Azurin, a member of the cupredoxin family of copper containing redox proteins, preferentially penetrates human cancer cells and exerts cytostatic and cytotoxic (apoptotic) effects with no apparent activity on normal cells. Amino acids 50 to 77 (p28) of azurin seem responsible for cellular penetration and at least part of the antiproliferative, proapoptotic activity of azurin against a number of solid tumor cell lines. We show by confocal microscopy and fluorescence-activated cell sorting that amino acids 50 to 67 (p18) are a minimal motif (protein transduction domain) responsible for the preferential entry of azurin into human cancer cells. A combination of inhibitors that interfere with discrete steps of the endocytotic process and antibodies for caveolae and Golgi-mediated transport revealed that these amphipathic, α-helical peptides are unique. Unlike the cationic cell-penetrating peptides, α-helical antennapedia-like, or VP22 type peptides, p18 and p28 are not bound by cell membrane glycosaminoglycans and preferentially penetrate cancer cells via endocytotic, caveosome-directed, and caveosome-independent pathways. Once internalized, p28, but not p18, inhibits cancer cell proliferation initially through a cytostatic mechanism. These observations suggest the azurin fragments, p18 and p28, account for the preferential entry of azurin into human cancer cells and a significant amount of the antiproliferative activity of azurin on human cancer cells, respectively. [Cancer Res 2009;69(2):537–46]

Cell-penetrating peptides (CPP) are short amphipathic and cationic peptides and peptide derivatives, usually containing multiple lysine and arginine residues (1). They form a class of small molecules receiving significant attention as potential transport agents for a variety of cargoes including cytotoxic drugs (2, 3), antisense oligonucleotides (4), in gene therapy (5, 6), and as decoy peptides (7). Small arginine-rich and other cationic peptides generated from phage-displayed peptide libraries, initially characterized by the RGD (Arg-Gly-Asp) sequence that recognizes the integrin family of cell surface receptors important to the invasion of tumor cells (8), are also potential carriers for imaging (9) and therapeutic agents. These peptides enter cells in an energy-dependent manner (9) and can distinguish between normal and malignant vasculature (10, 11) and lymphatic tissue in murine systems (9). The latter also induce apoptosis in tumor types, which also bind the peptide (9). However, even these recent additions to the CPP armamentarium seem limited to binding to tumor-associated endothelial or lymphatic cells rather than directly and preferentially penetrating a wide variety of malignant cells. Various mechanisms for entry have been proposed; it now seems that the majority of CPPs enter cells via adsorptive-mediated endocytosis rather than direct penetration of the plasma membrane (1). Whatever the mechanisms of cell entry and intracellular disposition, the overriding question regarding the potential pharmacologic application of CPPs is whether or not their intracellular concentration in a target cell, in addition to that of any attendant cargo, is sufficient to elicit a pharmacologic response at levels that are not toxic to nontarget cells (1). We have recently suggested amino acids (aa) 50 to 77 of azurin, a cupredoxin secreted by Pseudomonas aeruginosa, as a putative protein transduction domain (PTD) responsible for the penetration of azurin into cancer cells (12), although we did not identify a route of cellular entry. The present study refines the PTD of azurin from aa 50 to 77 (p28) to aa 50 to 67 (p18) and provides evidence for an endocytotic and nonendocytotic-mediated entry into normal and cancer cells that is not dependent on membrane bound glycosaminoglycans. Moreover, the COOH-terminal 12 aa of p28 accounts for a significant amount of the antiproliferative activity of azurin on human cancer cells (13, 14).

Cell culture and cell lines. Human cancer and noncancer (immortalized and nonimmortalized) cell lines were obtained from American Type Culture Collection [lung cancer (A549 and NCI-H23 adenocarcinoma), normal lung (CCD-13Lu), prostate cancers (DU145 and LN-CAP), normal prostate (CRL11611), breast cancer (MCF-7), normal breast (MCF-10A), colon cancer (HCT116), normal colon (CCD33Co), fibrosarcoma (HT1080), and ovarian cancer (SK-OV3 adenocarcinoma)]. Normal fibroblasts isolated from skin were established in our laboratory. Normal ovarian cells (HOSE6-3) were a generous gift from Dr. S.W. Tsao (University of Hong Kong, Hong Kong, China). Melanoma lines (UISO-Mel-2, 23, 29) were established and characterized in our laboratory (15). All cells except UISO-Mel-2 (MEM-H) were cultured in MEM-E (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Atlanta Biological, Inc.), 100 units/mL penicillin, and 100 μg/mL streptomycin at 37°C in 5% CO2 or air.

Proliferation assays/cell growth. Melanoma cells were seeded (4 replicates) in flat-bottomed 24-well plates (Becton Dickinson) at a density of 12 × 103 cells per well. After 24 h, medium was changed and fresh p18, p28, azurin, or a similar volume of medium without peptide (8 replicates) added daily for 72 h. Cells were then counted in a Beckman Coulter (Z1 coulter particle counter). Values represent the mean ± SD of four replicates.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Melanoma cells were seeded at a density of 2,000 cells per well and allowed to attach for 24 h. Freshly prepared peptide (10 μL) or culture medium was then added to each well. After 24 h, medium was changed and p18, p28, or azurin were added daily. After 72 h of incubation, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (Trevigen) was added to each well, the samples incubated for 3 h at room temperature, 100 μL of detergent added to each well, and incubated for an additional 3 h at 37°C. Absorbance was measured with a SpectraMax 340 plate reader (Molecular Devices Corporation) and percent change in the absorbance at 570 nm in treated cells relative to untreated controls determined. Values represent the mean ± SD. Significance between control and treated groups was determined by Student's t test.

