Up-regulation of hepatic alpha-2-HS-glycoprotein transcription by testosterone via androgen receptor activation.

BACKGROUND/AIMS: Fetuin-A (alpha-2-HS-glycoprotein, AHSG), a liver borne plasma protein, contributes to the prevention of soft tissue calcification, modulates inflammation, reduces insulin sensitivity and fosters weight gain following high fat diet or ageing. In polycystic ovary syndrome, fetuin-A levels correlate with free androgen levels, an observation pointing to androgen sensitivity of fetuin-A expression. The present study thus explored whether the expression of hepatic fetuin-A is modified by testosterone. METHODS: HepG2 cells were treated with testosterone and androgen receptor antagonist flutamide, and were silenced with androgen receptor siRNA. To test the in vivo relevance, male mice were subjected to androgen deprivation therapy (ADT) for 7 weeks. AHSG mRNA levels were determined by quantitative RT-PCR and fetuin-A protein abundance by Western blotting. RESULTS: In HepG2 cells, AHSG mRNA expression and fetuin-A protein abundance were both up-regulated following testosterone treatment. The human alpha- 2-HS-glycoprotein gene harbors putative androgen receptor response elements in the proximal 5 kb promoter sequence relative to TSS. The effect of testosterone on AHSG mRNA levels was abrogated by silencing of the androgen receptor in HepG2 cells. Moreover, treatment of HepG2 cells with the androgen receptor antagonist flutamide in presence of endogenous ligands in the medium significantly down-regulated AHSG mRNA expression and fetuin-A protein abundance. In addition, ADT of male mice was followed by a significant decrease of hepatic Ahsg mRNA expression and fetuin-A protein levels. CONCLUSIONS: Testosterone participates in the regulation of hepatic fetuin-A expression, an effect mediated, at least partially, by androgen receptor activation.

Impact of anesthetics on 3'-[18F]fluoro-3'-deoxythymidine ([18F]FLT) uptake in animal models of cancer and inflammation.

The aim of this study was to evaluate the impact of different anesthetics on 3'-[18F]fluoro-3'-deoxythymidine ([18F]FLT) uptake in carcinomas and arthritic ankles. To determine the amount of [18F]FLT uptake in subcutaneous CT26 colon carcinomas or arthritic ankles, spontaneously room air/medical air-breathing mice were anesthetized with isoflurane, a combination of medetomidine/midazolam, or ketamine/xylazine. Mice were kept conscious or anesthetized during [18F]FLT uptake before the 10-minute static positron emission tomographic (PET) investigations. [18F]FLT uptake in CT26 colon carcinomas and arthritic ankles was calculated by drawing regions of interest. We detected a significantly reduced (4.4 ± 0.9 %ID/cm3) [18F]FLT uptake in the carcinomas of ketamine/xylazine-anesthetized mice compared to the [18F]FLT-uptake in carcinomas of medetomidine/midazolam- (7.0 ± 1.5 %ID/cm3) or isoflurane-anesthetized mice (6.4 ± 1.5 %ID/cm3), whereas no significant differences were observed in arthritic ankles regardless of whether mice were anesthetized or conscious during tracer uptake. The time-activity curves of carcinomas and arthritic ankles yielded diverse [18F]FLT accumulation related to the used anesthetics. [18F]FLT uptake dynamics are different in arthritic ankles and carcinoma, and the magnitude and pharmacokinetics of [18F]FLT uptake are sensitive to anesthetics. Thus, for preclinical in vivo [18F]FLT PET studies in experimental tumor or inflammation models, we recommend the use of isoflurane anesthesia as it yields a stable tracer uptake and is easy to handle.

Preclinical evaluation of a novel c-Met inhibitor in a gastric cancer xenograft model using small animal PET.

