Anti-Hsp90 and anti-Hsp70 antibodies, human recombinant Hsp90α, and Hop (p60) were purchased from Stressgen Bioreagents. Anti-Akt and anti-CDK4 antibodies were purchased from Cell Signaling. Anti-survivin antibody was purchased from Thermo Scientific. Human recombinant FKBP5 and PP5 were purchased from Abnova. Anti-β-actin antibody and human recombinant Hsp70 were purchased from SIGMA. All reagents were of reagent-grade quality.

Escherichia coli AD494 (DE3) {Δara, leu 7697, ΔlacX74, ΔphoA, PvuII, PhoR, ΔmalF3, F' [lac^-, (lacI^q), pro], trxB::kan (DE3)} and pET-15b (Novagen Inc.) were used for expression of the TPR2A domain of human Hop.

The following human tumor and normal cell lines were obtained from the American Type Culture Collection (ATCC): human breast cancer (BT-20, T47D, and MDA-MB-231), lung cancer (A549), kidney cancer (Caki-1), prostate cancer (LNCap), gastric cancer (OE19) and lung fibroblast (MRC5). Human pancreatic cancer cell line (BXPC3) was purchased from the European Collection of Cell Culture (ECACC). Human embryonic kidney cell line (HEK293T) and human normal pancreatic epithelial (PE) cell line ACBRI 515 were purchased from RIKEN cell bank and DS Pharma Biomedical, respectively. Cells were cultured in RPMI-1640 (BT-20, MDA-MB-231, T47D, LNCap, OE19, and BXPC3), MEM (MRC5 and A549), DMEM (HEK293T and Caki-1) or CSC (PE) containing 10% fetal bovine serum (FBS), 100 μg/ml penicillin, and 100 μg/ml streptomycin.

Peptides used in this study were synthesized by Invitrogen or SIGMA. All peptides were synthesized by use of solid-phase chemistry, purified to homogeneity (i.e. >90% purity) by reversed-phase high-pressure liquid chromatography, and assessed by mass spectrometry. Peptides were dissolved in water and buffered to pH 7.4. The TPR sequence 301K-312K (KAYARIGNSYFK; TPR), TPR mutant 1 (KAYA

AA

GGG

RQIKIWFQNRRMKWKK

RQIKIWFQNRRMKWKK

RQIKIWFQNRRMKWKK

RQIKIWFQNRRMKWKK

The TPR2A domain (223K-352L) of human Hop was cloned in-frame into the XhoI and BamHI sites of pET-15b for expression in E. coli AD494 (DE3), and purified using a nickel-chelating resin column as described previously 30. To confirm the presence of purified protein, SDS/PAGE was performed according to the method of Laemmli 31.

SPR experiments were performed with the Biacore biosensor 3000 system as described previously 3032. Human recombinant Hop, FKBP5, PP5, and purified TPR2A domain of Hop proteins were immobilized on the surface of CM5 sensor chips via N-hydroxysuccinimide and N-ethyl-N'-(dimethylaminopropyl)carbodiimide activation chemistry according to the manufacturer's instructions. Biotin-conjugated TPR peptide (biotin-TPR) was immobilized on the surface of streptavidin (SA) sensor chip. As the analyte, several concentrations of Hsp90 or Hsp70 were injected over the flow-cell at a flow rate 30 μl/min at 25°C. HBS-EP buffer (0.01 M Hepes/0.15 M NaCl/0.005% Tween 20/3 mM EDTA, pH 7.4) was used as a running buffer during the assay to inhibit non-specific binding. Data analysis was performed using BIA evaluation version 4.1 software. Competition experiments were performed by preincubating Hsp90 or Hsp70 with short, defined peptides or combinatorial peptide mixtures according to the method of Brinker et al. 30. Briefly, protein/peptide mixtures were passed over the immobilized Hop, FKBP5, PP5 or TPR2A domain of Hop, and bindings of Hsp90 or Hsp70 to these proteins were followed. SPR signals obtained in the absence of competing peptides were used as a reference (100% binding) to normalize values obtained in the presence of peptides. For competition experiments involving defined peptides the concentration of TPR protein was kept constant, whereas the peptide concentration of the protein/peptide mixtures was increased systematically.

Western-blot analyses were carried out as described previously 33. Briefly, protein extracts were prepared from cells lysed with buffer containing 1% (v/v) Triton X-100, 0.1% (w/v) SDS, and 0.5% (w/v) sodium deoxycholate, separated by SDS/PAGE, and transferred to nitrocellulose filters. Quenched membranes were probed with antibodies and analyzed using enhanced chemiluminescence reagent (GE Healthcare) with an LAS-3000 LuminoImage analyzer (Fujifilm).

Cells were seeded on to 96-well plates at 2000-3000 cells/well and incubated with the test peptide. After incubation, an assay for cell viability was carried out using Living Cell Count Reagent SF (Nacalai Tesque) according to the manufacturer's protocol. Absorbance was measured at a wavelength of 450 nm using a 96-well microplate reader (GE Healthcare).