Peptide synthesis. All azurin-derived peptides including p18, Leu50-Gly67 LSTAADMQGVVTDGMASG, p28 Leu50-Asp77 LSTAADMQGVVTDGMASGLDKDYLKPDD, p18b Val60-Asp77 VTDGMASGLDKDYLKPDD, p12 Gly66-Asp77 SGLDKDYLKPDD, mitogen-activated protein, Mastoparan-7, and poly arginine (Arg8) were synthesized by CS Bio, Inc., as >95% purity and mass balance.

Predictive modeling for azurin peptides. We used GENETYX software (ver. 6.1) to generate Robson structure models for azurin derived peptides (16). The MAPAS software was used to identify strong membrane contacts and define regions of the protein surface that form such contacts (17). If a protein, i.e., azurin, has a membranephilic residue score (MRS) of >3, membranephilic area score (MAS) of >60%, and coefficient of membranephilic asymmetry (Kmpha) of >2.5, there is a high probability that the protein has a true membrane-contacting region.

Peptide/protein labeling. Peptides were dissolved in 1 mL PBS mixed with Alexa Fluor 568 dye (Molecular Probes) at a 1:2 protein/dye ratio, 100 μL sodium bicarbonate were added, and the mixture were incubated overnight at 4°C with continuous stirring. Labeled peptide was dialyzed against cold-PBS using Slide-A-Lyzer Dialysis Cassettes 1000 MWCO for p12 and 2000 MWCO for others (Pierce Biotechnology).

Cell penetration/confocal analysis. Cells were seeded overnight on glass coverslips at 37°C under 5% CO2, rinsed with fresh medium, and incubated at 37°C for 2 h in prewarmed medium containing Alexa Fluor 568–labeled azurin peptides (20 μmol/L), Arg8 (5 μmol/L), or medium alone. After incubation, coverslips were rinsed 3× with PBS, fixed in 2.5% formalin for 5 min, and washed 2× in PBS, once in d.i.H2O, and mounted in medium containing 1.5 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) to counter stain nuclei (VECTASHIELD; Vector Laboratories). Cellular uptake and distribution were photographed under an inverted confocal laser scanning microscope (Model LC510; Carl Zeiss Inc.).

Peptide colocalization with lysosomes or mitochondria was determined by incubating cells growing on a glass coverslip for 2 h at 37°C with Alexa Fluor 568–labeled azurin or peptides. Mitrotracker (MitroTracker Green FM) or lysotracker (LysoTracker Green DND-26; Invitrogen Corporation) was added (final concentration, 1 μmol/L) for the last 30 min of incubation. Cells were rinsed 3× with PBS, fixed in 2.5% formalin for 5 min, washed 2× with PBS, and incubated in 0.1% Triton-X100 in PBS for 15 min. Cells were then incubated with 1 μg/mL rabbit anti-human golgin 97 or anti-human caveolin 1 (Abcam) in PBS with 1% bovine serum albumin. After 1 h of incubation at 4°C, coverslips were washed once with PBS, incubated 10 min in PBS containing Alexa Fluor 468–conjugated goat anti-rabbit antibody, washed 2× in PBS, and once in d.i.H2O. Coverslips were mounted in medium containing 1.5 μg/mL DAPI. Colocalization (yellow) of Alexa Fluor 568 (red) and Alexa Fluor 468 (green) was analyzed and photographed.

UISO-Mel-2 cells on coverslips were preincubated in MEM-H containing 100 μg/mL heparin sulfate (HS; Sigma-Aldrich) for 30 min and p18, p28, or Arg8 were added to bring the final concentration to 20 μmol/L. After 1 h, coverslips were washed, fixed, and analyzed as described.

Cell penetration by fluorescence-activated cell sorting. Cells (1.0 × 106/500 μL PBS) were incubated for 2 h at 37°C with Alexa Fluor–568 labeled p18 or p28 (20 μmol/L), Arg8 (5 μmol/L), or medium alone, washed 3× in PBS, fixed in 2.5% formalin for 5 min, washed twice in PBS, resuspended in 200 μL PBS, passed through a screen to obtain a single-cell suspension, and analyzed with a MoFlo Cell Sorter (Dako) λex 568 nm and λem 603 nm. The fold increase of the mean fluorescence intensity (MFI) over background levels represents mean fluorescence of three separate experiments.

Entry inhibitors. UISO-Mel-2 cells (3 × 105 per 300 μL) maintained in phenol red–free, serum-free MEM-H at 37°C, were pretreated with the following inhibitors (Sigma-Aldrich): chlorpromazine (CPZ), amiloride, nystatin, filipin, methyl-β-cyclodextrin (MβCD), brefeldin A (BFA), Taxol, sodium azide, cytochalasin D, staurosporine, ouabain, wortmannin, monensin, nocodazole, benzyl-2-acetamido-2-deoxy-α-d-galactopyranoside, (BnGalNac), tunicamycin, and neuraminidase. Final concentrations were derived from the dose response curves of individual inhibitors. Alexa Fluor 568–labeled p18 or p28 (20 μmol/L) was then added, incubated for 1 h, and the cells washed, fixed, and prepared for flow cytometric analysis.

Cell membrane toxicity assays/lactate dehydrogenase leakage assay. A lactate dehydrogenase (LDH) leakage assay was performed according to the manufacturer's instructions (CytoTox-One; Promega) with 100 μL of UISO-Mel-2 cells (5 × 103). Cells without peptides/proteins were used as a negative control. Experiments were carried out in triplicate (data represent mean ± SE).

