Delivery of a Pro-apoptotic Peptide to EGFR-positive Cancer Cells by

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Delivery of a Pro-apoptotic Peptide to EGFR-positive Cancer Cells by a Cyclic Peptide Mimicking the Dimerization Arm Structure of EGFR Kei Toyama, Wataru Nomura, Takuya Kobayakawa, and Hirokazu Tamamura Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00250 • Publication Date (Web): 15 May 2018 Downloaded from http://pubs.acs.org on May 17, 2018

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Bioconjugate Chemistry

Delivery of a Pro-apoptotic Peptide to EGFR-positive Cancer Cells by a Cyclic Peptide Mimicking the Dimerization Arm Structure of EGFR

Kei Toyama, Wataru Nomura, Takuya Kobayakawa, and Hirokazu Tamamura*

Department of Medicinal Chemistry, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan

Keywords: cyclic peptide, dimerization arm, EGF receptor, intracellular delivery

Correspondence should be addressed to [email protected].

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Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: A cyclic decapeptide, CQTPYYMNTC (1), which mimics the dimerization arm of the EGF receptor (EGFR), was previously found to be captured into cells. We have sought to investigate the promising potential of this peptide as an intracellular delivery vehicle directed to EGFR-positive cells. The selectivity of Peptide 1 to the EGFR was confirmed by a positive correlation between the expression level of the receptor and the cellular uptake of Peptide 1 as shown by siRNA knockdown of the EGFR. The pro-apoptotic domain (PAD) peptide, ([KLAKLAK]2), has limited use due to a deficiency of cell membrane permeability resulting from cationic sequences and lack of specificity for cancer cells. As a proof-of-concept study, the cellular delivery of the PAD peptide was challenged by conjugation with Peptide 1. The cellular uptake of a conjugated Peptide 2, which was composed of Peptide 1, the PAD peptide and a linker cleavable with a protease, was evaluated by treatment of an EGFR-positive lung carcinoma cell line, A549. Significant suppression of proliferation by Peptide 2 was shown in the results of a cell viability assay. The PAD peptide alone had no effect on the cells. The results suggest that Peptide 1 is a promising lead compound as a new intracellular delivery vehicle for therapeutically effective peptides.

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INTRODUCTION In the development of anticancer agents, cationic antimicrobial peptides (AMPs) have attracted attention because of their toxicity to cells and their anticancer activity in drug-resistant cancer cells1. A membrane impermeable pro-apoptotic domain (PAD) peptide ([KLAKLAK]2) is known as an antimicrobial agent and causes mitochondrial membrane disruption, followed by cell apoptosis in eukaryotic cells.2,3 Most AMPs, including PAD are positively charged and have inhibitory activity against bacteria. AMPs have antitumor functions because of a multifunctional host defense system of multicellular organisms4–6 but they cannot penetrate cell membranes and alone, they have little cytotoxicity. There are several reports of PAD peptides entering cells by conjugating with cell-penetrating peptides (CPPs).7,8 Several PAD conjugates with a CPP selectively bind to a specific protein in carcinoma cells and achieve induction of apoptosis. Cationic AMPs are also attractive for their anticancer activity against drug-resistant cancer cells and consequently, selective delivery of AMPs into cancer cells should enhance the development of new anticancer therapeutic disciplines. The epidermal growth factor receptor (EGFR) is one of the receptor tyrosine kinases that are involved in cellular signal transductions and oncogenesis.9,10 Binding of the ligand to the ectodomain of the EGFR triggers its activation.11–13 In many cancer cells, overexpression of the receptor is observed, and its unregulated activation causes the aberrant cell growth.14,15 Ligand binding and downstream signaling result in endocytosis and intracellular trafficking of the EGFR, terminating its signaling.16–18 The activated EGFR is internalized to endosomes by clathrin-dependent and -independent pathways, and the signal continues until the receptor is recycled to the membrane or is degraded in lysosomes.19,20 Since EGFR has a fundamental function of internalization, it is useful in this way for intracellular drug delivery. Efficient delivery to cancer cells is one of the most critical issues for cancer cell-specific treatments because reduction of the side effects of anticancer drugs is crucial for patients.21