BXPC3 cells were plated in a glass-bottomed dish at 1 × 10⁶ cells per ml of medium, and small aliquots of labeled-peptides, Antp-TPR-TAMRA-OH or TPR-TAMRA-OH (Invitrogen) (15 μl) were added directly into the dish at a final concentration of 10 μM. After 2 hr incubation, intracellular penetration of the peptides was visualized by an Olympus FV1000 confocal laser scanning microscope (Olympus).

After incubation with or without Antp-TPR peptide, cells were collected and washed twice with PBS. Following this, the cell pellets were resuspended. Flow cytometry (Becton Dickinson) analysis was performed using the Annexin V-Fluorescein Staining Kit (Wako) or carboxyfluorescein FLICA caspase 3 & 7 assay kit (immunochemistry Technologies) according to the manufacturer's protocol. Data were analyzed using CellQuest Software.

Animal experiments were carried out in accordance with the guidelines of the Kyoto University School of Medicine. Cells of the pancreatic cancer cell line BXPC3 (5×10⁶ cells), resuspended in 150 μl of PBS, were transplanted subcutaneously into the flank region of 7-9-week-old athymic nude mice weighing 17-21 g. When tumors reached around 50 mm³ in volume, animals were randomized into three groups, and PBS (control) or Antp-TPR peptide (1 or 5 mg/kg) was injected intravenously (50 μl/injection) three times a week for a total of nine doses. Tumors were measured with a caliper, and the tumor volume (in mm³) was calculated using the following formula: length×width²×0.5. All values are expressed as the mean ± SD and statistical analysis was calculated by a one-way ANOVA with Dunnett test. Differences were considered to be significance at P < 0.05.

Immunohistochemical staining was performed as described previously 34. Briefly, BXPC3 tumor from animals treated either with saline or Antp-TPR peptide (5 mg/kg) intravenously were harvested at the end of treatment, and subsequently embedded in paraffin after fixation with 10% formaldehyde in PBS. After deparaffinized and hydrated, tumor sections were treated with antibodies, and then peroxidase activity was detected by incubation in 0.05% of 3, 3'-diaminobenzidine tetrachloride in PBS (pH7.2) containing 0.012% of H₂O₂.

It is well known that the functional form of Hsp90 is a complex in which the chaperones Hsp90 and Hsp70 are brought together by binding to Hop 14 and assembly of this multiprotein complex is achieved by means of two independent TPR1 and TPR2A domains on Hop. In the complex of TPR2A and C-terminal region of Hsp90, Lys 301 and Arg 305 in helix A3 of TPR2A donate hydrogen bonds to the respective side chains of Asp and Glu of the Hsp90 C-terminal region 17. In addition, Arg 305 in helix A3 is highly conserved among other TPR domains 17, and mutation of this Arg residue to Ala in the TPR domain of Tom70 is critical for binding to Hsp90 35. Based on these information, we designed a new TPR peptide, KAYARIGNSYFK, which includes the significant and highly conserved amino acids Lys 301 and Arg 305 for binding to Hsp90, using structural information obtained from the TPR2A-Hsp90 complex (Figure 1A). As shown in Figure 1(B), both Hsp90 and Hsp70 bind to the immobilized TPR peptide, and with similar K_D values, 1.42 × 10^-6 (M) and 0.68 × 10^-6 (M) at increasing ligand concentrations, respectively, but the relative binding ability of Hsp70 to TPR peptide for Hsp90 was 49.9% (data not shown). In addition, the K_D value of the interaction of Hsp90 with Hop was also similar (4.43 × 10^-6 (M), data not shown). It was found that the TPR peptide did not inhibit the interaction of Hsp70 with Hop protein as assessed by Biacore biosensor (Figure 1C), and that this peptide also did not affect the interaction of Hsp90 with FKBP5 or PP5 proteins (Figure 1D), which also have Hsp90-binding TPR domain as described previously 17. However, it was shown that TPR peptide inhibited the interaction of Hsp90 with Hop protein (Figure 1D). The designated TPR peptide was further fused by its N-terminus to helix III of the Antennapedia homeodomain protein 29 to generate a cell-permeable variant, hybrid Antp-TPR peptide, as described in the Materials and Methods section.

Based on analysis of the interaction of the designed hybrid Antp-TPR peptide with human Hsp90 protein, we then examined cancer-cell viability to assess the selectivity of this peptide in discriminating between normal and cancer cells. As shown in Figure 2(A), the Antp-TPR peptide caused a concentration-dependent loss of human cancer cell viability (in the Caki-1, BXPC3, T47D, and A549 cell lines); however, identical concentrations of this peptide did not apparently reduce the viability of normal human cell lines (HEK293T, MRC5, and PE) (Figure 2A), and TPR peptide without Antp, the cell-permeable peptide, had no effect on normal or cancer cells (Figure 2B). Confocal microscopy analysis also demonstrated that Antp-TPR peptide labeled with TAMRA penetrated the cancer cells, whereas TPR-TAMRA peptide without Antp sequence did not penetrate to cancer cells (Additional file 1). In addition, Antp-scramble peptide had no effect on these cell lines (data not shown). For the cancer cell lines tested, Antp-TPR peptide showed IC₅₀ values of between 20 and 60 μM. On the other hand, TPR peptide showed no cytotoxicity towards either these cancer cell lines or normal cells (Table 1). These results demonstrate that the TPR peptide combined with Antp, a cell-permeable peptide, has selective anticancer activity that discriminates between normal and tumor cells. In addition, as shown in Figure 2(C) and 2(D), Antp-TPR mutant 1 and 2 peptides did not show selective antitumor activities when these peptides were tested with both normal and cancer cell lines. This suggests that the mutated amino acids in Antp-TPR mutants 1 and 2 are indispensable for the selective antitumor activity of Antp-TPR.