Hemolysis assay. Human whole blood samples (2–3 mL) were centrifuged for 10 min at 1,000 × g, the pellets washed once with PBS, once with HKR buffer (pH7.4; ref. 18), resuspended in HKR buffer to 4% erythrocytes, and 50 μL transferred to a 1.5-mL tube with 950 μL of peptides, azurin (5, 50, and 100 μmol/L) or 0.1% Triton X-100 in HRK buffer to disrupt the RBC membrane. MAP and Mastoparan7 (Bachem California, Inc.) were used as positive controls. After 30 min at 37°C with rotation, tubes were centrifuged for 2 min at 1,000 × g, 300 μL of supernatants transferred to a 96-well plate, and absorbance recorded at 540 nm.

Kinetics of entry. UISO-Mel-2 cells (5 × 105 cells) were suspended in MEM-H without phenol red. Reactions were started by adding either Alexa Fluor 568-conjugated p18 at 0, 10, 20, 50, 100, 150, and 200 μmol/L for 5, 10, 15, and 20 s, or Alexa Fluor 568–conjugated p28 at 1, 10, 25, 50, 100, 150, and 200 μmol/L for 30, 60, 90, and 120 s on ice. After incubation, 1 mL of cold-PBS was added to the 250 μL reaction in mixture. Cells were centrifuged twice at 600 × g for 2 min at 4°C. At least 10,000 fixed cells were analyzed by flow cytometry in each reaction, and their background and relative fluorescence were calculated.

I125 labeling of azurin and competition assays. Peptide binding and entry was determined using a whole cell assay with UISO-Mel-2 cells in HEPES solution (50,000 cells/mL), were incubated for 30 min at 37°C with increasing concentrations (0–175 nmol/L) of radiolabeled azurin in the presence/absence of 1,000-fold excess of unlabeled p18, p28, or azurin, then washed thrice with ice cold PBS, and radioactivity remaining in the cell pellet counted. Radioactivity in cells incubated with 125I azurin alone was considered total binding; radioactivity in the presence of unlabeled azurin, p18, or p28 was considered nonspecific binding. Specific binding was determined by subtracting nonspecific binding from total binding and Scatchard plots generated.

The NH2-terminal domain of p28 is responsible for preferential entry into cancer cells. We initially defined aa 50 to 77 of azurin as a putative PTD for cell penetration (12), which fits well with structural evidence for an α-helical region encompassing residues 54 to 67 of azurin stabilizing the azurin molecule (19). Confocal analyses initially suggested that p28 and p18 of p28/azurin (Fig. 1A) penetrated human cancer cells with similar efficiency but did not penetrate histologically matched normal cell lines to the same degree (Fig. 1A; ref. 12). A singular exception was CCD13-Lu, a cell line derived from lung fibroblasts. The cationic Arg8 was rapidly and efficiently taken up into fibroblasts (Fig. 1A) and all other normal cell lines tested (data not shown). These observations were confirmed by a more sensitive fluorescence-activated cell sorting (FACS) analyses (Fig. 1B) where p28 fluorescence was ∼0.5 to 6 and p18 ∼0.5- to 3-fold higher than the corresponding normal cell line, with the exception of lung cancer. A similar pattern in intracellular fluorescence intensity was observed within a melanomas, where the relative intensity of p18 was ∼50% of that observed with p28 (Fig. 1C). Fluorescence intensity over background was also consistently lower in cell pairs exposed to p18 than p28 (data not shown), again suggesting less p18 entered individual cells. In all cases, the degree of entry of p18 and p28 into either cancer or normal cells was less than that observed with Arg8, where no preference for entry was observed (Fig. 1A). The predicted Robson structure (data not shown) of p18 suggests that the COOH-terminal aas form a partial β-sheet. This and the shorter length of p18, which lacks the hydrophilic COOH-terminal 10 aas (aa 68–77) of p28, suggests that p18, as a putative PTD for azurin, may have a more rapid entry into cancer and normal cells via a nonendocytotic over an endocytotic or membrane receptor mediated process. MAPAS data (MRS, 3.74; MAS, 87.1; Kmpha, 2.37) predict that aas 69, 70, 75, 76, and 85 of azurin provide the best opportunity for membrane contact, suggesting the COOH-terminal region of p28, not present on p18 (aa, 50–67), is most likely to contact specific residues on the cell membrane, irrespective of the status of a cell.

Figure 1.

Penetration of azurin-derived peptides, p18 and p28, into cancer cell lines of diverse histogenesis and their normal counterparts. Penetration of Alexa Fluor 568–labeled p28 or p18 after 2 h at 37°C (A). The cationic Arg8 was used as a control; red, Alexa Fluor–labeled peptide; blue, DAPI (nucleus); green, autofluorescence was detected in some cell lines. Flow cytometric analysis of the penetration of Alexa Fluor 568–labeled p28 or p18 into the same cell lines after 2 h at 37°C (B). The increase over fluorescence from normal cells was calculated. C, similar observations of p28 or p18 entry into 4 melanoma cell lines show a several-fold increase (1.5–6-fold) over fluorescence from normal cells.

Figure 1.

Penetration of azurin-derived peptides, p18 and p28, into cancer cell lines of diverse histogenesis and their normal counterparts. Penetration of Alexa Fluor 568–labeled p28 or p18 after 2 h at 37°C (A). The cationic Arg8 was used as a control; red, Alexa Fluor–labeled peptide; blue, DAPI (nucleus); green, autofluorescence was detected in some cell lines. Flow cytometric analysis of the penetration of Alexa Fluor 568–labeled p28 or p18 into the same cell lines after 2 h at 37°C (B). The increase over fluorescence from normal cells was calculated. C, similar observations of p28 or p18 entry into 4 melanoma cell lines show a several-fold increase (1.5–6-fold) over fluorescence from normal cells.