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Endocytosis mediated by cancer cell-specific receptors is also useful in development of new therapeutics, which can permit reduction of side effects on normal cells thus utilizing drug candidates.22–24 Thus, the EGFR is attractive as a target for intracellular drug delivery agents. An antibody drug, cetuximab, which targets the extracellular domain of the EGFR, has been reported to inhibit receptor activation by binding to the ligand-binding site of the EGFR, and causing induction of the receptor internalization.25–27 Previously, we reported a fluorescein-labeled cyclic decapeptide (fluorescein-CQTPYYMNTC, FAM-Peptide 1), which has an intra-disulfide bond between two cysteine residues at both N- and C-termini. Derived from the dimerization arm (residues 242–259) of the EGFR ectodomain, it has inhibitory activity against EGFR autophosphorylation.28 Confocal microscopy imaging and flow cytometry analysis have shown that Peptide 1 can be captured inside EGFR-positive cancer cell lines, A431 and A549. Consequently, Peptide 1 has potential as an intracellular delivery vehicle directed to EGFR-positive cells. In the present study, the selectivity of Peptide 1 to EGFR was verified and it was used as a vehicle for intracellular delivery to cells by conjugation with a membrane-impermeable apoptotic peptide (PAD) via an enzymatically cleavable linker.

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RESULTS AND DISCUSSION The cellular uptake of Peptide 1 to EGFR-positive cells. To examine whether Peptide 1 can be selectively captured by EGFR-positive cells, two EGFR-positive cell lines, A431 and A549, and a normal cell line, HEK293, were treated with a fluorescein-labeled Peptide 1 (FAM-Peptide 1, Figure 1A) and observed with a confocal laser scanning microscope. The cyclic form of Peptide 1 is more suitable compared to the corresponding linear form because FAM-Peptide 1 has even higher inhibitory activity against EGFR autophosphorylation than a fluorescein-labeled linear derivative of Peptide 1 (FAM-linear QTPYYMNT) (Supporting Information). The significant fluorescence of FAM-Peptide 1 was observed inside EGFR-positive cells but not in HEK293, a normal cell line with minimal expression of the receptor (Figure 1B).29 Next, the influence of temperature in the cellular uptake of Peptide 1 was assessed. Temperature is known to be a key factor for cellular uptake via an endocytosis pathway. It has been reported that various CPPs are internalized inside cells by endocytosis, and their cellular uptake is largely suppressed at lower temperatures.30,31 The cellular uptake of FAM-Peptide 1 was reduced at 4 °C (Figure 1C) showing that internalization of Peptide 1 into cells is dependent on cellular activity.

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Figure 1. Evaluation of cell selectivity and influence of temperature on the cellular uptake of Peptide 1. (A) Structure of FAM-Peptide 1. (B) Cellular uptake of FAM-Peptide 1 (10 µM) in EGFR-positive cell lines, A431 and A549, and an EGFR-negative cell line, HEK293, for 15 min at 37 °C. Nuclei were counterstained with Hoechst 33258. Scale bars represent 20 µm. (C) Cellular uptake of FAM-Peptide 1 (10 µM) in A549 cells for 30 min at 37 °C or at 4 °C. Cellular uptake was observed by flow cytometry (left) and confocal microscopy (right). Flow cytometry (left): the results are shown as the mean ± standard deviation values of three independent experiments

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performed in triplicate. The fluorescence intensity of FAM-Peptide 1 at 37 °C in A549 is defined as standard. Confocal microscopy (right): scale bars represent 20 µm.

In addition, the intracellular localization of Peptide 1 was investigated using three organelle markers, MitoTracker® Red, CellLight® Lysosomes-RFP and CellLight® Golgi-RFP all of which were obtained from Thermo Fisher Scientific Inc. Co-localization of FAM-Peptide 1 with each marker in A549 cells was observed with a confocal microscope. As shown in Figure 2, co-localization was observed between the fluorescence from FAM-Peptide 1 and the lysosome marker in vesicles inside the cells (Figure 2). No apparent co-localization of FAM-Peptide 1 with mitochondria or Golgi markers was observed. These results indicate that Peptide 1 can localize in lysosomes after the cellular uptake.

Figure 2. Analysis of the accumulation sites of Peptide 1 in A549 cells. Cells were treated with FAM-Peptide 1 (10 µM) at 37 °C for 30 min. Red fluorescence indicates mitochondria, lysosome and Golgi-apparatus stained with Mitotracker Red, CellLight Lysosome-RFP, and CellLight Golgi-RFP, respectively. Scale bars represent 20 µm. White arrows indicate the co-localization of 7



FAM-Peptide 1 (green) with lysosomes (magenta). A high resolution data stained with Mitotracker Red is shown as Figure S1 in the Supporting Information.