PURPOSE: Here, we describe the efficacy of the novel small molecule c-Met inhibitor BAY 853474 in reducing tumor growth in the Hs746T gastric cancer xenograft model and tested the suitability of 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) versus 3'-deoxy-3'-18F-fluorothymidine ([(18)F]FLT) for response monitoring in a gastric cancer xenograft mouse model using small animal PET. PROCEDURES: The c-Met inhibitor or vehicle control was administered orally at various doses in tumor-bearing mice. Glucose uptake and proliferation was measured using PET before, 48 and 96 h after the first treatment. The PET data were compared to data from tumor growth curves, autoradiography, Glut-1 and Ki-67 staining of tumor sections, and biochemical analysis of tissue probes, i.e., c-Met and ERK phosphorylation and cyclin D1 levels. RESULTS: BAY 853474 significantly reduces tumor growth. [(18)F]FDG uptake in Hs746T tumors was significantly reduced in the groups receiving the drug, compared with the control group. The [(18)F]FLT uptake in the tumor tissue was completely absent 96 h after treatment. Autoradiographic, immunohistochemical, and biochemical analyses confirmed the PET findings. Treatment with the c-Met inhibitor did not affect body weight or glucose levels, and no adverse effects were observed in the animals. CONCLUSION: These preclinical findings suggest that clinical PET imaging is a useful tool for early response monitoring in clinical studies.

Oxygen breathing affects 3'-deoxy-3'-18F-fluorothymidine uptake in mouse models of arthritis and cancer.

UNLABELLED: Noninvasive in vivo imaging of biologic processes using PET is an important tool in preclinical studies. We observed significant differences in 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) uptake in arthritic ankles and carcinomas between dynamic and static PET measurements when mice breathed oxygen. Thus, we suspected that air or oxygen breathing and the anesthesia protocol might influence (18)F-FLT tracer uptake. METHODS: We injected arthritic, healthy, and CT26 colon carcinoma-bearing mice with (18)F-FLT before static or dynamic small-animal PET measurements. The spontaneously oxygen- or air-breathing mice were kept conscious or anesthetized with ketamine and xylazine during (18)F-FLT uptake before the 10-min static PET measurements. For dynamic PET scans, mice were anesthetized during the entire measurement. (18)F-FLT uptake was reported in percentage injected dose per cubed centimeter by drawing regions of interest around ankles, carcinomas, and muscle tissue. Additionally, venous blood samples were collected before (18)F-FLT injection and after PET measurement to analyze pH, carbon dioxide partial pressure (pCO(2)), and lactate values. RESULTS: A significantly reduced (18)F-FLT uptake was measured in arthritic ankles and in CT26 colon carcinomas when the mice breathed oxygen and were conscious during tracer uptake, compared with mice that were anesthetized during (18)F-FLT uptake. Breathing air completely abolished this phenomenon. Analysis of blood samples that were obtained from the mice before (18)F-FLT injection and after the PET scan implicated respiratory acidosis that was induced by oxygen breathing and consciousness during tracer uptake. Acidosis was found to be the primary factor responsible for the reduced (18)F-FLT uptake, as reflected by increased pCO(2) and reduced pH and lactate values. CONCLUSION: Oxygen-breathing conscious mice sustained respiratory acidosis and, consequently, reduced cell proliferation and (18)F-FLT uptake in arthritic ankles and CT26 colon carcinomas. Thus, we suggest the use of air instead of oxygen breathing for (18)F-FLT PET measurements.

An evaluation of 2-deoxy-2-[18F]fluoro-D-glucose and 3'-deoxy-3'-[18F]-fluorothymidine uptake in human tumor xenograft models.