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The preferential penetration of p18 and p28 was confirmed by exposing the same cell lines to azurin 60 to 77 (p18b), or aa 66 to 77 (p12), the COOH-terminal 12 aa of p28 (Fig. 2A and B). Here, the preferential penetration observed with p18 and p28 was completely abolished. p18b (theoretical pI 4.13) has a short α-helix and partial β-sheet (16), and is extremely hydrophilic, which together may negate preferential entry. p12 (theoretical pI 4.33) lacks a secondary α-helical structure but is also hydrophilic, suggesting overall hydrophilcity may be a major contributor to the decrease in selectivity of cell penetration.

Figure 2.

Entry of azu 60 to 77 (p18b) and azu 66 to 77 (p12) into cancer and normal cells. Cells were incubated with Alexa Fluor 568–labeled p18b (A) or p12 (B) at 37°C for 2 h and images recorded by confocal microscopy. Red, Alexa Fluor–labeled peptide; blue, API (nucleus). Green or yellow, Autofluorescence in normal cell lines (green+red).

Figure 2.

Entry of azu 60 to 77 (p18b) and azu 66 to 77 (p12) into cancer and normal cells. Cells were incubated with Alexa Fluor 568–labeled p18b (A) or p12 (B) at 37°C for 2 h and images recorded by confocal microscopy. Red, Alexa Fluor–labeled peptide; blue, API (nucleus). Green or yellow, Autofluorescence in normal cell lines (green+red).

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Cell penetration is not a result of membrane disruption. Cell penetration by azurin, p28, and p18 does not result from membrane disruption. An LDH assay (Fig. 3A) suggested that neither peptide nor azurin entered cells by altering plasma membrane integrity (18). The lack of membrane disruption was confirmed by determining the hemolytic activity of azurin, p28, and p18 on human erythrocytes against the receptor mimetic MAP and mastoparan 7, which translocates cell membranes as an amphipathic α-helix, and activates heterotrimeric G proteins (18). Mastoparan 7 caused complete cell lysis at 25 μmol/L, whereas azurin, p28, and p18 had no effect when compared with control (Fig. 3B).

Figure 3.

Cellular membrane toxicity of azurin and its peptides. A, LDH leakage assay of UISO-Mel-2 cells exposure for 10 min to different concentrations of p28, p18, and azurin at 37°C. A standard lysis buffer (cytotox-one reagent) was included as a positive control. Changes in fluorescence after exposure were measured at λex 560 nm and λem 590 nm. Lysis buffer was defined as 100% LDH release. Data represent % of positive fluorescence of control. Columns, mean; bars, SE. B, hemoglobin leakage from human erythrocytes incubated with p28, p18, and azurin. Human erythrocytes were incubated with peptide for 30 min at 37°C, and absorbance at 540 nm were determined. Hemoglobin release after 0.1% Triton X-100 was defined as 100% hemoglobin release. Columns, mean of triplicate determinations; bars, SE.

Figure 3.

Cellular membrane toxicity of azurin and its peptides. A, LDH leakage assay of UISO-Mel-2 cells exposure for 10 min to different concentrations of p28, p18, and azurin at 37°C. A standard lysis buffer (cytotox-one reagent) was included as a positive control. Changes in fluorescence after exposure were measured at λex 560 nm and λem 590 nm. Lysis buffer was defined as 100% LDH release. Data represent % of positive fluorescence of control. Columns, mean; bars, SE. B, hemoglobin leakage from human erythrocytes incubated with p28, p18, and azurin. Human erythrocytes were incubated with peptide for 30 min at 37°C, and absorbance at 540 nm were determined. Hemoglobin release after 0.1% Triton X-100 was defined as 100% hemoglobin release. Columns, mean of triplicate determinations; bars, SE.

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p18/p28 penetration is energy dependent and saturable. The penetration of p28 (Fig. 4A) and p18 (Fig. 4B) into UISO-Mel-2 cells is temperature dependent. Cell penetration and intracellular transport occurs relatively slowly over 3 hours at 4°C, whereas entry and intracellular transport through various compartments is rapid at 22 and 37°C as p18 and p28 were present in the nucleus of UISO-Mel-2 cells within 2 hours postexposure. The penetration of 5 μmol/L p28 (Fig. 4C) or p18 (Fig. 4D) into UISO-Mel-2 cells after 30 minutes in the presence of a 200-fold excess of unlabeled peptide was severely curtailed, suggesting that entry was a saturable process and specific receptors or cell surface proteins or specific residues were, at least in part, responsible for initial entry.

Figure 4.

Temperature-dependent and competitive internalization of p28 and p18 into UISO-Mel-2 cells. Penetration of Alexa Fluor 568–labeled p28 (A) or p18 (B) at 20 μmol/L was evaluated by confocal microscopy at different temperatures. Red, Alexa Fluor–labeled peptide; blue, DAPI (nucleus). C and D, confocal analysis of entry of Alexa Fluor 568–labeled p28 (C) or p18 (D) at 5 μmol/L into UISO-Mel-2 cells after 30 min at 37°C in the presence/absence of unlabeled peptide (200-fold excess). RT, room temperature.

Figure 4.

Temperature-dependent and competitive internalization of p28 and p18 into UISO-Mel-2 cells. Penetration of Alexa Fluor 568–labeled p28 (A) or p18 (B) at 20 μmol/L was evaluated by confocal microscopy at different temperatures. Red, Alexa Fluor–labeled peptide; blue, DAPI (nucleus). C and D, confocal analysis of entry of Alexa Fluor 568–labeled p28 (C) or p18 (D) at 5 μmol/L into UISO-Mel-2 cells after 30 min at 37°C in the presence/absence of unlabeled peptide (200-fold excess). RT, room temperature.

