Domain IV of Annexin A5 Is Critical for Binding Calcium and Guarantees Its Maximum Binding to the Phosphatidylserine Membrane.

Background: Although domain IV of annexin A5 (anxA5) may be less effective in binding phosphatidylserine (PS), the four domains together may guarantee the maximum binding of anxA5 to the PS membrane. Additionally, previous research has shown that annexin mutants lacking one or more domain(s) have different biological activities compared to the wild-type. The present research mainly aims to study the role of domain IV in the crucial PS-binding function of anxA5. Methods: The domain IV-truncated anxA5 protein was constructed and purified. Isothermal titration calorimetry, flow cytometry and activated partial thromboplastin time were adopted to examine the function of domain IV in anxA5-PS binding directly or indirectly. Results: The domain IV-truncated form of anxA5 is impaired in binding PS liposome and apoptotic cells, and anticoagulation activity. The mutant cannot bind calcium, but binds PS only in the presence of calcium. Conclusions: Truncation of domain IV of anxA5 destroys its calcium-binding ability and impairs its PS-binding activity. Truncation of domain IV may induce conformation change of anxA5 or reduce the hydrophobic interactions between protein and membrane, which may explain the decrease of PS-binding affinity of the mutant.

Preliminary biological evaluation of 18F-AlF-NOTA-MAL-Cys-Annexin V as a novel apoptosis imaging agent.

A novel annexin V derivative (Cys-Annexin V) with a single cysteine residue at its C-terminal has been successfully labeled site-specifically with NOTA-maleimide aluminum [18F]fluoride complexation and evaluated it as a novel apoptosis agent in vitro and in vivo. The total synthesis time of 18F-AlF-NOTA-MAL-Cys-Annexin V from [18F]fluoride was about 65 min. The tracer was stable in vitro and it was excreted through renal in normal mice. The rate of the tracer bound to erythrocytes with exposed phosphatidylserine was 89.36±0.61% and this binding could be blocked by unlabeled Cys-Annexin V. In rats treated with cycloheximide, there were 6.23±0.23 times (n=4) increase in hepatic uptake of the tracer as compared to normal rats at 1h p.i. The uptake of the tracer in liver also could be blocked by co-injection of unlabeled Cys-Annexin V. These results indicated the favorable characterizations such as convenient synthesis and specific apoptotic cells targeting of18F-AlF-NOTA-MAL- Cys-Annexin V were suitable for its further investigation in clinical apoptosis imaging.

Preliminary biological evaluation of ¹8F-FBEM-Cys-Annexin V a novel apoptosis imaging agent.

A novel annexin V derivative (Cys-Annexin V) with a single cysteine residue at its C-terminal has been developed and successfully labeled site-specifically with 18F-FBEM. 18F-FBEM was synthesized by coupling 18F-fluorobenzoic acid (18F-FBA) with N-(2-aminoethyl)maleimide using optimized reaction conditions. The yield of 18F-FBEM-Cys-Annexin V was 71.5% ± 2.0% (n = 4, based on the starting 18F-FBEM, non-decay corrected). The radiochemical purity of 18F-FBEM-Cys-Annexin V was >95%. The specific radioactivities of 18F-FBEM and 18F-FBEM-Cys-Annexin V were >150 and 3.17 GBq/µmol, respectively. Like the 1st generation 18F-SFB-Annexin V, the novel 18F-FBEM-Cys-Annexin V mainly shows renal and to a lesser extent, hepatobiliary excretion in normal mice. In rat hepatic apoptosis models a 3.88 ± 0.05 (n = 4, 1 h) and 10.35 ± 0.08 (n = 4, 2 h) increase in hepatic uptake of 18F-FBEM-Cys-Annexin V compared to normal rats was observed after injection via the tail vein. The liver uptake ratio (treated/control) at 2 h p.i. as measured via microPET correlated with the ratio of apoptotic nuclei in liver observed using TUNEL histochemistry, indicating that the novel 18F-FBEM-Cys-Annexin V is a potential apoptosis imaging agent.

PET imaging of apoptosis in tumor-bearing mice and rabbits after paclitaxel treatment with (18)F(-)Labeled recombinant human His10-annexin V.