PURPOSE: The aim of this study is to assess the variability of 2-deoxy-2-[(18)F]fluoro-D: -glucose ([(18)F]-FDG) and 3'-deoxy-3'-[(18)F]-fluorothymidine ([(18)F]-FLT) uptake in pre-clinical tumor models and examine the relationship between imaging data and related histological biomarkers. PROCEDURES: [(18)F]-FDG and [(18)F]-FLT studies were carried out in nine human tumor xenograft models in mice. A selection of the models underwent histological analysis for endpoints relevant to radiotracer uptake. Comparisons were made between in vitro uptake, in vivo imaging, and ex vivo histopathology data using quantitative and semi-quantitative analysis. RESULTS: In vitro data revealed that [1-(14)C]-2-deoxy-D: -glucose ([(14)C]-2DG) uptake in the tumor cell lines was variable. In vivo, [(18)F]-FDG and [(18)F]-FLT uptake was highly variable across tumor types and uptake of one tracer was not predictive for the other. [(14)C]-2DG uptake in vitro did not predict for [(18)F]-FDG uptake in vivo. [(18)F]-FDG SUV was inversely proportional to Ki67 and necrosis levels and positively correlated with HKI. [(18)F]-FLT uptake positively correlated with Ki67 and TK1. CONCLUSION: When evaluating imaging biomarkers in response to therapy, the choice of tumor model should take into account in vivo baseline radiotracer uptake, which can vary significantly between models.

Assessment of PET tracer uptake in hormone-independent and hormone-dependent xenograft prostate cancer mouse models.

UNLABELLED: The pharmacokinetics of (18)F-fluorodeoxythymidine (FLT), (18)F-FDG, (11)C-choline, and (18)F-fluoroethylcholine (FEC) in 2 hormone-independent (PC-3, DU145) and 2 hormone-dependent (CWR22, PAC120) prostate cancer xenograft mouse models were evaluated by PET and compared by immunohistochemistry. Further investigation was performed to determine whether PET can detect early changes in tumor metabolism after androgen ablation therapy through surgical castration. METHODS: PET was performed on 4 consecutive days. In addition, the CWR22 and PAC120 tumor models were surgically castrated after the baseline measurement and imaged again after castration. The tracer uptake was analyzed using time-activity curves, percentage injected dose per volume (%ID/cm(3)), and tumor-to-muscle ratio (T/M). RESULTS: Regarding the hormone-independent prostate tumor models, (18)F-FLT showed the best T/M and highest %ID/cm(3) in PC-3 (2.97 ± 0.63 %ID/cm(3)) and DU145 (2.06 ± 0.75 %ID/cm(3)) tumors. (18)F-FDG seemed to be the tracer of choice for delineation of the PC-3 tumors but not for the DU145 tumors. Using (11)C-choline (PC-3: 1.33 ± 0.29 %ID/cm(3), DU145: 1.60 ± 0.27 %ID/cm(3)) and (18)F-FEC, we did not find any significant uptake in the tumors, compared with muscle tissue. Regarding the hormone-dependent prostate tumor models, the CWR22 model showed a highly significant (P < 0.01) decrease in tumor (18)F-FDG uptake from 4.11 ± 1.29 %ID/cm(3) to 2.19 ± 1.45 %ID/cm(3) after androgen ablation therapy. However, the (18)F-FLT, (11)C-choline, or (18)F-FEC tracers did not provide sufficient uptake or reliable information about therapy response in CWR22 tumors. The PAC120 model showed a significant increase in (18)F-FLT tumor uptake (P = 0.015) after androgen ablation therapy. The accumulation of (18)F-FEC (before: 2.32 ± 1.01 %ID/cm(3), after: 1.36 ± 0.39 %ID/cm(3)) was found to be the next highest after (18)F-FDG (before: 2.45 ± 0.93 %ID/cm(3), after: 2.18 ± 0.65 %ID/cm(3)) in PAC120 tumors before castration and is better suited for monitoring therapy response. CONCLUSION: This comprehensive study in 2 hormone-dependent and 2 hormone-independent prostate tumor mouse models shows that (18)F-FLT and (18)F-FDG are the most appropriate tracers for delineation of PC-3, DU145 (except (18)F-FDG), and CWR22 tumors, but not for PAC120 tumors. (18)F-FEC and (11)C-choline, in particular, revealed insufficient T/M ratio in the prostate tumor models. The results may indicate that radiolabeled choline and choline derivatives compete with a high concentration of the precursor dimethylaminoethanol, resulting in reduced uptake in small-rodent tumor models, a hypothesis that is currently under investigation in our laboratory.