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Kinetics of p28 and p18. The kinetics of p28 and p18 entry into UISO-Mel-2 cells relative to human fibroblasts was calculated using MFI. The Km and Vmax of each peptide were calculated by plotting peptide concentration (μmol/L) versus velocity (MFI/seconds) or by Scatchard analysis. Although the penetration of azurin fragments 50 to 67 (p18: Vmax, 2.46; Km, 101.6) and 50 to 77 (p28: Vmax, 1.87; Km, 159.1) into cancer and normal cells (Vmax, 2.88; Km, 102.1; and Vmax, 1.89; Km, 166.0, respectively) differs from each other, with p18 entering ∼42% faster, the rate of the entry of each peptide into normal and cancer cells is virtually identical. The increase in amount of fluorescence after exposure of cancer cells to p28 relative to p18 is likely due to the increase in the amount of p28 entering malignant cells. 125I azurin and p18 bound to UISO-Mel-2 cells with a similar affinity. In contrast, more p28 [Kd, 2.5 μm; maximum number of binding sites (Bmax), 3.0 pm] bound to UISO-Mel-2 cells with a higher affinity when exposed for a longer period of time (20 versus 2 min) at a higher temperature (37°C versus 4°C) than either p18 (Kd, 18 nm; Bmax, 0.51 pm) or azurin (Kd, 10 nm and 0.48 pm). These results suggest that azurin, p28, and p18 all bind with relatively high affinity and capacity to a site on the cancer and normal cell surface before entry, but may enter via more than one mechanism.

p18/p28 penetration involves Caveolae and the Golgi Complex. As a class, cationic CPPs such as pTat and Arg8 enter cells by initially binding to anionic, sulfated proteoglycans before endocytosis (1). Incubation of p28 and p18 and Arg8 with UISO-Mel-2 cells under serum-free conditions in the presence/absence of (HS) reduced the amount of intracellular Arg8 but did not alter the entry of either p28 or p18 (Fig. 5A). The penetration of p18, and p28 (Fig. 5B) into UISO-Mel-2 cells in the presence or absence of a specific inhibitor of O-linked glycosylation, BnGalNac, and neuraminidase, which cleaves sialic acid residues, was also inhibited. However, tunicamycin, an inhibitor of N-linked glycosylation, significantly reduced the penetration of p18 and p28 across the cell membrane (P < 0.05).

Figure 5.

A, peptide entry and trafficking. Confocal analysis of p28, p18 (20 μmol/L), and Arg8 (10 μmol/L) entry into UISO-Mel-2 cells after 1 h at 37°C in the presence/absence of HS (100 μg/mL). B, flow cytometric analysis of p28 or p18 entry in the presence of inhibitors. UISO-Mel-2 cells were pretreated with inhibitor and incubated with 20 μmol/L of Alexa Fluor 568–labeled p28 or p18 for 1 h. Specific inhibitors included the following: CPZ (inhibitor of clathrin-mediated endocytosis; 10 μg/mL; 60 min); Amiloride (macropinocytosis inhibitor; 50 μmol/L; 30 min); Nystatin (50 μg/mL; 30 min); Methly-β-cyclodextrin (MβCD; 5 mmol/L; 60 min); Filipin (inhibitors of caveolae-mediated endocytosis; 3 μg/mL; 60 min); Taxol (microtubule stabilizer; 20 μmol/L; 30 min); Staurosporine (cell cycle inhibitor; 250 nmol/L; 10 min); Sodium azide (metabolic inhibitor; 1 mmol/L; 60 min); Ouabain (ATPase-dependent Na+/K+ pump inhibitor; 50 mmol/L; 60 min); BFA (Golgi apparatus disruptor; 100 μmol/L; 60 min); Wortmannin (early endosome inhibitor; 100 nmol/L; 30 min); Monensin (inhibits at late endosome/lysosome; 10 μmol/L; 60 min); Nocodazole (inhibits caveosome formation; 10 μmol/L; 60 min); Cytochalasin D (actin filament and microtubule disruptor; 5 μmol/L; 30 min); Benzyl 2-acetamido-2-deoxy-α-D-galactopyranoside (BnGalNac; O-linked glycosylation inhibitor; 3 mmol/L; 48 h); Tunicamycin (N-linked glycosylation inhibitor; 20 μg/mL; 48 h); and Neuraminidase (cleaves sialic acid residues from proteins; 1 U/mL; 30 min). Cell fluorescence intensity in the absence of inhibitor (control) was considered as 100%. C, FACS analysis of p28 and p18 entry into fibroblasts in presence of inhibitors. D, colocalization of p18 and p28 with caveolin 1 (1). UISO-Mel-2 cells were incubated with Alexa Fluor 568–labeled p18 or p28 (20 μmol/L) or medium for 2 h at 37°C. Cells were fixed and processed for anticaveolin 1 immunostaining. Red, Alexa Fluor–labeled peptide; blue, DAPI (nucleus); green, caveolin; yellow, colocalization of p18 or p28 (red) with caveolin 1 (green). Confocal analysis of entry of Alexa Fluor 568–labeled p18 or p28 (20 μmol/L) into UISO-Mel-2 cells after 2 h at 37°C followed by antigolgin 97 antibodies (2). Red, Alexa Fluor–labeled peptide; blue, DAPI (nucleus); green, golgi; yellow, colocalization of p18 or p28 (red) with golgi (green). Colocalization of Alexa Fluor 568–labeled azurin, p28, and p18 (red) with mitotracker (green; 3) and Lysotracker (green; 4) dyes in UISO-Mel-2 cells. Cells were incubated at 37°C with 20 μmol/L azurin, p28, p18, or medium only. After 90 min incubation, mitotracker/lysotracker probes were added and cells incubated for 30 min. Cells were counterstained with DAPI (blue). Colocalization of azurin, p28, or p18 seems as a yellow fluorescence.