Determination of specificity of Peptide 1 by siRNA knockdown of the EGFR. A correlation between the expression level of the receptor and the amount of FAM-Peptide 1 entering into the cells was investigated using the EGFR knockdown of A549 cells to verify the selectivity of Peptide 1 to EGFR. The knockdown cells were prepared by transfecting siRNA-EGFR as described in the experimental section. The expression level of the receptor in the knockdown cells decreased to approximately 27% of that in the native A549 cells (Figure 3A). Judging by flow cytometry analysis, the amount of FAM-Peptide 1 captured in the knockdown cells was reduced to 70% of that in the cells treated with a negative control (Figure 3B).

(A) 1.2 1.0 0.8 0.6 0.4 0.2 0.0 No siRNAtransfection negative control l

Relative EGFR expression

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siRNAEGFR

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FAMAlexa Fluor 488Peptide 1 cetuximab

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Figure 3. EGFR knockdown using siRNA in A549 cells. (A) The total EGFR expression levels were detected by western blotting using anti-EGFR. The EGFR expression level was quantified by densitometry. The band intensity of “no transfection” is set as a standard. The results are shown as the mean ± standard deviation from three independent experiments. (B) The effects of the EGFR knockdown on the cellular uptake of FAM-Peptide 1 (10 µM), Alexa Fluor 488-cetuximab (5 nM) and FAM-R8 (10 µM), were analyzed by flow cytometry. The results are shown as the mean ± standard deviation values of three independent experiments performed in triplicate. The fluorescence intensity of each test peptide in an siRNA-negative control is defined as a standard. Semi-quantitative assessment of the uptake of the peptides, FAM-Peptide 1 and FAM-R8, in A549 cells (no transfection of siRNA-EGFR) were shown as Figure S2 in the Supporting Information.

Alexa Fluor® 488-cetuximab, which is a labeled anti-EGFR antibody, also showed a decreased level of uptake in the knockdown cells, which was 22% of that in the cells treated with the negative control. A fluorescein-labeled octa-arginine peptide (FAM-R8), which has membrane-permeability independent of the EGFR, was taken into the knockdown cells at the level similar to that in the negative control. These results indicate that the EGFR is responsible for the cellular uptake of Peptide 1, and suggest that the peptide could be a promising candidate for an intracellular delivery molecule which targets EGFR-positive cells. Cetuximab is a monoclonal anti-EGFR antibody approved by FDA, and reduction of the amount of cellular uptake of cetuximab was linearly and 9



wholly correlated with the expression level of the EGFR. However, the amount of cellular uptake of Peptide 1 was shown to be correlated at a certain levels with the expression level of the EGFR. The lower affinity of Peptide 1 to the EGFR compared to that of cetuximab might be a possible reason for this effect. In addition, since the dimerization arm is conserved in the other ErbB family receptors HER2, ErbB3 and ErbB4,32–34 which are frequently overexpressed in cancer cells,35,36 it is possible that Peptide 1 could bind to the cell surface and be captured inside the cells. Furthermore, a complete reduction of cellular uptake was not accomplished because Peptide 1 might enjoy other uptake mechanisms such as macropinocytosis.

Peptide 1 as an intracellular delivery molecule. To demonstrate the potency of Peptide 1 as a vehicle for intracellular delivery, conjugates of Peptide 1 with a membrane-impermeable pro-apoptotic domain (PAD) peptide were designed and synthesized (Figure 4). As shown in Figure 4, conjugate 2 with a peptide linker (GFLG), which can be cleaved by a lysosomal cysteine protease cathepsin B,37 was designed and synthesized. Conjugate 3, containing a Gly linker was prepared as a negative control for conjugate 2.

Figure 4. Amino acid sequences of PAD and conjugates 2 and 3.

