissues when compared with that of corresponding normal tissues, suggesting that HSP27 may be used as a cancer predictor in lung cancer tissues. When we examined HSP27 expression in human breast tissues using tissue array embedded 25 cases of malignant cancer, similar effects were observed (Supplementary Fig. S1A). Moreover, HSP25 expression in rat mammary tumors was greater in more advanced malignant tumors (grade 2), which was induced by dimethylbenz(a)anthracene, than in normal mammary gland tissues or less advanced malignant tumors (grade 1; Supplementary Fig. S1B).


Figure 1
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  		Figure 1. HSP27 expression in lung cancer tissues. Immunohistochemical analysis for HSP27 was done using tissue microarray. Antibody was detected by the diaminobenzidine method that produces a brown color. All lung cancers in this array were shown to be strongly HSP27 positive when compared with nonneoplastic tissue.

 
Expression levels of HSP27 correlate with resistance and with interaction with PKC{delta} in lung carcinoma cell lines. There have been several reports showing that high expression of HSP27 correlates with resistance to DNA-damaging agents such as radiation or chemotherapeutic agents (24, 29, 30). Therefore, lung cancer cells with different levels of HSP27 expression were examined. Expression levels of other heat shock proteins such as HSP70, HSP90, and HSP110 were not largely different. Indeed, expression levels of HSP27 were found to correlate with survival after radiation damage (Fig. 2A ); NCI-H358 and NCI-H1299 cells with higher HSP27 expression had less cell death, whereas NCI-H23 and NCI-H460 cells with lower HSP27 expression experienced more cell death. When NCI-H1299 cells with higher HSP27 expression were treated with siRNA targeting HSP27, radiation-induced clonogenic survival by HSP27 was significantly reduced to the similar level of NCI-H460 cells, which have lower HSP27 expression. In addition, the MTT assay also revealed that, after cisplatin treatment, significantly increased survival corresponding to high levels of HSP27 was inhibited by siRNA of HSP27 (Fig. 2B). Furthermore, data on cell death indicated that HSP27 was involved in resistance to radiation or chemotherapeutic drug treatment (Fig. 2C). HSP27 inhibited radiation- or cisplatin-mediated cleavage of caspase-3, caspase-9, and poly(ADP-ribose) polymerase (PARP; caspase-dependent apoptosis markers), whereas siRNA of HSP27 restored these phenomena (Supplementary Fig. S2A). On the other hand, when we overexpressed HSP27 to NCI-H460 cells which showed lower HSP27 expression, clonogenic assay upon irradiation or MTT assay by cisplatin treatment indicated increased survival than in control NCI-H460 cells. The increase was equivalent to that of NCI-H1299 cells with higher endogenous HSP27 expression (Supplementary Fig. S3). These data suggest that HSP27 is involved in cellular resistance in lung cancer cells. When we examined these phenomena using breast cancer cells lines such as MCF-7 or MDA-MD231 cells, higher expression of HSP27 and increased radioresistance or chemoresistance were shown in MCF-7 cells when compared with those of MDA-MD231 cells (Supplementary Fig. S4).


Figure 2
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  		Figure 2. Correlation between expression levels of HSP27 and radioresistance or chemoresistance. A, cell death of the human lung carcinoma cell lines NCI-H23, NCI-H358, NCI-H460, NCI-H596, and NCI-H1299 after exposure to 10 Gy of {gamma}-radiation for 72 h was measured by flow cytometric analysis after propidium iodide staining. Columns, mean of three independent experiments; bars, SD. Cell lysates were immunoblotted (IB) with the indicated antibodies and immunoprecipitated (IP) with anti-PKC{delta}. The resulting precipitates were analyzed using anti-HSP27. B, radiosensitivity or chemosensitivity of NCI-H460 and NCI-H1299 cells after transfection with control siRNA (siControl) or HSP27 siRNA (siHSP27), with or without exposure to {gamma}-radiation or cisplatin, was measured by clonogenic survival (15 d after radiation treatment) and MTT assay (48 h after cisplatin treatment). Western blotting was done with anti-HSP27 antibody. Points, mean of three independent experiments; bars, SD. *, P < 0.05, versus siControl-transfected NCI-H1299 cells. C, cell death with or without exposure to 10-Gy {gamma}-radiation for 72 h or 50 µmol/L cisplatin for 48 h was measured by flow cytometry after propidium iodide staining. Columns, mean of three independent experiments; bars, SD. *, P < 0.05. D, cell lysates were immunoblotted with the indicated antibodies and immunoprecipitated with anti-PKC{delta}. The resulting precipitates were analyzed by anti-HSP27 and PKC{delta} kinase assay. Representative results of three independent experiments.

