Apoptosis imaging studies in various animal models using radio-iodinated peptide.

Apoptosis has a role in many medical disorders and treatments; hence, its non-invasive evaluation is one of the most riveting research topics. Currently annexin V is used as gold standard for imaging apoptosis. However, several drawbacks, including high background, slow body clearance, make it a suboptimum marker for apoptosis imaging. In this study, we radiolabeled the recently identified histone H1 targeting peptide (ApoPep-1) and evaluated its potential as a new apoptosis imaging agent in various animal models. ApoPep-1 (CQRPPR) was synthesized, and an extra tyrosine residue was added to its N-terminal end for radiolabeling. This peptide was radiolabeled with (124)I and (131)I and was tested for its serum stability. Surgery- and drug-induced apoptotic rat models were prepared for apoptosis evaluation, and PET imaging was performed. Doxorubicin was used for xenograft tumor treatment in mice, and the induced apoptosis was studied. Tumor metabolism and proliferation were assessed by [(18)F]FDG and [(18)F]FLT PET imaging and compared with ApoPep-1 after doxorubicin treatment. The peptide was radiolabeled at high purity, and it showed reasonably good stability in serum. Cell death was easily imaged by radiolabeled ApoPep-1 in an ischemia surgery model. And, liver apoptosis was more clearly identified by ApoPep-1 rather than [(124)I]annexin V in cycloheximide-treated models. Three doxorubicin doses inhibited tumor growth, which was evaluated by 30-40% decreases of [(18)F]FDG and [(18)F]FLT PET uptake in the tumor area. However, ApoPep-1 demonstrated more than 200% increase in tumor uptake after chemotherapy, while annexin V did not show any meaningful uptake in the tumor compared with the background. Biodistribution data were also in good agreement with the microPET imaging results. All of the experimental data clearly demonstrated high potential of the radiolabeled ApoPep-1 for in vivo apoptosis imaging.

Vivid tumor imaging utilizing liposome-carried bimodal radiotracer.

By developing a new bimodal radioactive tracer that emits both luminescence and nuclear signals, a trimodal liposome for optical, nuclear, and magnetic resonance imaging is efficiently prepared. Fast clearance of the radiotracer from reticuloendothelial systems enables vivid tumor imaging with minimum background.

Synthesis and evaluation of a radioiodinated bladder cancer specific peptide.

Bladder cancer is the second most common cancer of the urinary tract, however the invasive cystoscopy is still the standard technique for diagnosis and surveillance of bladder cancer. Herein, we radiolabel bladder cancer specific peptide with radioactive iodine ((131/124)I) and evaluate its potential as a new radiopharmaceutical for the non-invasive diagnosis of bladder cancer. A 9-mer bladder cancer specific peptide (BP) was conjugated with tyrosine and cyclized by disulfide bond formation to give Y-BP, which was further radioiodinated to give [(131/124)I]Y-BP in good radiochemical yield. The biodistribution data showed the high selectivity of [(124)I]Y-BP in HT1376 human bladder cancer xenograft models with a tumor-to-muscle ratio of 6.2. This tumor targeting was not observed in control B16F10 melanoma tumor models. In microPET studies, while the control scrambled peptide, [(124)I]Y-sBP, did not accumulate in either the bladder cancer or melanoma, [(124)I]Y-BP showed high tumor uptake only in animals with HT1376 bladder cancer cells. Furthermore, [(124)I]Y-BP showed superior bladder cancer uptake even compared to most commonly used cancer imaging tracer, [(18)F]FDG. The experimental results suggest the potential of [(124)I]Y-BP as a new radiopharmaceutical for the non-invasive diagnosis of bladder cancer with high binding affinity and selectivity.

Evaluation of kidney repair capacity using 99mTc-DMSA in ischemia/reperfusion injury models.

