Mechanistic studies of a peptidic GRP78 ligand for cancer cell-specific drug delivery

Abstract

Major obstacles in the development of new therapeutic anticancer drugs are the low bioavailability of hydrophilic substances and the nonspecific toxicity toward healthy tissues. As such, cell-targeting oligopeptides have emerged as attractive drug delivery vehicles for a variety of different types of cargo. The recently identified peptide Pep42 binds to the glucose-regulated protein 78 (GRP78), which is overexpressed on the cell surface of human cancer cells and internalizes into these cells. Herein, we demonstrate how Pep42 can be utilized as a carrier for different types of cytotoxic drugs to specifically target human cancer cell lines in vitro in a strictly GRP78-dependent manner. Furthermore, the mechanism of internalization of Pep42 was elucidated and found to involve clathrin-mediated endocytosis. Pep42 subsequently colocalizes within the lysosomal compartment. Importantly, we also provide evidence that Pep42-conjugated quantum dots have the ability to selectively enrich in tumor tissue in a xenograft mouse model. Our results suggest that the highly specific GRP78-Pep42 interaction can be utilized for the generation of Pep42-drug conjugates as a powerful anticancer drug delivery system that could substantially enhance the efficacy of chemotherapy by increasing the drug-tumor specificity, thus minimizing the adverse side effects associated with conventional cancer therapeutics.

Figure 1

Pep42-displaying phage particles bind specifically to GRP78. A, GRP78, HSP70 and BSA were coated on microtiter wells at 10 μg/mL and incubated with either Pep42-phage or control phage. The control phage displays an unrelated sequence. Phage input was 10⁹ cfu per well. Results are expressed as mean ± standard error of the mean (SEM) of triplicate wells. B, The inhibition of binding of Pep42-phage to immobilized GRP78 was obtained by the addition of increasing concentrations of Pep42 or a scrambled Pep42. *Student’s t test, p<0.05.

Figure 2

Pep42 binding to tumor cells is strictly dependent on GRP78 cell surface expression. A, Cells were incubated with either 50 nM FITC-conjugated Pep42 (red curve) or FITC-conjugated control peptide (green curve) for 25 min. at 4 °C with slow rotation. The black curves correspond to cells incubated in the absence of any peptide. B, Pep42 binding to tumor cells was estimated using photoaffinity-labeling. Western blot of Pep42 with its interacting proteins is shown. Here, cells were incubated with 50 μM Pep42-Bpa-Biotin for 1 h at 4 °C in the dark. The Pep42 protein interactions found on the cell surface were purified by avidin-agarose affinity chromatography, separated by SDS-PAGE, and transferred onto a nitrocellulose membrane. Upon incubation with streptavidin-HRP the membrane was developed with luminal and analyzed by chemoluminescence. C, Inhibition of binding of Pep42 to HepG2 cells was evaluated by the addition of a monoclonal anti-GRP78 antibody and the subsequent incubation of cells with Pep42 or a control peptide. **Difference of cells treated with Pep42 only, Student’s t-test, p<0.01. D, Effect of silencing the GRP78 gene expression on binding of Pep42 to Me6652/4 cells. After 48 h of transfection with GRP 78 siRNA or control siRNA, cells were incubated with Pep42-Bpa-Biotin. The detection was carried out as described for Fig. 2B.

Figure 3

Selective killing and induction of apoptosis in surface GRP78 expressing cancer cells mediated by Pep42-D-(KLAKLAK)₂. A, Cells were incubated with increasing concentrations of the peptides Pep42-D-(KLAKLAK)₂ or B, a mixture of free Pep42 and D-(KLAKLAK)₂ in 100 μL 1% FBS or supplemented medium for 24 h at 37 °C. The viability of the cells was assessed by a MTT assay. Results are expressed as percentages of cells still viable relative to non-treated cells. C, SJSA-1 cells cultured in the presence of Pep42-D-(KLAKLAK)₂ or negative controls (free D-(KLAKLAK)₂, free Pep42 or scrambled Pep42) for 6 h at 37 °C were washed in cold PBS and resuspended in staining buffer prior to the addition of FITC-labeled Annexin V and propidium iodide for 15 min. at room temperature. Flow cytometry was performed on SJSA-1 cells gated on the basis of their forward and side light scatter with any cell debris excluded from the analysis. Cell staining with Annexin V-FITC, but no propidium iodide were regarded as early apoptotic cells. Early apoptotic cells (1) Pep42-D-(KLAKLAK)₂ 35.8%; (2) Free Pep42 2.97%; (3) Free D-(KLAKLAK)₂ 1.82%; (4) Medium 1.35%; (5) scrambled Pep42-D-(KLAKLAK)₂ 2.78%. D, The role of surface GRP78 siRNA or control siRNA using Lipofectamine 2000 reagent. After 48 hours, cells were treated with 30 μM Pep42-D-(KLAKLAK)₂ for 3 h at 37 °C. *Difference from Pep42-D-(KLAKLAK)₂ treated cells, student’s t test, p<0.05.

Figure 4

Pep42 partially co-localizes with known markers of endocytosis. A, A549cells were preincubated with Pep42-QDots 525 for 1 h at 37 °C and then added 10 μg/mL AF-594 transferrin or 10 μg/mL AF-594 cholera toxin subunit B for 30 min. at 37 °C. The cells were then washed with PBS and analyzed by laser scanning confocal microscopy. B, Weighted co-localization coefficients were measured with Zeiss LSM Image examiner software. Red in the legend means the co-localized coefficient that red channel co-localizes with the green channel. Green in the legend means the co-localized coefficient that the green channel co-localizes with the red channel.

Figure 5

Inhibition of Pep42 uptake using known endocytosis inhibitors. A549 cells were pretreated with 25 μM Nystatin, 30 μM Chlorpromazine or 5 mM methyl-β-cyclodextrin (MCD) for 10 min. and then Pep42-Qdots 525, AF-594-transferrin or AF-594- cholera toxin subunit B was added, respectively.

Figure 6

Pep42-QDot conjugates co-localize with the lysosomal compartment. A, Co-localization of Pep42-Qdots with Lysotracker Red in A549 cells. Cells were treated with 12 nM Lysotracker Red for 30 min, 20 nM ER-tracker Red for 20 min or 25 nM Mitotracker Red for 15 min after incubation of the peptide for 1 h at 37 °C. B, Weighted co-localization coefficients were measured with Zeiss LSM Image examiner software. Red in the legend means the co-localized coefficient that red channel co-localizes with the green channel. Green in the legend means the co-localized coefficient that the green channel co-localizes with the red channel.

Figure 7

Phototoxicity of Pep42-hematoporphyrin conjugates toward surface expressing GRP78 cancer cells. The viability of the cells was assessed by a MTT assay. Results are expressed as percentages of cells still viable relative to non-treated control cells. A, Phototoxicity of Pep42-hematoporphyrin conjugate on A549 cells and HepG2 cells. HDFa and CCD-11Lu cells served as controls. B, An equimolar mixture of 10 μM hematoporphyrin and Pep42 had no effect on HepG2 and A549 cells.

Figure 8

In vivo targeting of Pep42-QDot conjugates. Peptide-coated QDots were injected into the tail vein of tumor bearing CB17Scid male mice, and the presence of QDots in the tumor and control tissue was assessed by examining sections under a confocal microscope. Accumulation of conjugate was observed in the tumor tissue with cyclic peptide QDots (A), less material was found in the liver (B) and the spleen (C). Linear peptide coated QDots did not accumulate in the tumor (D), and also not in the control organs liver (E) and spleen (F). Blood vessels were stained with anti-CD31 (red).