 
In our previous studies, interaction between HSP27 and PKC{delta} was found to be involved in HSP27-mediated radioresistance (23, 30). Therefore, using NCI-H1299 and NCI-H460 cells, the interaction between these two proteins was examined by coimmunoprecipitation. As shown in Fig. 2D, greater interaction between PKC{delta} and HSP27 and lower PKC{delta} kinase activity were found in NCI-H1299 than in NCI-H460. When NCI-H1299 cells were treated with siRNA of HSP27, the HSP27-PKC{delta} interaction disappeared and PKC{delta} kinase activity was restored. When we overexpressed HSP27 to NCI-H460 cells which normally exhibit lower expression of HSP27, radiosensitivity or chemosensitivity was inhibited and greater interaction between HSP27 and PKC{delta} was observed when compared with those of control NCI-H460 cells. Moreover, higher PKC{delta} kinase activity in NCI-H460 cells was also suppressed, which was similar to the level of NCI-H1299 cells (Supplementary Fig. S3), further suggesting that the interaction between HSP27 and PKC{delta} was involved in HSP27-mediated radioresistance or chemoresistance.

Overexpression of the catalytic domain of PKC{delta} overcomes HSP27-mediated chemoresistance or radioresistance. Our earlier studies together with the above result indicate that interaction of HSP27 with PKC{delta} inhibits PKC{delta} kinase activity. Indeed, it was shown that the PKC{delta}-CAT domain is the binding site for HSP27, and kinase activity is important for this interaction (23, 30). Therefore, to confirm the involvement of PKC{delta} in radiation- or cisplatin-mediated cell death, several constructs of PKC{delta} were generated, including a wild-type (PKC{delta}-WT) construct and deletion constructs of the catalytic domain of PKC{delta} (PKC{delta}-CAT) and a kinase-defective dominant negative mutant of the catalytic domain of PKC{delta} (PKC{delta}-CAT-KR). As seen in Fig. 3A , the interaction of HSP27 and PKC{delta} was evident in PKC{delta}-WT– and PKC{delta}-CAT–transfected NCI-H1299, whereas no interaction was observed in CAT-KR–transfected cells. In addition, interaction of HSP27 with PKC{delta}-WT or PKC{delta}-CAT was not observed when NCI-H1299 cells were cotransfected with HSP27 siRNA, suggesting specific binding between HSP27 and PKC{delta}. In addition, PKC{delta} kinase activity was increased by transfection of PKC{delta}-WT when compared with the control pcDNA–transfected NCI-H1299 cells, and transfection of PKC{delta}-CAT augmented PKC{delta} kinase activation. Moreover, when we cotransfected siHSP27 to PKC{delta}-WT– or PKC{delta}-CAT–transfected cells, these phenomena were more potentiated (Fig. 3A), suggesting that the HSP27-PKC{delta} interaction inhibited PKC{delta} kinase activity. PKC{delta}-WT and PKC{delta}-CAT transfection increased cell death due to DNA-damaging agents; PKC{delta}-CAT seemed to mediate this effect because PKC{delta}-CAT-KR, which does not have PKC{delta} kinase activity, did not show increased radiation- or cisplatin-mediated cell death (Fig. 3B). Moreover, caspase-3, caspase-9, and PARP cleavage were increased in both PKC{delta}-WT– and PKC{delta}-CAT–transfected NCI-H1299 cells, whereas PKC{delta}-CAT-KR inhibited this effect (Supplementary Fig. S2B). When HSP27 siRNA was cotransfected, an increase in cell death was observed. To elucidate whether the function of PKC{delta} is important in HSP27-mediated resistance, knockdown of PKC{delta} using siRNA (siPKC{delta}) was applied, and then HSP27 was overexpressed to NCI-H460 cells. Radiation- or cisplatin-induced cell death was significantly inhibited by HSP27 transfection or by siPKC{delta}, suggesting that PKC{delta} knockdown as well as HSP27 overexpression contributed to resistance to cisplatin or radiation. In addition, concomitant siPKC{delta} and HSP27 transfection showed comparable cell death to that of HSP27- or siPKC{delta} alone–transfected cells, suggesting that without PKC{delta}, HSP27 exhibited no more increased resistance to DNA-damaging agents (Supplementary Fig. S5A). Increased binding activity between HSP27 and PKC{delta} and reduction of PKC{delta} kinase activity were also shown in HSP27-transfected NCI-H460 cells when compared with the control NCI-H460 cells (Supplementary Fig. S5B). Therefore, these data suggest the correlation between binding of HSP27 with PKC{delta} and resistance to radiation or cisplatin. Extrinsic transfection of PKC{delta}-CAT or intervention of binding activity between HSP27 and PKC{delta} has the capability to overcome HSP27-mediated resistance.