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To examine the bioactivity of the conjugates 2 and 3 in vitro, their inhibitory activity against cell viability was evaluated by their treatment of EGFR-positive cell lines, A549 and A431, and a normal cell line, HEK293, which expresses lower levels of the EGFR. It was found that conjugate 2 significantly suppressed the cell viability in EGFR-positive cell lines, A549 and A431 (Figure 5A, B). In contrast, conjugate 3 with a Gly linker, or the PAD peptide alone failed to show any substantial inhibitory activity. However, in the assay with HEK293, conjugate 2 failed to show any significant effect on the cell viability (Figure 5C). Further, simultaneous treatment of A431 cells with both Peptide 1 and PAD instead of conjugate 2 showed essentially zero effect on the cell viability (Figure S4). These results indicate that conjugate 2 inhibits the cell viability of EGFR-positive cancer cells, A549 and A431, although, at 60 µM, conjugate 2 decreased cell viability to 70% in HEK293 cells. Thus, conjugate 2 might be captured inside HEK293 cells as a result of the slight expression of the EGFR. In addition, Peptide 1 could bind to the cellular membrane or some other proteins on the surface without specificity at higher concentrations and could be captured inside the cells by other mechanisms such as macropinocytosis. Therefore, while the complete selectivity of conjugate 2 to the EGFR has not been demonstrated to date, Peptide 1 can certainly deliver a membrane-impermeable PAD peptide into cells by conjugation, and conjugate 2 is potentially useful for the development of new anti-cancer therapeutic disciplines. From this result, it can be seen that a cleavable linker plays an essential role in the bioactivity of conjugate peptides. PAD is known to form a helical structure, but Peptide 1 with its cyclic structure, might hinder this helix formation. Conjugate 2 might be taken up into cells and cleaved by cathepsin B in endosomes at a cleavable linker site, and then PAD could form a helical structure and escape from lysosomes to the cytoplasm by barrel-stave mechanism or in a carpet-like manner.38

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Peptide (µM) Figure 5. Cell viability of A549 (A), A431 (B) and HEK293 (C) in the presence or absence of each test peptide. The viability with H2O alone (0 µM) was taken as 100% cell viability. The results are shown as the mean ± standard deviation values of three independent experiments performed in triplicate. Curve fitting data to calculate IC50 values of conjugate 2 using A549 (A) and A431 (B) are shown in as Figure S3 in the Supporting Information.

Detection of apoptosis by assessment of exposed-phosphatidylserine on the outside membrane. To verify the apoptotic activity of conjugate 2 in A549 cells, the exposed phosphatidylserine (PS) on the cellular surface was detected using FITC-labeled Annexin V, which specifically binds to PS. In apoptotic cells, PS is exposed on the cytoplasmic membrane.39 As shown in Figure 6, significant fluorescence of Annexin V could be detected on the membrane of A549 cells treated with conjugate 2. The same phenomenon was also observed in staurosporine-treated cells (Figure 6). Staurosporine is a protein kinase inhibitor with apoptotic activity to cells,40 and was used as a positive control. These results indicate that conjugate 2 can induce apoptosis in A549 cells. In cells treated with conjugate 3 or PAD peptide alone, the fluorescence of Annexin V was barely detectable,

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suggesting these peptides do not induce apoptosis. Therefore, as in Figure 5A, the viability of A549 might be suppressed by the apoptotic activity of conjugate 2.

Figure 6. Detection of apoptosis in A549 cells using Annexin V-FITC. Cells were treated with each peptide (50 µM) at 37 °C for 24 h or with staurosporine (1 µM) at 37 °C for 4 h. Scale bars represent 50 µm.

CONCLUSION Confocal microscopy analysis showed that FAM-Peptide 1 is captured inside the cell by EGFR-positive cell lines, but not by the normal cell line, HEK293. The cellular uptake of FAM-Peptide 1 was remarkably decreased at 4° C, indicating that internalization of Peptide 1 in cells proceeds via endocytosis. In addition, analysis of co-localization with organelle-specific markers showed that after cellular uptake, FAM-Peptide 1 is localized in lysosomes. The involvement of EGFR in the cellular uptake of Peptide 1 was verified by siRNA knockdown of EGFR using A549 cells. These results indicate that Peptide 1 could be useful as a vehicle for 14


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intracellular delivery with a certain selectivity to EGFR. To investigate the ability of Peptide 1 to behave as a vehicle for intracellular delivery, conjugate 2, in which Peptide 1 is conjugated with a PAD peptide via a cleavable linker, was synthesized and its bioactivity was evaluated. Conjugate 2 showed clear suppression of the viability of A549 and A431 cells, whereas conjugate 3, containing a Gly linker had no significantly inhibitory activity in these cell lines. Furthermore, conjugate 2, at an optimum concentration for intracellular delivery fails to show any significant effect on the HEK293 cell viability. In the apoptosis detection experiments in A549 using Annexin V-FITC, cells treated with conjugate 2 showed apoptotic behavior. These results indicate that Peptide 1 might become a lead compound for a vehicle for intracellular drug delivery with selectivity for EGFR-positive cells. Furthermore, a new apoptotic peptide, conjugate 2, with apoptosis induction activity could contribute to the development of anticancer reagents based on the AMP functions.