Figure 5.

A, peptide entry and trafficking. Confocal analysis of p28, p18 (20 μmol/L), and Arg8 (10 μmol/L) entry into UISO-Mel-2 cells after 1 h at 37°C in the presence/absence of HS (100 μg/mL). B, flow cytometric analysis of p28 or p18 entry in the presence of inhibitors. UISO-Mel-2 cells were pretreated with inhibitor and incubated with 20 μmol/L of Alexa Fluor 568–labeled p28 or p18 for 1 h. Specific inhibitors included the following: CPZ (inhibitor of clathrin-mediated endocytosis; 10 μg/mL; 60 min); Amiloride (macropinocytosis inhibitor; 50 μmol/L; 30 min); Nystatin (50 μg/mL; 30 min); Methly-β-cyclodextrin (MβCD; 5 mmol/L; 60 min); Filipin (inhibitors of caveolae-mediated endocytosis; 3 μg/mL; 60 min); Taxol (microtubule stabilizer; 20 μmol/L; 30 min); Staurosporine (cell cycle inhibitor; 250 nmol/L; 10 min); Sodium azide (metabolic inhibitor; 1 mmol/L; 60 min); Ouabain (ATPase-dependent Na+/K+ pump inhibitor; 50 mmol/L; 60 min); BFA (Golgi apparatus disruptor; 100 μmol/L; 60 min); Wortmannin (early endosome inhibitor; 100 nmol/L; 30 min); Monensin (inhibits at late endosome/lysosome; 10 μmol/L; 60 min); Nocodazole (inhibits caveosome formation; 10 μmol/L; 60 min); Cytochalasin D (actin filament and microtubule disruptor; 5 μmol/L; 30 min); Benzyl 2-acetamido-2-deoxy-α-D-galactopyranoside (BnGalNac; O-linked glycosylation inhibitor; 3 mmol/L; 48 h); Tunicamycin (N-linked glycosylation inhibitor; 20 μg/mL; 48 h); and Neuraminidase (cleaves sialic acid residues from proteins; 1 U/mL; 30 min). Cell fluorescence intensity in the absence of inhibitor (control) was considered as 100%. C, FACS analysis of p28 and p18 entry into fibroblasts in presence of inhibitors. D, colocalization of p18 and p28 with caveolin 1 (1). UISO-Mel-2 cells were incubated with Alexa Fluor 568–labeled p18 or p28 (20 μmol/L) or medium for 2 h at 37°C. Cells were fixed and processed for anticaveolin 1 immunostaining. Red, Alexa Fluor–labeled peptide; blue, DAPI (nucleus); green, caveolin; yellow, colocalization of p18 or p28 (red) with caveolin 1 (green). Confocal analysis of entry of Alexa Fluor 568–labeled p18 or p28 (20 μmol/L) into UISO-Mel-2 cells after 2 h at 37°C followed by antigolgin 97 antibodies (2). Red, Alexa Fluor–labeled peptide; blue, DAPI (nucleus); green, golgi; yellow, colocalization of p18 or p28 (red) with golgi (green). Colocalization of Alexa Fluor 568–labeled azurin, p28, and p18 (red) with mitotracker (green; 3) and Lysotracker (green; 4) dyes in UISO-Mel-2 cells. Cells were incubated at 37°C with 20 μmol/L azurin, p28, p18, or medium only. After 90 min incubation, mitotracker/lysotracker probes were added and cells incubated for 30 min. Cells were counterstained with DAPI (blue). Colocalization of azurin, p28, or p18 seems as a yellow fluorescence.

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Inhibitors of energy-dependent transport mechanisms, i.e., ATP. Sodium azide (Fig. 5B) and ouabain (Na+K+ ATPase pump) did not inhibit the penetration of either peptide, suggesting nonendocytotic pathways might also be involved in the penetration of these peptides. CPZ, which inhibits clathrin mediated endocytosis, also had no effect on penetration, nor did the macropinocytosis inhibitor amiloride (Fig. 5B). Stabilization of microtubules with Taxol had no effect on penetration, but disruption of actin filaments and macropinocytosis with Cytochalasin D produced a small (∼20%), reproducible inhibition of p18 and p28 penetration. The lack of effect of amiloride suggests that the inhibitory activity of Cytochalasin D is probably through its effect on actin filaments. Inhibition of the cell cycle with staurosporine did not block penetration, suggesting that penetration was not cell cycle specific. The lack of effect of staurosporine on p18 and p28 penetration of the cancer cell plasma membrane also suggests that a Src kinase/tyrosine kinase–dependent pathway was not involved in penetration (20), was dynamin independent, and hence independent of caveolae budding (21). In contrast, nocodazole, which disrupts caveolae transport and inhibitors of cholesterol mobilization and, hence, caveolae-mediated endocytosis, inhibited penetration 50% to 65%.

Wortmannin, an inhibitor of early endosome formation, did not block the intracellular accumulation of either p18 or p28 suggesting that, unlike cholera toxin (22), a caveolae to early endosome pathway is not involved in the intracellular trafficking of p18 and p28. The lack of early endosome involvement in the intracellular trafficking of p18 and p28 also suggests that clathrin-mediated endocytosis is not involved in internalization of these peptides (22, 23).

