Abstract
1292
Background: Arg-Arg-Leu (RRL) selected from peptide display library is a potent tumor-homing tripeptide, which could noninvasively trace the malignant tumors in vivo. However, its binding target is still unclear, which hinders its development and application. In previous study, we utilized immunoprecipitation-mass spectrometry (IP-MS) method to screen the binding proteins and we speculated the heat shock protein 70 (HSP70) family is our target protein. So in this study, we intend to dig out the relationship between RRL and HSPs.
Methods: Two-photon imaging was conducted to determine the subcellular localization of 5-TAMRA-RRL. Western blotting was utilized to verify the existence of HSP70 by RRL-biotin-streptavidin beads and to evaluate the HSP70 expression of HepG2, MCF-7, PC-3, HeLa, and U87MG. Cell fluorescence staining was verified the co-localization between RRL and HSP70. VER-155008, a potent HSP70 family inhibitor, was deployed to prove the relationship between RRL and HSP70 on cellular, tissue and in vivo levels.
Results: In two-photon imaging, 5-TAMRA-RRL was quickly assembled into tumor microvessels and gradually concentrated by tumor cells in the membrane and cytoplasm. Western blotting supported the existence of HSP70, HSC70 and GRP75 in the RRL-biotin captured protein lysate. It also showed all cell lines expressed above mentioned HSPs, but 5-TAMRA-RRL strong positive cell (HepG2) had high expression than weak positive cell U87MG (P < 0.05).Cell fluorescence staining suggested that there were no co-localization signals between 5-TAMRA-RRLand GRP75, but strong co-localization signals between RRL and HSC70 and HSP70, especially in cytoplasm with Pearson's R value of 0.80±0.07 (HSC70) and 0.79±0.04 (HSP70).VER-155008 can effectively inhibited the uptake of 99mTc-MAG3-RRL on cellular (P < 0.05)with Spearman's rank correlation coefficient of 0.98 (HepG2), 0.97 (MCF-7), 0.89 (PC-3) and 0.91 (HeLa).VER-155008 can inhibitedthe uptake of 5-TAMRA-RRL on hepatoma xenografts frozen tissue section (80μM,P < 0.05) with Spearman's rank correlation coefficientof 0.62. In γ-camera imaging, VER-155008 also can reduce the accumulation of 99mTc-MAG3-RRL in hepatoma xenografts modelafter 2 and 4h i.v. (T/NT: 8.23±1.14 VS 6.10 ±0.68, p < 0.05; 7.92 ± 0.61VS 3.38±0.91, P < 0.05). In biodistribution, VER-155008 also can reduce the T/NT value at all time point (n=5, P < 0.05).
Conclusions: 5-TAMRA-RRL, biotin-RRL and 99mTc-MAG3-RRL tracers are successfully prepared to visualized tumor cells and hepatoma xenografts models. HSP70 may be the target protein of RRL in all likelihood. Detection the tumor expression of HSPs would be a meaningful application for RRL related probes. Key words: 5-TAMRA-RRL, Biotin-RRL, 99mTc-MAG3-RRL,Tumor-homing peptide, Heat shock protein