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MATERIALS AND METHODS General. Fmoc amino acid derivatives, Thr(But), Tyr(But), Pro, Met, Cys(Trt), Asn(Trt), Lys(Boc), Leu, Ala, Phe, Gly, Arg(Pbf) and Gln(Trt), were purchased from Merck Japan Ltd. (Tokyo, Japan) or KOKUSAN CHEMICAL Co., Ltd. (Tokyo, Japan). 5(6)-Carboxyfluorescein was purchased from Sigma-Aldrich Co. LLC. (St. Louis MO, USA). Dulbecco’s modified eagles medium (DMEM), Cell Count Reagent SF and Annexin V-FITC Apoptosis Detection Kit were purchased from Nacalai Tesque Inc. (Kyoto, Japan). Fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific Inc. (Rockford, IL, USA). The reagents were used without further purification. Analytical HPLC was performed using a C18 reverse phase column (4.6 × 250 mm; a COSMOSIL Packed column; Nacalai Tesque Inc.) with a binary solvent system; a linear gradient of CH3CN in 0.1% aqueous TFA at a flow rate of 1.0 mL/min with detection at 220 nm. Preparative HPLC was carried out using a C18 reverse phase column (20 × 250 mm; Nacalai Tesque Inc.) with a binary solvent system; a linear gradient of CH3CN in 0.1% aqueous TFA at a flow rate of 5 mL/min with detection at 220 nm. The solvents were of HPLC grade. The purified products were lyophilized and identified using a Bruker Daltonics micrOTOF focus (ESI-MS) spectrometer. For confocal laser scanning fluorescence images, cells were observed with a FluoView FV10i laser scanning confocal microscope (OLYMPUS, Tokyo, Japan). Cell lines and culture conditions. Human epidermoid carcinoma cells, A431, were obtained from RIKEN (Tsukuba, Japan) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/L glucose, L-glutamine, and sodium pyruvate supplemented with 10% FBS and 1% penicillin–streptomycin (v/v; 10,000 units/mL and 10,000 µg/mL, respectively) at 37 °C under a humidified atmosphere of 5% CO2. The human lung adenocarcinomic human alveolar basal epithelial cells, A549, and the human embryonic kidney cells, HEK293, were obtained from RIKEN

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and were cultured in essentially the same conditions described above. Peptide synthesis Preparation of an amino acid-loaded 2-chlorotrityl resin: A 2-chlorotrityl chloride resin (1.62 mmol/g, 2.16 g, 3.26 mmol) was agitated in dry DMF (5.0 mL) at rt for 2 h. Then Fmoc-Cys(Trt)-OH (678.8 mg, 1.16 mmol) and diisopropylethylamine (DIPEA) (808 µL, 4.64 mmol) were added, and the mixture was stirred at rt for 60 min. After washing with dry DMF (×5), non-reacted trityl chloride group on the resin was capped with MeOH (1 mL) and DIPEA (404 µL) in dry DMF for 20 min at rt. Synthesis of a PAD-derived fragment: A PAD-derived fragment (KLAKLAKKLAKLAKG) was synthesized by Fmoc-based solid phase peptide synthesis on Fmoc-Gly-(2-Cl)Trt-resin (0.905 mmol scale). Fmoc-protected amino acids (3 equiv.) were coupled using 1-hydroxybenzotriazole monohydrate (HOBt·H2O) (3 equiv.) and N,N’-diisopropylcarbodiimide (DIPCDI) (3 equiv.) in DMF. The Fmoc group was deprotected with 20% piperidine (v/v) in DMF for 15 min. Successive condensation of the corresponding Fmoc amino acid derivatives was carried out with the same deprotection/coupling protocol. The resulting protected PAD-derived fragment was cleaved from the resin with TFE/AcOH/DCM (1:1:3, v/v, 200 mL) for 2 h. The reaction mixture was filtered, the filtrate was evaporated under vacuum, and the peptide was precipitated as solid powder. The crude peptide was subjected to preparation of PAD-Peptide 1 conjugates 2 and 3. Conjugation of PAD and Peptide 1: The protected peptide chains composed of Peptide 1 with additional residues (Phe-Leu-Gly and Gly-Gly-Gly) at the N-terminus were constructed on a 2-chlorotrityl resin (0.19 mmol) as described above. After the elongation of the peptide chains, the protected peptide of the PAD-derived fragment (KLAKLAKKLAKLAKG, 1.5 equiv.) was reacted at the N-terminus of the protected peptide resins with HOAt (1.5 equiv.) and DIPCDI (1.5 equiv.) in N-methylpyrrolidone (MNP) by stirring for 4 d. The resins were washed with DMF, CH2Cl2, and