Monitoring response to chemo- or radiotherapy is of great importance in clinical practice. Apoptosis imaging serves as a very useful tool for the early evaluation of tumor response. The goal of this study was PET imaging of apoptosis with (18)F-labeled recombinant human annexin V linked with 10 histidine tag ((18)F-rh-His10-annexin V) in nude mice bearing an A549 tumor and rabbits bearing a VX2 lung cancer after paclitaxel therapy. (18)F-rh-His10-annexin V was prepared by conjugation of rh-His10-annexin V with N-succinimidyl 4-[(18)F]fluorobenzoate. Biodistribution was determined in mice by the dissection method and small-animal PET. Single-dose paclitaxel (175 mg/m(2)) was used to induce apoptosis in A549 and VX2 tumor models. (18)F-rh-His10-annexin V was injected into A549 mice and VX rabbits to acquire dynamic and static PET images 72 h after paclitaxel treatment. The uptake of (18)F-rh-His10-annexin V in apoptotic cells 4 h after induction was 6.45±0.52 fold higher than that in non-induced cells. High focal uptake of (18)F-rh-His10-annexin V was visualized in A549 (SUVmax: 0.35±0.13) and VX2 (0.41±0.23) tumor models after paclitaxel treatment, whereas lower uptake was found in the corresponding tumors before treatment (A549 SUVmax: 0.04±0.02; VX2: 0.009±0.002). The apoptotic index was 75.61±11.56% in the treated VX2 cancer, much higher than that in the untreated VX2 (8.03±2.81%). This study demonstrated the feasibility of (18)F-rh-His10-annexin V for the detection of apoptosis after chemotherapy in A549 and VX2 tumor models.

Annexin V-TRAIL fusion protein is a more sensitive and potent apoptotic inducer for cancer therapy.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent, which kills cancer cells selectively, while leaving normal cells unharmed. However, the emerging resistance of tumor cells and patients to TRAIL-induced apoptosis limits its further application. In this study, we developed a chimeric protein Annexin V-TRAIL (designated as TP8) with higher efficacy than TRAIL both in vitro and in vivo. In vitro, the EC50 of TP8 on a series of tumor cells was much lower than wild-type TRAIL. Annexin V provided this recombinant protein with higher efficacy, while leaving tumor specificity of TRAIL unchanged since TP8 had no effects on normal cells. In vivo, TP8 effectively suppressed tumor growth and prolonged tumor doubling time and tumor growth delay time in mouse xenografts involving multiple cancer cell types including A549, Colo205 and Bel7402. This study provides a new rational strategy to treat TRAIL-resistant cancers.

Preliminary biological evaluation of novel (99m)Tc-Cys-annexin A5 as a apoptosis imaging agent.

A novel annexin A5 derivative (cys-annexin A5) with a single cysteine residue at its C-terminal has been developed and successfully labeled in high labeling yield with (99m)Tc by a ligand exchange reaction. Like the 1st generation (99m)Tc-HYNIC-annexin A5, the novel (99m)Tc-cys-annexin A5 derivative shows in normal mice mainly renal and, to a lesser extent, hepatobiliary excretion. In rat models of hepatic apoptosis there was 283% increase in hepatic uptake of (99m)Tc-cys-annexin A5 as compared to normal mice. The results indicate that the novel (99m)Tc-cys-annexin A5 is a potential apoptosis imaging agent.

Nanoarchitectured electrochemical cytosensors for selective detection of leukemia cells and quantitative evaluation of death receptor expression on cell surfaces.

The variable susceptibility to the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment observed in various types of leukemia cells is related to the difference in the expression levels of death receptors, DR4 and DR5, on the cell surfaces. Quantifying the DR4/DR5 expression status on leukemia cell surfaces is of vital importance to the development of diagnostic tools to guide death receptor-based leukemia treatment. Taking the full advantages of novel nanobiotechnology, we have developed a robust electrochemical cytosensing approach toward ultrasensitive detection of leukemia cells with detection limit as low as ~40 cells and quantitative evaluation of DR4/DR5 expression on leukemia cell surfaces. The optimization of electron transfer and cell capture processes at specifically tailored nanobiointerfaces and the incorporation of multiple functions into rationally designed nanoprobes provide unique opportunities of integrating high specificity and signal amplification on one electrochemical cytosensor. The high sensitivity and selectivity of this electrochemical cytosensing approach also allows us to evaluate the dynamic alteration of DR4/DR5 expression on the surfaces of living cells in response to drug treatments. Using the TRAIL-resistant HL-60 cells and TRAIL-sensitive Jurkat cells as model cells, we have further verified that the TRAIL susceptibility of various types of leukemia cells is directly correlated to the surface expression levels of DR4/DR5. This versatile electrochemical cytosensing platform is believed to be of great clinical value for the early diagnosis of human leukemia and the evaluation of therapeutic effects on leukemia patients after radiation therapy or drug treatment.