A Small Peptide Increases Drug Delivery in Human Melanoma Cells

Abstract

Melanoma is the most fatal type of skin cancer and is notoriously resistant to chemotherapies. The response of melanoma to current treatments is difficult to predict. To combat these challenges, in this study, we utilize a small peptide to increase drug delivery to melanoma cells. A peptide library array was designed and screened using a peptide array-whole cell binding assay, which identified KK-11 as a novel human melanoma-targeting peptide. The peptide and its D-amino acid substituted analogue (VPWxEPAYQrFL or D-aa KK-11) were synthesized via a solid-phase strategy. Further studies using FITC-labeled KK-11 demonstrated dose-dependent uptake in human melanoma cells. D-aa KK-11 significantly increased the stability of the peptide, with 45.3% remaining detectable after 24 h with human serum incubation. Co-treatment of KK-11 with doxorubicin was found to significantly enhance the cytotoxicity of doxorubicin compared to doxorubicin alone, or sequential KK-11 and doxorubicin treatment. In vivo and ex vivo imaging revealed that D-aa KK-11 distributed to xenografted A375 melanoma tumors as early as 5 min and persisted up to 24 h post tail vein injection. When co-administered, D-aa KK-11 significantly enhanced the anti-tumor activity of a novel nNOS inhibitor (MAC-3-190) in an A375 human melanoma xenograft mouse model compared to MAC-3-190 treatment alone. No apparent systemic toxicities were observed. Taken together, these results suggest that KK-11 may be a promising human melanoma-targeted delivery vector for anti-melanoma cargo.

Keywords: melanoma; nNOS inhibitors; peptide; targeted delivery; tumor xenograft; uptake.

Figure 1

Structures of doxorubicin and MAC-3-190 used in this study.

Figure 2

Peptides for targeted delivery in human melanoma. (A) Schematic showing peptide array screening for melanoma specific peptides. The average CyQUANT fluorescence intensity of the peptide bound cells was used to estimate the relative cells bound to the peptides. (B,C) Relative fluorescence was determined compared to human embryonic kidney cells (HEK-293). Each bar represents one peptide synthesized in the library as listed in Table 1. The top-ranked peptides (such as 11, black) showing a better binding profile for melanoma cells (B, A375 cells; C, Sk-Mel-28 cells) were further labeled with FITC for cellular uptake analysis.

Scheme 1

Synthesis of VivoTag-KK-11. Steps for conjugating VivoTag 680 XL to D-aa KK-11 using N-terminal β-alanine as a spacer following the manufacturer’s protocol. Blue color and lower-case letters stand for D-amino acids, and X stands for norleucine.

Figure 3

Uptake and drug delivery of KK-11 in human melanoma cells. (A) A representative fluorescence microscopy image of A375 melanoma cells after incubation with FITC-KK-11 (0.5 µM) for 30 min at 37 °C. Cell nuclei were stained blue with DAPI. (B) Uptake of FITC-peptides KK-1, KK-11, KK-12, and KK-13 by human melanoma cells. The bar represents the average fluorescence density of three cell lines (A375, Sk-mel-28, and wm3211 cells) detected by fluorescence-activated cell sorting (FACS) analysis. The cells were incubated with FITC-labeled peptide for 30 min at 37 °C (0.5 µM, grey bar; 1 µM, dark bar). The control is cells were incubated with FITC alone. The bar graph shows mean fluorescence intensity (MFI) of three experiments ± SD; *, p < 0.05 compared to 0.5 µM FITC alone; #, p < 0.001 compared to 1 µM FITC alone. (C) Co-treatment of KK-11 peptide enhanced the cytotoxicity of DOX in melanoma A375 cells. Cells were either treated with DOX or co-treated with DOX and KK-11 (1 µM) for 72 h (KK-11 + DOX), or pretreated with KK-11 for 4 h, then washed away following a DOX 72-hour treatment (KK-11 to DOX). Control is DMSO or KK-11 alone. Cell viability was detected by an MTT colorimetric assay. Statistical analysis was performed with a Student’s t-test, and error bars denote mean ± SD; **, p < 0.01; ***, p < 0.001 compared to DOX alone. (D) Co-treatment with KK-11 did not enhance the cytotoxicity of nNOS inhibitor MAC-3-190 in A375 melanoma cells. Cells were treated with MAC-3-190 at various concentrations with or without 1 µM KK-11 for 72 h. Cell viability was detected by MTT assay. The assays were repeated independently at least three times.