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Et2O, and dried in vacuo. The protected peptide resins were treated with TFA/EDT/H2O/TIS (10:0.75:0.25:0.1, 10 mL) for 2 h. The reaction mixtures were filtered, the filtrates were evaporated under vacuum, and an excess volume of cold Et2O (40 mL) was added to the residues to precipitate the crude peptides. The crude peptide of conjugate 2 (14.8 mg) in NH4HCO3 buffer (pH 7.8, peptide concentration of 0.2 mg/mL) was stirred in air at rt. After 48 h, the solution was evaporated and purified by a preparative RP-HPLC [C18 reversed phase column (20 × 250 mm; Nacalai Tesque), 0.1% TFA/CH3CN in H2O, 5 mL/min] to obtain the desired peptide, conjugate 2. HPLC rt. 30.4 min [C18 reversed phase column (4.6 × 250 mm; a COSMOSIL Packed column), 1 mL/min, CH3CN (20-50%)/60 min]. The purified product was lyophilized and identified by ESI-TOF-MS; calcd. for C142H236N36O35S32+ [M/2+H]2+: 1550.9, found: 1550.9 (13% yield). Conjugate 3 was prepared similarly in 19% yield. HPLC rt. 25.4 min [C18 reversed phase column (4.6 × 250 mm; a COSMOSIL Packed column), 1 mL/min, CH3CN (15-45%)/60 min], ESI-TOF-MS; calcd. for C131H222N36O35S32+ [M/2+H]2+: 1477.8, found: 1477.8. H-[Lys-Leu-Ala-Lys-Leu-Ala-Lys]2-NH2 (PAD): HPLC rt. 32.4 min [C18 reversed phase column (4.6 × 250 mm; a COSMOSIL Packed column), 1 mL/min, CH3CN (10-40%)/60 min], ESI-TOF-MS; calcd. for C72H140N21O14+ [M+H]+: 1523.1, found: 1523.1 (27% yield). 5(6)-Carboxyfluorescein-Gly-Gly-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-NH2 (FAM-octa-arginine) (FAM-R8): HPLC rt. 12.9 min [C18 reversed phase column (4.6 × 250 mm; a COSMOSIL Packed column), 1 mL/min, CH3CN (15–45%)/60 min], ESI-TOF-MS; calcd. for C69H110N33O14+ [M+H]+: 1738.9, found: 1738.8 (13% yield). Fluorescence imaging by confocal laser scanning microscopy Cells (8 × 104 cells/mL) were seeded in 35 mm glass-bottom dishes (Greiner Bio-one Co., Ltd.) and cultured in DMEM at 37°C under 5% CO2 for 24 h. After removing the medium, the cells were