Monensin, which inhibits late endosome/lysosome, and BFA, a disruptor of the Golgi apparatus, reduced the intracellular accumulation of both peptides with a greater inhibitory effect on p28 (∼30%) than p18 (∼10%; Fig. 5B).

The penetration of p28 and p18 into fibroblasts was also inhibited by MβCD, nocodazole, monensin, and tunicamycin, but not by amiloride, sodium azide, and CPZ (Fig. 5C). This suggests that at least one mechanism of entry into cancer and normal cells may be similar, but additional preferential accumulation into cancer cells may be a function of the number of common membrane receptors or structures, i.e., caveolae (Fig. 5B,, 1 and 2). p18 and p28 colocalized with caveolin-1 and golgin 97 antibodies (Fig. 5D,, 1 and 2), confirming these organelles are involved in the intracellular trafficking of p18 and p28. Interestingly, azurin, but neither p18 nor p28, colocalized with mitochondrial specific fluorescence (Fig. 5D,, 3). In contrast, p28 and azurin, but not p18, colocalized with lysosomes, (Fig. 5D , 4).

Functional analysis of p28 and p18. Azurin inhibits the growth of several human cancer cell lines in vitro (14, 2426) and in vivo (13). Figure 6A and B illustrate the effect of p18 and p28 relative to azurin and dacarbazine (DTIC) on UISO-Mel-2 cells as determined by MTT and cell count. Azurin decreased (P < 0.05) cell survival at 100 and 200 μmol/L ∼15% (Fig. 6A). p28 inhibited cell survival 14% and 22% (P < 0.05) at 100 and 200 μmol/L, respectively. In contrast, p18 had no effect, whereas DTIC produced a significant dose-related decrease on UISO-Mel-2 survival. Azurin and p28 (200 μmol/L) also significantly decreased the survival of UISO-Mel-23 and 29 cells, whereas p18 had no significant effect. In contrast, azurin and p28 produced a dose-related decrease in cell number (Fig. 6B). p18 had no effect on UISO-Mel-2 cell proliferation. The apparent increase (∼30–35%; UISO-Mel-2) in p28 and azurin inhibition of melanoma cell proliferation, measured directly, suggests that the inhibitory effect may reside primarily at the level of cell cycle with apoptosis subsequent to any delay. Although p18 preferentially penetrated cancer cells, unlike p28, it did not inhibit cell proliferation, suggesting that the cytostatic and cytotoxic activity of p28 lies in the COOH-terminal 10 to 12 aa of the sequence.

Figure 6.

Anticell proliferative activity of azurin and its peptides. UISO-Mel-2 cells were incubated with increasing concentrations of azurin, p28, or p18 at 37°C for 72 h. MTT (A); direct cell count (B). Cell viability (MTT) or cell number in control wells were considered as 100%. Columns, mean; bars, SE.

Figure 6.

Anticell proliferative activity of azurin and its peptides. UISO-Mel-2 cells were incubated with increasing concentrations of azurin, p28, or p18 at 37°C for 72 h. MTT (A); direct cell count (B). Cell viability (MTT) or cell number in control wells were considered as 100%. Columns, mean; bars, SE.

Close modal

PTDs cluster into two groups based on their structural characteristics, cationic residues, or amphipathic α-helix, although several decrease into both classes. In general, cationic peptides initially interact with the cell membranes of prokaryotic and eukaryotic species (27) by binding to negatively charged surface glycoproteins, facilitating efficient entry into a broad range of normal and malignant cell lines (28). The binding of cationic peptides to HS is consistent with their high affinity for HS (Kd, ∼109 nmol/L; ref. 22).

The cytotoxic effect exerted by cationic, amphipathic α-helical diastereomeric peptides is not generally specific to cancer cells. Amphipathic CPPs cytolytic to cancer cells either disrupt the cancer cell membrane, alter mitochondrial permeability, or act through a specific receptor mediated mechanism (29). Synthetic magainins, a helical, channel-forming, or ionophore class of peptides including those exclusively composed of Lys, Ala, and Leu residues (30) rapidly and irreversibly lyse hematopoietic and solid tumor target cells at doses below those cytotoxic to normal cells (31, 32) but do not preferentially penetrate cancer cells.

Peptides generated through phage-display technology also do not induce cytotoxicity by direct penetration of cancer cells (8, 33). In contrast, azurin and the two peptides derived from it (p28 and p18) possess the unique property of preferentially entering cancer cells and inhibiting their proliferation through cytostatic and cytotoxic mechanisms.

Redox proteins are not normally classified as CPPs, or antiproliferative agents. The amphipathic, azurin fragments p18 and p28 contain the 54 to 67 aa α-helical structure of azurin as well as a partial β-sheet structure (16) and describe a minimal sequence for cancer cell entry and cell cycle inhibitory activity, respectively. The entry of azurin, p28, and p18 is distinct from that of cationic CPPs because sulfated proteoglycans are not involved in the penetration of either p28 or p18 into cancer cells. Mucin-type O-glycosylation, reportedly associated with somatic cell differentiation and altered expression on some cancer cells, is also not involved in the entry of p18 and p28 (34). However, this was not true for N-glycoslyated proteins. Aberrant N-glycosylation on several cell surface receptors, including integrins and cadherins, is associated with changes in progression and metastasis of cancers of diverse histogenesis (35, 36), suggesting a role for as yet unknown N-glycoslyated cell surface protein(s) in the initial steps of azurin, p18, and p28 penetration.