We further investigated whether the designed TPR peptide was able to compete specifically for the interaction of Hsp90 with the TPR2A domain of Hop, which is necessary for the correct folding of several oncogenic proteins in cancer cells 181920. Hsp90 was passed over a sensor chip carrying immobilized purified recombinant TPR2A domain of human Hop in either the absence or presence of increasing concentrations of TPR peptide (Figure 3). The SPR signal in the absence of peptide competitor was used as a reference (100% binding) to normalize the signals for Hsp90 binding recorded in the presence of peptide. The interaction of the TPR2A domain of Hop with Hsp90 was competed for by TPR peptide (Figure 3A and 3C). In contrast, the TPR scramble peptide and TPR mutant peptides 1 and 2 did not demonstrate any protein interaction when analyzed at up to millimolar concentrations (Figure 3B and 3C). These results indicate that the designed TPR peptide is a specific competitor capable of inhibiting the interaction between Hsp90 and the TPR2A domain of Hop, and that the amino acids targeted in our mutagenesis experiment are critical for this protein interaction to occur.

As mentioned previously, the interaction of Hsp90 with Hop in cancer cells is significant for folding of several oncogene proteins including survivin, which is a member of the inhibitor of apoptosis gene family 27. In addition, Antp-TPR has selective cytotoxic activity towards cancer cells and is an inhibitor of the interaction of Hsp90 with the TPR2A domain of Hop (Figures 2 and 3). These results prompted us to investigate whether Antp-TPR induces apoptosis in cancer cells. As assessed by flow cytometry analysis, annexin V or caspase 3 and 7 positive cells were found when Antp-TPR peptide was added to breast cancer T47D cells (Figure 4A, middle and right lane panels), suggesting that this peptide induces cancer cell death by apoptotic mechanism (Figure 4A, middle and right lane panels). On the other hand, there was no appearance of annexin V-labeled HEK293T cells after addition of this hybrid peptide (Figure 4A, left lane panels). Taken together with Figures 2 and 3, it was shown that the Antp-TPR peptide designed in this study provided selectivity to cancer cells, discriminating between normal and cancer cells.

When we examined the levels of Hsp90 client proteins after intracellular loading of Antp-TPR peptide, T47D cells treated with Antp-TPR exhibited loss of multiple Hsp90 client proteins, including survivin, CDK4, and Akt, as assessed by Western blotting (Figure 4B). In contrast, Antp-TPR peptide did not affect the levels of Hsp90 itself (Figure 4B). When normal and cancer cell lines (HEK293T, Caki-1, BXPC3, T47D, and A549) received heat shock, the up-regulation of Hsp90 and Hsp70 was observed in the cancer cells, but not in normal HEK293T cells (Additional file 2A). In addition, the up-regulation of Hsp70 after the treatment with this peptide was not observed in both cancer and normal cell lines (Additional file 2B). When we investigated the expression levels of Hsp90, Hsp70, and survivin in these cell lines using Western blotting, it was found that the expression of Hsp90 was almost equal between normal and cancer cells, however, survivin was highly expressed in cancer cell lines, and the expression level of Hsp70 was different among these cell lines (Figure 4C). These results suggest that the Antp-TPR peptide designed in this study would affect the cell-survival pathways in cancer cells by competing with cochaperone recruitment, which is indispensable for the correct folding of Hsp90 client proteins.

To assess the antitumor effect of Antp-TPR peptide in a xenograft model of human cancer, BXPC3 pancreatic cancer cells were implanted subcutaneously into athymic nude mice and the animals were treated with Antp-TPR peptide. The control group exhibited progressive tumor growth, reaching 749 mm³ at day 58 (Figure 5A). On the other hand, administration of Antp-TPR peptide (1 or 5 mg/kg, administered intravenously three times a week for 3 weeks) suppressed tumor growth remarkably. On day 58, mean tumor volume was 371 mm³ in 1 mg/kg dosage group and 204 mm³ in 5 mg/kg dosage group (P < 0.05 compared with control group) (Figure 5A). Immunohistochemical staining also demonstrated that Antp-TPR peptide caused loss of Hsp90 client protein (CDK4) in BXPC3 tumors in vivo after the treatment, although tumors from the saline group exhibited extensive labeling for this protein (Figure 5B). In addition, histologic examination of liver, kidney, and lung was equally unremarkable in the saline or hybrid peptide-treated mice (Figure 5C). These results suggest that the newly designed hybrid Antp-TPR peptide successfully induces tumor death via loss of Hsp90 client proteins in vivo.