Figure 3
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  		Figure 3. Increase of PKC{delta}-mediated cell death by the catalytic domain of PKC{delta}. A, cell lysates from NCI-H1299 cells after transfection with the indicated HA-tagged PKC{delta} mutants, with or without cotransfection of HSP27 siRNA (siHSP27), were immunoblotted with the indicated antibodies and immunoprecipitated with anti-HA. The resulting precipitates were analyzed by anti-HSP27 and PKC{delta} kinase assay. MBP, myelin basic protein. B, cell death with or without 10-Gy radiation for 72 h or 50 µmol/L cisplatin treatment for 48 h was followed by flow cytometry after propidium iodide staining. Representative results of three independent experiments. Columns, mean of two independent experiments; bars, SD. *, P < 0.05.

 
V5 region of PKC{delta} attenuates HSP27-mediated chemoresistance or radioresistance. Because the V5 region of PKC{delta}-CAT was found to be the binding site of PKC{delta} in interaction with HSP27 (23), we next examined whether ectopic expression of the V5 region of PKC{delta} could overcome HSP27-mediated resistance to radiation or cisplatin in NCI-H1299 cells. As shown in Fig. 4A , transfection of the V5 region of PKC{delta} increased PKC{delta} activity and interaction of the transfected V5 region with HSP27. Furthermore, PKC{delta} kinase activity in NCI-H1299 cells was increased by the transfection of the V5 region of PKC{delta}, and this activity was sustained under various treatment conditions (Fig. 4B). Radiation increased the PKC{delta} kinase activity at 1 h after irradiation followed by a decrease to the basal level (data not shown). However, when the V5 region of PKC{delta} was transfected into the cells, PKC{delta} kinase activity was higher and sustained longer than that of control vector–transfected cells. A similar phenomenon was observed following cisplatin treatment. Radiation or cisplatin-induced cell death (Fig. 4C) and caspase-3, caspase-9, and PARP cleavage (Supplementary Fig. S2C) were also increased by transfection of the V5 region of PKC{delta}. The results in nude mice grafted with NCI-H1299 cells transfected with PKC{delta}-CAT, PKC{delta}-CAT-KR, and PKC{delta}-V5 revealed that the V5 region of PKC{delta} significantly attenuated HSP27-mediated resistance to radiation and that the radiation effect was similar to that of mice grafted with NCI-H460 cells (Fig. 4D). The doubling time of NCI-H460 cells (23 h) was similar to that of NCI-H1299 cells (25 h), and without radiation, each mutant transfected cell line showed similar growth pattern after xenograft (data not shown).


