A high-affinity near-infrared fluorescent probe to target bombesin receptors.

PURPOSE: This study aimed to create new optical surgical navigation NIRF probes for prostate and breast cancers. PROCEDURES: IR800-linker-QWAVGHLM-NH2 with linker = GSG, GGG, and G-Abz4 were synthesized and characterized. IC50 for bombesin receptors (BBN-R) in PC-3 prostate and T47D breast cancer cells, fluorescence microscopy in PC-3 cells, and NIRF imaging in mice PC-3 tumor xenografts were studied. RESULTS: GGG, GSG, and G-Abz4 derivatives had IC50 (nM) for BBN-R+ PC-3 cells = 187 ± 31, 56 ± 5, and 2.6 ± 0.2 and T47D cells = 383 ± 1, 57.4 ± 1.2, and 3.1 ± 1.1, respectively. By microscopy the Abz4 derivative showed the highest uptake, was competed with by BBN, and had little to no binding to BBN-R- cells. In NIRF imaging the G-Abz4 probe was brighter than GGG probe in BBN-R+ tissues in vivo and tissues, tumors, and tumor slices ex vivo. Uptake could be partially blocked in BBN-R+ pancreas but not visibly in tumor. CONCLUSIONS: Linker choice can dominate peptidic BBN-R binding. The G-Abz4 linker yields a higher affinity and specific BBN-R binder in this series of molecules.

Site specific discrete PEGylation of (124)I-labeled mCC49 Fab' fragments improves tumor MicroPET/CT imaging in mice.

The tumor-associated glycoprotein-72 (TAG-72) antigen is highly overexpressed in various human adenocarcinomas and anti-TAG-72 monoclonal antibodies, and fragments are therefore useful as pharmaceutical targeting vectors. In this study, we investigated the effects of site-specific PEGylation with MW 2-4 kDa discrete, branched PEGylation reagents on mCC49 Fab' (MW 50 kDa) via in vitro TAG72 binding, and in vivo blood clearance kinetics, biodistribution, and mouse tumor microPET/CT imaging. mCC49Fab' (Fab'-NEM) was conjugated at a hinge region cysteine with maleimide-dPEG 12-(dPEG24COOH)3 acid (Mal-dPEG-A), maleimide-dPEG12-(dPEG12COOH)3 acid (Mal-dPEG-B), or maleimide-dPEG12-(m-dPEG24)3 (Mal-dPEG-C), and then radiolabeled with iodine-124 ((124)I) in vitro radioligand binding assays and in vivo studies used TAG-72 expressing LS174T human colon carcinoma cells and xenograft mouse tumors. Conjugation of mCC49Fab' with Mal-dPEG-A (Fab'-A) reduced the binding affinity of the non PEGylated Fab' by 30%; however, in vivo, Fab'-A significantly lengthened the blood retention vs Fab'-NEM (47.5 vs 28.1%/ID at 1 h, 25.1 vs 8.4%/ID at 5 h, p < 0.01), showed excellent tumor to background, better microPET/CT images due to higher tumor accumulation, and increased tumor concentration in excised tissues at 72 h by 130% (5.09 ± 0.83 vs 3.83 ± 1.50%ID/g, p < 0.05). Despite the strong similarity of the three PEGylation reagents, PEGylation with Mal-dPEG-B or -C reduced the in vitro binding affinity of Fab'-NEM by 70%, blood retention, microPET/CT imaging tumor signal intensity, and residual 72 h tumor concentration by 49% (3.83 ± 1.50 vs 1.97 ± 0.29%ID/g, p < 0.05) and 63% (3.83 ± 1.50 vs 1.42 ± 0.35%ID/g, p < 0.05), respectively. We conclude that remarkably subtle changes in the structure of the PEGylation reagent can create significantly altered biologic behavior. Further study is warranted of conjugates of the triple branched, negatively charged Mal-dPEG-A.

Localization of CaSR antagonists in CaSR-expressing medullary thyroid cancer.