Figure 3

Uptake and drug delivery of KK-11 in human melanoma cells. (A) A representative fluorescence microscopy image of A375 melanoma cells after incubation with FITC-KK-11 (0.5 µM) for 30 min at 37 °C. Cell nuclei were stained blue with DAPI. (B) Uptake of FITC-peptides KK-1, KK-11, KK-12, and KK-13 by human melanoma cells. The bar represents the average fluorescence density of three cell lines (A375, Sk-mel-28, and wm3211 cells) detected by fluorescence-activated cell sorting (FACS) analysis. The cells were incubated with FITC-labeled peptide for 30 min at 37 °C (0.5 µM, grey bar; 1 µM, dark bar). The control is cells were incubated with FITC alone. The bar graph shows mean fluorescence intensity (MFI) of three experiments ± SD; *, p < 0.05 compared to 0.5 µM FITC alone; #, p < 0.001 compared to 1 µM FITC alone. (C) Co-treatment of KK-11 peptide enhanced the cytotoxicity of DOX in melanoma A375 cells. Cells were either treated with DOX or co-treated with DOX and KK-11 (1 µM) for 72 h (KK-11 + DOX), or pretreated with KK-11 for 4 h, then washed away following a DOX 72-hour treatment (KK-11 to DOX). Control is DMSO or KK-11 alone. Cell viability was detected by an MTT colorimetric assay. Statistical analysis was performed with a Student’s t-test, and error bars denote mean ± SD; **, p < 0.01; ***, p < 0.001 compared to DOX alone. (D) Co-treatment with KK-11 did not enhance the cytotoxicity of nNOS inhibitor MAC-3-190 in A375 melanoma cells. Cells were treated with MAC-3-190 at various concentrations with or without 1 µM KK-11 for 72 h. Cell viability was detected by MTT assay. The assays were repeated independently at least three times.

Figure 4

In vivo and ex vivo organ distribution images of VivoTag 680 XL-labeled D-aa KK-11 in athymic nude mice bearing human melanoma A375 tumors. Saline or VivoTag-KK-11 in a dose of 8 µg per mouse was injected into the mice via the tail vein (n = 2 for each time point). (A) Representative images of CT-fluorescence scans at tumor area sections were captured in live mice at different times after injection (30 min, 1 h, or 25 h). The top panels represent the scanned images of the tumor section area in coronal views. The bottom-row pictures display the fluorescence image in transaxial views: arrows point to the spine, and circles mark the xenograft tumors. (B) At defined time intervals (5 min, 15 min, 30 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 24 h, and 48 h) following peptide injection, animals were euthanized. Organs, including tumor xenografts (T), liver (L), spleen (S), heart and lungs (H + L), kidneys (K), and brain (B), were excised and imaged using the IVIS Spectrum imaging system. Representative fluorescence images of different organs show peptide distribution at different time points as indicated.

Figure 5

Enhanced antitumor activity of nNOS inhibitor MAC-3-190 co-injected with D-aa KK-11 peptide in a xenograft mouse model. Mice bearing human melanoma A375 tumors were intravenously injected with either D-aa KK-11, MAC-3-90, or a mixture of MAC-3-190 (5 mg/kg) and D-aa KK-11 (1.75 mg/kg) or 1× PBS, once daily. (A) The tumors were measured three times a week using digital Vernier calipers. Tumor volume (mm³) was calculated as [Length × (Width²)]/2. (B) The tumors were harvested and weighed after 21 days of treatment (Control: n = 10; D-aa KK-11 alone: n = 5; MAC-3-190: n = 5; D-aa KK-11 + MAC-3-190: n = 10). (C) The body weights of the mice were measured after 21 days of treatment. Statistical analysis was performed with Student’s t-test, and error bars denote mean ± SD; *, p < 0.05 compared to control group.

Figure 5

Enhanced antitumor activity of nNOS inhibitor MAC-3-190 co-injected with D-aa KK-11 peptide in a xenograft mouse model. Mice bearing human melanoma A375 tumors were intravenously injected with either D-aa KK-11, MAC-3-90, or a mixture of MAC-3-190 (5 mg/kg) and D-aa KK-11 (1.75 mg/kg) or 1× PBS, once daily. (A) The tumors were measured three times a week using digital Vernier calipers. Tumor volume (mm³) was calculated as [Length × (Width²)]/2. (B) The tumors were harvested and weighed after 21 days of treatment (Control: n = 10; D-aa KK-11 alone: n = 5; MAC-3-190: n = 5; D-aa KK-11 + MAC-3-190: n = 10). (C) The body weights of the mice were measured after 21 days of treatment. Statistical analysis was performed with Student’s t-test, and error bars denote mean ± SD; *, p < 0.05 compared to control group.