Figure 4
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  		Figure 4. Abolishment of HSP27-mediated resistance to DNA-damaging agents by the V5 region of PKC{delta}. A, GST immunoprecipitates in lysates of NCI-H1299 cells transfected with the GST-tagged V5 region of PKC{delta} were analyzed with anti-HSP27. HSP27 or GST protein was detected with anti-HSP27 or anti-GST antibodies. Cellular proteins were extracted after lysis with PKC extraction buffer and analyzed by PKC{delta} kinase assay. B, at indicated time points, cellular proteins from NCI-H1299 cells, which were transfected with the GST-tagged V5 region of PKC{delta} with or without 10-Gy radiation (1 h) or 50 µmol/L cisplatin (30 min), were analyzed by PKC{delta} kinase assay. HSP27 or GST protein was detected with anti-HSP27 or anti-GST antibodies. C, cells with or without 10-Gy radiation for 72 h or 50 µmol/L cisplatin treatment for 48 h were followed by flow cytometry after propidium iodide staining. Columns, mean of three independent experiments; bars, SD. *, P < 0.05. D, exponentially growing NCI-H460 or NCI-H1299 cells (3 x 106) after stable transfection of the indicated PKC{delta} mutants were injected into the hind legs of nude mice. Radiation (12 Gy) with local regional application was applied when the tumor reached a minimal volume of 150 mm3. Three mice in each group were used. Points, mean; bars, SD. *, P < 0.05, versus control vector–transfected NCI-H1299 cells xenografted into mice. The transfection efficiencies of GST- or HA-tagged vectors in NCI-H1299 cells were confirmed by Western blotting with anti-GST or anti-HA (top). Representative results of three independent experiments.

 
Amino acid residues 668 to 674 in the V5 region of PKC{delta} are the binding site for HSP27. To further delineate the binding site on PKC{delta} for HSP27, several green fluorescent protein–labeled deletion mutants were produced. After cotransfection of these mutants and His-tagged HSP25 into L929 cells, the His-tag pull-down assay was done. The COOH-terminal seven-amino-acid sequence of the PKC{delta}-V5 region (668–674) was the binding site involved in the interaction with HSP27 (Supplementary Fig. S6). To confirm this, we made a deletion mutant devoid of the COOH-terminal seven amino acids (PKC{delta}V5{Delta}668–674). Histidine pull-down assay revealed that PKC{delta}V5{Delta}668–674 did not bind HSP27, whereas PKC{delta}V5 did (Fig. 5A ). Next, we prepared peptides containing the seven amino acids from residues 668 to 674 of the V5 region (p668) and a control peptide containing residues 606 to 618 of the V5 region (p606). When FITC-conjugated peptides were transfected to NCI-H1299 cells, increased fluorescence was observed in both p606 and p668 peptide–transfected NCI-H1299 cells (Fig. 5B, left). Interaction of p668 with HSP27 was apparent in NCI-H1299 cells; on the other hand, control peptide p606 did not interact with HSP27. The binding of PKC{delta} or cytochrome c to HSP27 was also reduced by p668 transfection, which suggested that p668 interacted predominantly with HSP27, thus blocking interaction of endogenous PKC{delta} or cytochrome c with HSP27 (Fig. 5B, right). The transfection of p668 increased PKC{delta} kinase activity compared with the untransfected control or p606-transfected cells. In addition, the results of cell death experiments indicated that p668 significantly increased radiation- or cisplatin-induced cell death to a similar or more extent as found in NCI-H460 cells; the control peptide p606 alone did not induce any apoptosis (Fig. 5C). Mouse xenograft data revealed that when tumor reached a minimal volume of 250 mm3, three times treatments with FITC-conjugated p668 or p606 to xenograft NCI-H1299 tumors showed sufficient amount of accumulation in tumors. The treatment with FITC-conjugated p668 in NCI-H1299 tumor tissue increased PKC{delta} kinase activity compared with the FITC-conjugated p606-treated cells (Supplementary Fig. S7). In addition, treatment with p668 in combination with radiation dramatically inhibited the growth of NCI-H1299 tumor cells, whereas p606 did not (Fig. 5D). However, intratumor treatment with p668 or p606 to tumor site without radiation did not induce any tumor growth inhibition (data not shown).