The temperature-dependent entry of azurin and aa fragment 50 to 77 (p28; ref. 12) extends to aa 50 to 67 of azurin (p18), whose entry into normal and malignant cells seems accelerated relative to p28. The lower Km and higher Vmax of p18 suggest that aa 50 to 67 define an amphipathic structure when associated with phospholipid membranes that more closely represents the actual PTD of azurin. However, an energy-dependent endocytotic or pore-related process does not seem to be the only entry mechanism available to these peptides. Sodium azide and ouabain (Na+K+ ATPase inhibitor), which inhibit the entry of cationic peptides (37), did not impair the entry of either p18 or p28 into UISO-Mel-2 cells or fibroblasts (Fig. 5B and C), suggesting that either peptide may also penetrate the cell membrane directly. However, the nonendocytotic mechanism described for penetratin and related peptides requires a peptide more basically charged than either p18 or p28.

Clathrin-mediated endocytosis, which underlies cellular penetration of a wide variety of cationic peptides (38, 39), was also not a route of p18 and p28 entry as CPZ had no effect on cancer cell membrane penetration (Fig. 5B and C). Azurin-derived peptides also distance themselves from the proposed routes of cellular penetration of virtually all other cationic CPPs, i.e., macropinocytosis, distribution to late endosomes or lysosomes along actin filaments or microtubules, and penetration at specific cell cycle stages, as inhibitors of each of these routes were singularly ineffective (Fig. 5B and C). p18, p28, and perhaps azurin seem to penetrate the plasma membrane and reach late endosmes, lysosomes, and the golgi associated with caveolae in what has been described as a dynamin-independent clathrin-independent carrier-mediated manner (21). The striking inhibition of penetration by nocodazole and relative lack of inhibition by cytochalasin-D, which disrupts actin filaments, supports the idea of caveolae-mediated entry, but the lack of effect of staurosporine again suggests that dynamin does not play a large role in the penetration of either peptide (21). This route of entry has been described for integral cell surface components and a broad range of pathogens or their products that also use caveolae to bypass classic endocytic pathways. Depletion of cholesterol from the plamsa membrane with β-methylcylodextran, filipin, or nystatin to disrupt lipid rafts, plasma membrane domains that provide fluid platforms to segregate membrane components and compartmentalize membranes (1), significantly inhibited the penetration of p18 (50%) and p28 (∼60%) into UISO-Mel-2 cells and fibroblasts (35% and 42%, respectively), suggesting a significant percentage (∼60%) of p18 and p28 penetrates the plasma membrane via caveolae. Caveolae are a 50- to 100-nm ω-shaped subset of lipid raft invaginations of the plasma membrane defined by the presence of caveolin-specific proteins (caveolin-1, caveolin-2, or caveolin-3; ref. 40) that function as regulators of signal transduction.

Because BFA disrupts the Golgi apparatus and inhibited p18 accumulation, we speculate that this pathway is also used in p18 and p28 cell entry and intracellular transport. Cell penetration of p18 and p28 via caveolae also fits well with our observation that inhibitors of N-glycosylation reduce cell entry by ∼60% in UISO-Mel-2 cells and 25% and 35%, respectively, in fibroblasts. The percentile differences between p18 and p28 entry may relate to the numbers of N-glycosylation membrane structures in cancer versus normal cells and the relative route of entry of p28 and p28 via this mechanism (Fig. 5B and C). Azurin, p28, and p18 all bind to cancer cells with high affinity and high capacity relative to other potential anticancer peptides (41), suggesting this protein/receptor complex localizes in caveolae and is internalized, eventually moving (via caveosomes) to the golgi, endoplasmic reticulum, and nucleus. Macropinocytosis does not seem to be involved nor does any entry mechanism that includes peptide transport to early endosomes. In addition to caveolar-mediated entry, our kinetic analysis also suggests that p28 and p18 penetrate the plasma membrane via a nonclathrin-caveolae–mediated process. A clathrin- and caveolin-independent pathway can exist as a constitutive internalization mechanism, such as for the interleukin 2 receptor (42) and for certain glycosyl-phosphatidylinositol–anchored proteins (43). Clathrin- and caveolin-independent endocytosis is also used by pathogens to invade cells, either exclusively, as for the murine polyoma virus (44), or in combination with a conventional pathway, as is the case for the influenza virus (45). An increase in caveolin-1 expression in cancer cells over normal cells is not likely to be the sole basis for the preferential entry of azurin, p28, and p18 into cancer cells. Fibroblasts and a number of other normal cells also have significant numbers of caveolae on their surface (23). However, it provides an avenue for additional investigation into the preferential penetration of these peptides into cancer cells.

Our results suggest that the cellular penetration of aa 50 to 67 and 50 to 77 of azurin is unique relative to all current CPPs in its preference for cancer cells, and that the COOH-terminal 10 to 12 aas of p28, aa 50 to 77 of azurin, contain the domain responsible for cell cycle inhibition and apoptotic activity. As such, these novel peptides could potentially serve as either a novel chemotherapeutic agent, adjunct to extant drugs or as CPPs to transport cargos including imaging agents into a broad range of cancer cells.

This work was supported through a sponsored research agreement between CDG Therapeutics, Inc. and UIC. CDG Therapeutics, Inc. has an exclusive licensing agreement for the development and commercialization of cupredoxin-derived peptides. A.M. Chakrabarty and T.K. Das Gupta are cofounders of and shareholders in CDG Therapeutics, Inc. T. Yamada is one of the inventors and potentially will receive a share of the royalty received by UIC through the licensing of the technology. C.W. Beattie is CSO of CDG Therapeutics, Inc. All terms of this arrangement are managed by UIC in accordance with its conflict of interest policies.

Note: Current address for B.N. Taylor: Caliper Life Sciences, 68 Elm Street, Hopkinton, MA 01748.

Grant support: A sponsored research agreement between CDG Therapeutics, Inc., and UIC.

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.

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