OBJECTIVE: Image-based localization of medullary thyroid cancer (MTC) and parathyroid glands would improve the surgical outcomes of these diseases. MTC and parathyroid glands express high levels of calcium-sensing receptor (CaSR). The aim of this study was to prove the concept that CaSR antagonists specifically localize to CaSR-expressing tumors in vivo. DESIGN: We synthesized two isomers of a known CaSR calcilytic, Calhex 231, and four new analogs, which have a favorable structure for labeling. Their antagonistic activity was determined using immunoblots demonstrating decreased ERK1/2 phosphorylation after calcium stimulation in human embryonic kidney cells overexpressing CaSR. Compound 9 was further radiolabeled with (125)I and evaluated in nude mice with and without heterotransplanted xenografts of MTC cell lines, TT and MZ-CRC-1, that do and do not express CaSR, respectively. RESULTS: Two newly synthesized compounds, 9 and 11, exhibited better antagonistic activity than Calhex 231. The half-life of (125)I-compound 9 in nude mice without xenografts was 9.9 hours. A biodistribution study in nude mice bearing both tumors demonstrated that the uptake of radioactivity in TT tumors was higher than in MZ-CRC-1 tumors at 24 hours: 0.39 ± 0.24 vs 0.18 ± 0.12 percentage of injected dose per gram of tissue (%ID/g) (P = .002), with a ratio of 2.25 ± 0.62. Tumor-to-background ratios for TT tumors, but not MZ-CRC-1 tumors, increased with time. Tumor-to-blood values increased from 2.02 ± 0.52 at 1 hour to 3.29 ± 0.98 at 24 hour (P = .015) for TT tumors, and 1.7 ± 0.56 at 1 hour to 1.48 ± 0.33 at 24 hour (P = .36) for MZ-CRC-1 tumors. CONCLUSIONS: Our new CaSR antagonists specifically inhibit CaSR function in vitro, preferentially localize to CaSR-expressing tumors in vivo, and therefore have the potential to serve as scaffolds for further development as imaging pharmaceuticals.

NER initiation factors, DDB2 and XPC, regulate UV radiation response by recruiting ATR and ATM kinases to DNA damage sites.

ATR and ATM kinases are central to the checkpoint activation in response to DNA damage and replication stress. However, the nature of the signal, which initially activates these kinases in response to UV damage, is unclear. Here, we have shown that DDB2 and XPC, two early UV damage recognition factors, are required for the damage-specific ATR and ATM recruitment and phosphorylation. ATR and ATM physically interacted with XPC and promptly localized to the UV damage sites. ATR and ATM recruitment and their phosphorylation were negatively affected in cells defective in DDB2 or XPC functions. Consequently, the phosphorylation of ATR and ATM substrates, Chk1, Chk2, H2AX, and BRCA1 was significantly reduced or abrogated in mutant cells. Furthermore, UV exposure of cells defective in DDB2 or XPC resulted in a marked decrease in BRCA1 and Rad51 recruitment to the damage site. Conversely, ATR- and ATM-deficiency failed to affect the recruitment of DDB2 and XPC to the damage site, and therefore did not influence the NER efficiency. These findings demonstrate a novel function of DDB2 and XPC in maintaining a vital cross-talk with checkpoint proteins, and thereby coordinating subsequent repair and checkpoint activation.

Differential contributory roles of nucleotide excision and homologous recombination repair for enhancing cisplatin sensitivity in human ovarian cancer cells.

BACKGROUND: While platinum-based chemotherapeutic agents are widely used to treat various solid tumors, the acquired platinum resistance is a major impediment in their successful treatment. Since enhanced DNA repair capacity is a major factor in conferring cisplatin resistance, targeting of DNA repair pathways is an effective stratagem for overcoming cisplatin resistance. This study was designed to delineate the role of nucleotide excision repair (NER), the principal mechanism for the removal of cisplatin-induced DNA intrastrand crosslinks, in cisplatin resistance and reveal the impact of DNA repair interference on cisplatin sensitivity in human ovarian cancer cells. RESULTS: We assessed the inherent NER efficiency of multiple matched pairs of cisplatin-sensitive and -resistant ovarian cancer cell lines and their expression of NER-related factors at mRNA and protein levels. Our results showed that only the cisplatin-resistant ovarian cancer cell line PEO4 possessed an increased NER capacity compared to its inherently NER-inefficient parental line PEO1. Several other cisplatin-resistant cell lines, including CP70, CDDP and 2008C13, exhibited a normal and parental cell-comparable NER capacity for removing cisplatin-induced DNA intrastrand cross-links (Pt-GG). Concomitant gene expression analysis revealed discordance in mRNA and protein levels of NER factors in various ovarian cancer cell lines and NER proteins level were unrelated to the cisplatin sensitivity of these cell lines. Although knockdown of NER factors was able to compromise the NER efficiency, it only caused a minimal effect on cisplatin sensitivity. On the contrary, downregulation of BRCA2, a critical protein for homologous recombination repair (HRR), significantly enhanced the efficacy of cisplatin in killing ovarian cancer cell line PEO4. CONCLUSION: Our studies indicate that the level of NER factors in ovarian cancer cell lines is neither a determinant of their NER capacity nor of the sensitivity to cisplatin, and suggest that manipulation of the HRR but not the NER factor expression provides an effective strategy for sensitizing cisplatin-resistant tumors to platinating agents.

Stem cell protein Piwil2 modulates chromatin modifications upon cisplatin treatment.

