Targeting glucose regulated protein 78 using a cell penetrating peptide, Pep42, for glioblastoma imaging and therapy
Tae Moon Chung, Cancer Research Institute; Seoul National University College of Medicine
Glucose regulated protein 78 (GRP78) is a multifunctional chaperone protein which is commonly overexpressed in malignant cancer cells. Because GRP78 has an important role in radio- and chemo-resistance of cancer, therapy targeting GRP78 may be useful in cancer treatment. In this study, we developed theranostic probe using GRP 78 targeting peptide (Pep42).
Pep42 is a cyclic 13-mer GRP78 targeting and cell penetrating peptide developed by phage display. In human glioma cell lines (U87MG, U251) and primary glioma cells from one patient (GBM28, GBM37; before and after Temozolomide-radiation therapy), GRP78 expression levels were measured by microarray, RT-PCR and immunoblotting. After the glioma cells were incubated with red fluorescence labeled Pep42 (Pep42-R) for 2 hours, targeting by Pep42 were observed using a confocal fluorescence microscope. Pep42 labeled with I-125 was used to investigate the GRP78 targeting and cellular uptake. For in vivo GRP78 targeted imaging, Pep42-R was conjugated with Cu64 labeled human serum albumin (Cu64–HSA-Pep42). Cu64–HAS-Pep42 was intravenously injected into the mice xenografted U87MG, and images were obtained by an animal PET-BOX.
Confocal microscope showed the specific binding of Pep42-R at the cytoplasm as well as membrane of glioma cells. GRP 78 targeted miRNA expression in U87 and GBM37 was lower than in U251 and GBM28. RT-PCR and immunoblotting assay revealed higher GRP78 expression in U87MG cells than in U251 cells. GRP78 expression was also higher in GBM37 than in GBM28. Pep42-R images showed that U87MG fluorescent signals were 1.98 times more intense than U251 and GBM37 fluorescent signals were 2.04 times more intense than GBM28. Therefore, Pep42-R image successfully showed that the expression of GRP78 in U87MG and GBM37 was higher than that of U251 and GBM28. Labeling efficiency of 125I-Pep42 and Cu64–HSA-Pep42 were over 95%, and in vitro uptake test showed its specificity in U87MG cells. Serial PET imaging revealed that liver uptake of Cu64–HSA-Pep42 was reduced dramatically and GRP78 positive tumor uptake was increased significantly. Therefore, Cu64–HSA-Pep42 successfully visualized targeting GRP78 positive tumors in U87MG xenograft model using animal PET-BOX.
We could evaluate the expression level of GRP78 on glioma cell lines and primary cultured glioma cells. Pep42 imaging probes specifically visualize GRP78 expression in glioma models, and could validate the malignant glioma cells. Therefore, this imaging probe could be useful as a theranostic probe for malignant cancers.