Figure 5
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  		Figure 5. Characterization of binding site of PKC{delta}-V5 in interaction with HSP27. A, the indicated GST-tagged deletion mutant of the PKC{delta}-V5 region and wild-type PKC{delta}-V5 region were transfected in L929 cells, and cell lysates were incubated with immobilized His-HSP25. GST proteins were detected by Western blotting with an anti-GST antibody. His-HSP25 proteins are also shown. The transfection efficiencies of GST-tagged vectors were confirmed by Western blotting with anti-GST. B, sequences of peptides (p668 and p606) used in this study. After transfection with FITC-conjugated p668 and p606 (10 µmol/L), the cells were incubated for 24 h and analyzed by confocal microscopy. Control means nontransfected NCI-H1299 cells (left). HSP27 was detected in immunoprecipitates from lysates of NCI-H1299 cells after transfection with biotin-conjugated p668 and p606 using anti-biotin, anti–cytochrome c, or anti-PKC{delta} antibodies. Cellular proteins were extracted after lysis with PKC extraction buffer and analyzed by PKC{delta} kinase assay (right). C, cell death of NCI-H460 and NCI-H1299 cells, which were transfected with biotin-conjugated p668 or p606 with or without exposure to 10-Gy {gamma}-radiation for 72 h or 50 µmol/L cisplatin for 48 h, was measured by flow cytometry after propidium iodide staining. Columns, mean of three independent experiments; bars, SD. *, P < 0.05. D, exponentially growing NCI-H460 or NCI-H1299 cells (3 x 106) were injected into the hind legs of nude mice. Radiation (12 Gy) with local regional application was applied when the tumor reached a minimal volume of 250 mm3. FITC-conjugated peptide 668 or p606 in 50-µL saline (three applications, 100 µmol/L per mouse) was injected at the tumor site from the day following irradiation. Three mice were used in each group. Points, mean; bars, SD. *, P < 0.05, versus p606-treated mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
HSP27 is constitutively expressed in many cancer cells and negatively regulates apoptotic induction. Therefore, neutralization of HSP27 is an attractive strategy for anticancer therapy. Clinical trials using a specific inhibitor of HSP90, 17-allylamino-17-demethyoxygeldanamycin, are in progress and seem to be providing encouraging results (31). The fact that 17-allylamino-17-demethoxygeldanamycin selectively kills cancer cells has been rationalized by assuming that tumor cells, when compared with their normal counterparts, exhibit a stressed phenotype with an enhanced dependency on the cytoprotective action of HSP90 (32). Similarly, it has been suggested that HSP27 antisense and interference RNA of HSP27 have chemosensitizing and radiosensitizing properties (14, 18–21). Unlike HSP90, however, no small molecules are thus far available that selectively inhibit HSP27.

HSP27 protein was highly expressed in various lung or breast cancer tissues (Fig. 1 and Supplementary Fig. S1A) that often show radioresistance or chemoresistance. In rat mammary tumorigenesis models, malignant tumors showed higher expression of HSP27 than normal tissues and malignant grade was well correlated with HSP27 expression pattern (Supplementary Fig. S1B), suggesting the possibility of HSP27 as a cancer marker in lung and breast. To determine the physiologic relevance of HSP27 expression and radioresistance or chemoresistance, siRNA for HSP27 was applied to NCI-H1299 or MCF-7 cells, which have high levels of HSP27 and increased radioresistance or chemoresistance (Fig. 2 and Supplementary Fig. S4), and the result showed highly significant physiologic relevance of HSP27 in cancer treatment (i.e., down-regulation of HSP27 resulted in sensitization to DNA-damaging agents in a caspase-dependent manner).