Piwil2 (mili in mouse or hili in humans), a member of the PIWI/Argonaute gene family, plays important roles in stem cell self-renewal, RNA silencing, and translational regulation in various organisms. Recent demonstration of stable Piwil2 expression in pre-cancerous stem cells and in various human and animal tumor cell lines suggests its association in tumorigenesis. Here, we show that cisplatin induces chromatin relaxation in Mili-wild type (WT) mouse embryonic fibroblasts (MEFs), but not in Mili-knockout (KO) MEFs. Moreover, in contrast to Mili-WT MEFs, Mili-KO MEFs showed a discernable H3 hypoacetylation response upon cisplatin treatment. Levels of the histone acetyltransferase (HAT), p300, were dramatically different due to a consistent cisplatin post-treatment decrease in Mili-WT and an increase in Mili-KO MEFs. Concomitant reduction of specific HAT activity of p300 could explain the decrease of H3 acetylation in Mili-KO MEFs. Our data also shows Mili is required for maintaining the euchromatic marks in MEFs upon cisplatin treatment. In addition, Mili-KO MEFs exhibited a significant deficiency in repairing cisplatin-induced DNA damage and displayed higher sensitivity to cisplatin. Further analysis revealed that Piwil2 was also enhanced in two completely different cisplatin-resistant ovarian cancer cell lines. Interestingly, knockdown of Piwil2 expression in these two cell lines also resulted in their enhanced sensitivity to cisplatin and decreased their efficiency for removing cisplatin-induced DNA intrastrand crosslinks (Pt-GG). The overall data showed that Piwil2 is a key factor in regulating chromatin modifications especially in response to cisplatin. To conclude, the overexpression of Piwil2 in some cancers could lead to cellular cisplatin resistance, possibly due to enhanced chromatin condensation affecting normal DNA repair.

Overexpression of DDB2 enhances the sensitivity of human ovarian cancer cells to cisplatin by augmenting cellular apoptosis.

Cisplatin is one of the most widely used anticancer agents, displaying activity against a wide variety of tumors. However, development of drug resistance presents a challenging barrier to successful cancer treatment by cisplatin. To understand the mechanism of cisplatin resistance, we investigated the role of damaged DNA binding protein complex subunit 2 (DDB2) in cisplatin-induced cytotoxicity and apoptosis. We show that DDB2 is not required for the repair of cisplatin-induced DNA damage, but can be induced by cisplatin treatment. DDB2-deficient noncancer cells exhibit enhanced resistance to cell growth inhibition and apoptosis induced by cisplatin than cells with fully restored DDB2 function. Moreover, DDB2 expression in cisplatin-resistant ovarian cancer cell line CP70 and MCP2 was lower than their cisplatin-sensitive parental A2780 cells. Overexpression of DDB2 sensitized CP70 cells to cisplatin-induced cytotoxicity and apoptosis via activation of the caspase pathway and downregulation of antiapoptotic Bcl-2 protein. Further analysis indicates that the overexpression of DDB2 in CP70 cells downregulates Bcl-2 expression through decreasing Bcl-2 mRNA level. These results suggest that ovarian cancer cells containing high level of DDB2 become susceptible to cisplatin by undergoing enhanced apoptosis.

Modulation of nucleotide excision repair by mammalian SWI/SNF chromatin-remodeling complex.

Accessibility within chromatin is an important factor in the prompt removal of UV-induced DNA damage by nucleotide excision repair (NER). Chromatin remodeling by the SWI/SNF complex has been shown to play an important modulating role in NER in vitro and yeast in vivo. Nevertheless, the molecular basis of cross-talk between SWI/SNF and NER in mammalian cells is not fully understood. Here, we show that knockdown of Brg1, the ATPase subunit of SWI/SNF, negatively affects the elimination of cyclobutane pyrimidine dimers (CPD), but not of pyrimidine (6, 4)pyrimidone photoproducts (6-4PP) following UV irradiation of mammalian cells. Brg1-deficient cells exhibit a lower chromatin relaxation as well as impaired recruitment of downstream NER factors, XPG and PCNA, to UV lesions. However, the assembly of upstream NER factors, DDB2 and XPC, at the damage site was unaffected by Brg1 knockdown. Interestingly, Brg1 interacts with XPC within chromatin and is recruited to UV-damaged sites in a DDB2- and XPC-dependent manner. Also, postirradiation decrease of XPC levels occurred more rapidly in Brg1-deficient than normal cells. Conversely, XPC transcription remained unaltered upon Brg1 knockdown indicating that Brg1 affects the stability of XPC protein following irradiation. Thus, Brg1 facilitates different stages of NER by initially modulating UV-induced chromatin relaxation and stabilizing XPC at the damage sites, and subsequently stimulating the recruitment of XPG and PCNA to successfully culminate the repair.