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washed three times with PBS(+) (PBS containing Ca2+ and Mg2+ ), and then treated with 10 µM labeled peptides in 200 µL of DMEM at 37°C for 15 min. Nuclei were counterstained with Hoechst 33258 (Dojindo Co., Ltd.). Cell imaging was performed with a confocal laser scanning microscope FV10i (Olympus Co., Tokyo, Japan.). Fluorescence image were acquired with an FITC filter (Ex: 473 nm, Em: 490-540 nm) and a Hoechst 33258 filter (Ex: 405 nm, Em: 420-470 nm). Intracellular localization of Peptide 1 was performed with organelle markers. MitoTracker® Red (Thermo Fisher Scientific Inc.), CellLight® Lysosomes-RFP (Thermo Fisher Scientific Inc.) and CellLight® Golgi-RFP (Thermo Fisher Scientific Inc.) were used as organelle markers for mitochondria, lysosomes and Golgi-apparatus, respectively. EGFR knockdown The synthetic siRNA used for the EGFR knockdown was purchased from Ambion® (Thermo Fischer Scientific Inc.). The target sequence was 5’-CCAUAAAUGCUACGAAUAUtt-3’ (ID: s564). Negative control #1 was used as a control. A549 cells (1.5 × 106 cells/10 cm diameter dish) in complete DMEM were grown at 37 °C for 24 h under 5% CO2, then trypsinized, and washed once with PBS(-). For transfection using Neon Transfection System (Thermo Fischer Scientific Inc.), A549 cells (1 × 106 cells total) and 50 nM siRNA were resuspended in 100 µL buffer R, followed by electroporation (1200 V, 30 ms, 2 Pulse). After electroporation, cells were resuspended in DMEM with 10% FBS, and plated at a concentration of 1 × 105 cells/mL. Knockdown of EGFR was confirmed by western blotting. Western blot analysis was performed after SDS-PAGE (NuPAGE® 4–12% Bis-Tris Gel, Life Technologies Corporation) using a rabbit polyclonal anti-EGFR antibody, 1005 (Santa Cruz Biotechnology, Inc.) in combination with a peroxidase-linked anti-rabbit IgG secondary antibody NA934 (GE Healthcare Ltd.). Cellular uptake of FAM-Peptide 1 by EGFR-knockdown cells After incubation in 24-well plates for 72 h after siRNA transfection, A549 cells were washed once

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with DMEM then incubated with 10 µM peptides or 5 nM Alexa Fluor® 488-C225 (cetuximab) (Novus biologicals, LLC.) in 150 µL DMEM at 37 °C for 30 min. Cells were then washed three times with PBS then trypsinized at 37 °C for 5 min. After incubation, cells were centrifuged at 800 ×g for 3 min at 4 °C, and washed twice with 200 µL cold PBS. After washing, cells were analyzed on a NovoSampler® Pro (ACEA Biosciences, Inc.), using 488 nm laser excitation and a 530 nm emission filter. Each sample was analyzed for 10,000 events. For observation using a confocal laser microscope, A549 cells (4 × 104 cells/mL) were plated on 35 mm glass-bottom dishes (Greiner Bio-one Co., Ltd.) and incubated for 72 h after siRNA transfection. After removing the medium, the cells were washed three times with PBS(+) (PBS containing Ca2+ and Mg2+ ), and then treated with 10 µM labeled peptides in 200 µL of DMEM at 37°C for 30 min. After that, the cells were washed three times with PBS(+). The images were acquired with Olympus FV10i (Olympus). Cell viability assay A549, A431 and HEK293 (3000 cells/well) cells in DMEM supplemented with 10% FBS were seeded in 96-well plates and incubated at 37 °C for 24 h under 5% CO2. After removal of the medium, the cells were treated with test peptides in media at 37 °C under 5 % CO2 for 72 h. Cell viability was determined with Cell Count Reagent SF (Nacalai Tesque Inc.). Absorption values at 450 nm (reference: 650 nm) were measured. The viability of H2O alone was defined as 100% cell viability. Apoptosis assay A549 (1 × 105 cells/dish) cells in DMEM supplemented with 10% FBS were seeded in 35 mm glass-bottom dishes (Greiner Bio-one Co., Ltd.) and cultured at 37 °C under 5% CO2 for 24 h. After removal of the medium, the cells were treated with test compounds in media at 37 °C under 5% CO2 for 24 h. For treatment with staurosporine (Wako Pure Chemical Industries, Ltd.), incubation time

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was 4 h. Cells were then washed three times with PBS(+) (PBS containing Ca2+ and Mg2+ ), and stained with AnnexinV-FITC (Nacalai Tesque Inc.) according to the manufacturer’s protocol. The cells were then washed three times with PBS(+). The images were acquired with Olympus FV10i (Olympus).

Acknowledgements This work was supported by JSPS KAKENHI Grant Numbers JP15H04652 to H.T., and JP17J08315 to K.T., the Platform for Drug Discovery, Informatics, and Structural Life Science of MEXT, Japan, and the Cooperative Research Project of Research Center for Biomedical Engineering.

Supporting Information Available Evaluation of linear QTPYYMNT, high resolution imaging data, cellular uptake data, additional cell viability data, and analytical data of EGFR expression levels, and HPLC charts. This material is available free of charge via the Internet at http://pubs.acs.org.

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overexpressing

ErbB2,

ErbB3,

or

specific

ErbB4

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Graphical Table of Contents

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Delivery of a Pro-apoptotic Peptide to EGFR-positive Cancer Cells by

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