PKC{delta} activity plays an essential role in apoptosis, and the catalytic domain of PKC{delta} was found to be involved in this process (Fig. 3). Therefore, with knockdown of PKC{delta} using siRNA technology, induction of cell death by DNA-damaging agents was inhibited (Supplementary Fig. S5). Chemotherapeutic agents or ionizing radiation induces translocation of PKC{delta} to the membrane in an open conformation, allowing its pseudosubstrate region to be released from its substrate-binding cavity and enabling the COOH terminus to access the substrate-binding site. Autophosphorylation at Ser643 and Ser662 by intramolecular mechanisms then activates downstream signaling (23). Our earlier studies indicated that PKC{delta} interacts with HSP25 and that phosphorylation-deficient mutants at Ser643 and Ser662, but not phosphorylation-mimicking mutants of the V5 region of PKC{delta}, can bind to HSP25. Moreover, HSP25 inhibits PKC{delta} translocation to the particulate fraction, suggesting that the primed activation of PKC{delta} permits the V5 region to be exposed, autophosphorylated, and fully activated. As soon as the V5 region is exposed, but before autophosphorylation at Ser643 and Ser662, HSP25 may bind to the V5 region of PKC{delta}, thus stabilizing PKC{delta} and inhibiting rephosphorylation and reactivation. Therefore, PKC{delta} activity was inhibited by the PKC{delta}-HSP25 interaction, and HSP27 siRNA treatment restored PKC{delta} activity (Fig. 2D).

As shown in the present study, rationally engineered PKC{delta}-V5 mutant target for HSP27 could sensitize cancer cells to apoptosis induction. Whereas PKC{delta}-V5 has no intrinsic apoptotic stimulatory activity, PKC{delta}-V5 exerted its radiosensitizing and chemosensitizing effect through the neutralization of HSP27 (Fig. 4). Moreover, PKC{delta}-V5 increased PKC{delta} kinase activity after radiation or cisplatin treatment. Because only seven amino acids of the V5 region constituted the binding site of PKC{delta}, we prepared hydrophobic, cell-permeable peptide containing these seven amino acids (p668) and found that this heptapeptide was able to sensitize the cancer cells to radiation or cisplatin in both in vitro and in vivo animal models by sequestering HSP27 and increasing PKC{delta} activity (Fig. 5). Interestingly, HSP27 has also been reported to interact with another apoptotic molecule, and our present results showed that p668 transfection inhibited the interaction of HSP27 with both PKC{delta} and cytochrome c, thereby neutralizing HSP27 (Fig. 5B).

Targeting protein-protein interactions that occur within the apoptotic pathways using peptide-based cancer therapies could provide novel, nongenotoxic alternatives or adjuvants to current treatment protocols. Optimally, the peptide should be preferentially toxic to cancer cells and/or work through a defined genetic pathway that is hyperactive in malignant cells. p53-targeted peptides, such as BH3 peptides, Smac peptides, and peptidomimetics, are good candidates for these studies (33). In this regard, the heptapeptide of PKC{delta}-V5 region would be beneficial for cancer therapy in cells that show high HSP27 protein expression.

In summary, PKC{delta} activity plays an essential role in the apoptosis of cells because knockdown of PKC{delta} inhibited cell death induced by DNA-damaging agents, and HSP27 is constitutively expressed in many cancer cells to negatively regulate apoptotic induction. Therefore, the HSP27-PKC{delta} interaction could indicate the physiologic importance of this small heat shock protein. Treatment of cancer cells with a heptapeptide that contains the binding sequence for PKC{delta} interaction with HSP27 induced DNA-damaging agents, and these data provide a novel strategy for selective neutralization of HSP27 (Fig. 6 ).


Figure 6
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  		Figure 6. Hypothetical scheme of sensitization to DNA-damaging agents by a novel PKC{delta}-V5 heptapeptide in HSP27 overexpressed cancer cells. HSP27 is constitutively expressed in many cancer cells to negatively regulate radiation (IR) or cisplatin-mediated cell death through direct interaction with PKC{delta}. Interaction between HSP27 and PKC{delta} inhibited PKC{delta} kinase activity, which resulted in suppression of PKC{delta}-mediated cell death. Treatment of cancer cells with a novel heptapeptide that contains the binding sequence for PKC{delta} interaction with HSP27 induced chemosensitization or radiosensitization because this heptapeptide leaves the cell endogenous PKC{delta} kinase activity intact.

 

   Acknowledgments
 
Grant support: Korea Institute of Science and Technology Evaluation and Planning and the Ministry of Science and Technology, Korea, through its National Nuclear Technology Program.

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.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Y-J. Lee and Y-S. Lee contributed equally to this work.

Received 12/ 4/06. Revised 3/30/07. Accepted 5/ 2/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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