Quantitative (99m)Tc-DMSA renal uptake was studied in different renal ischemia/reperfusion (I/R) mice models for the assessment of renal repair capacity. Mice models of nephrectomy, uni- and bi-lateral I/R together with sham-operated mice were established. At 1h, 1d, 4d, 1, 2 and 3 wk after I/R, (99m)Tc-DMSA (27.7 ± 1.3 MBq) was injected via tail vein and after 3h post-injection, the mice were scanned for 30 min with pinhole equipped gamma camera. Higher uptake of (99m)Tc-DMSA was measured in normal kidneys of uni-lateral I/R model and nephrectomized kidney I/R model at 3 wk post-surgery. Comparing the restoration capacities of the affected kidneys of nephrectomy, uni- and bi-lateral I/R models, higher repair capacity was observed in the nephrectomized model followed by bi-lateral then uni-lateral models. The normal kidney may retard the restoration of damaged kidney in uni-lateral I/R model. Moreover, 3 wk after Uni-I/R, the size of injured kidney was significantly smaller than non-ischemic contralateral and sham operated kidneys, while nephrectomy I/R kidneys were significantly enlarged compared to all others at 3 wk post-surgery. Very strong correlation between (99m)Tc-DMSA uptake and weight of dissected kidneys in I/R models was observed. Consistent with (99m)Tc-DMSA uptake results, all histological results indicate that kidney recovery after injury is correlated with the amount of intact tubules and kidney sizes. In summary, our study showed good potentials of (99m)Tc-DMSA scan as a promising non-invasive method for evaluation of kidney restoration after I/R injuries. Interestingly, mice with Bi-I/R injury showed faster repair capacity than those with uni-I/R.

In vivo imaging of tumor apoptosis using histone H1-targeting peptide.

In vivo imaging of apoptosis could allow monitoring of tumor response to cancer treatments such as chemotherapy. Using phage display, we identified the CQRPPR peptide, named ApoPep-1(Apoptosis-targeting Peptide-1), that was able to home to apoptotic and necrotic cells in tumor tissue. ApoPep-1 also bound to apoptotic and necrotic cells in culture, while only little binding to live cells was observed. Its binding to apoptotic cells was not dependent on calcium ion and not competed by annexin V. The receptor for ApoPep-1 was identified to be histone H1 that was exposed on the surface of apoptotic cells. In necrotic cells, ApoPep-1 entered the cells and bound to histone H1 in the nucleus. The imaging signals produced during monitoring of tumor apoptosis in response to chemotherapy was enhanced by the homing of a fluorescent dye- or radioisotope-labeled ApoPep-1 to tumor treated with anti-cancer drugs, whereas its uptake of the liver and lung was minimal. These results suggest that ApoPep-1 holds great promise as a probe for in vivo imaging of apoptosis, while histone H1 is a unique molecular signature for this purpose.

Combination therapy and noninvasive imaging with a dual therapeutic vector expressing MDR1 short hairpin RNA and a sodium iodide symporter.

UNLABELLED: We investigated the feasibility of using combination gene therapy and noninvasive nuclear imaging after expression of the human sodium iodide symporter (hNIS) and inhibition of the multidrug resistance (MDR1) gene in colon cancer cells. METHODS: HCT-15 cells were stably transfected with a dual expression vector, in which the hNIS gene, driven by a constitutive cytomegalovirus promoter, has been coupled to an MDR1 short hairpin RNA (shRNA) cassette. Cell lines stably expressing the hNIS gene and MDR1 shRNA (designated MN-61 and MN-62) were produced, and the expression of the NIS gene and MDR1 shRNA was examined by Western blotting, reverse transcription-polymerase chain reaction, and immunostaining. The functional activities of MDR1 shRNA were determined by paclitaxel uptake and sensitivity to doxorubicin. Functional NIS expression was confirmed by the uptake and efflux of (125)I and the cytotoxicity of (131)I. The effect of the combination of (131)I and doxorubicin was determined by an in vitro clonogenic assay. In vivo NIS expression was examined by small-animal PET with (124)I. RESULTS: The shMDR-NIS-expressing cells showed a significant decrease in the expression of MDR1 messenger RNA and its translated product, P-glycoprotein. The inhibition of P-glycoprotein expression by shRNA enhanced the intracellular accumulation of paclitaxel, the cellular retention of which is mediated by P-glycoprotein, thereby increasing sensitivity to the anticancer drug. The shMDR-NIS-expressing cells showed a significant increase of (125)I uptake, which was completely inhibited by KClO(4). Although the iodide efflux rate was rapid, the cell survival rate was markedly reduced by (131)I treatment. Interestingly, the combination of doxorubicin and a radioiodide ((131)I) displayed synergistic cytotoxicity that correlated with MDR1 inhibition and NIS expression in shMDR-NIS-expressing cells. Furthermore, in mice with shMDR-NIS-expressing tumor xenografts, small-animal PET with (124)I clearly visualized shMDR1-NIS-expressing tumors. CONCLUSION: We developed a dual expression vector with the NIS gene and MDR1 shRNA. This study represents a promising first step in investigations of the potential use of a combination of the NIS gene and MDR1 shRNA as a new therapeutic strategy allowing RNA interference-based gene therapy, NIS-based radioiodine therapy, and in vivo monitoring based on NIS imaging.