Male Nude Mice Research Articles

Abstract 3220: The effects of cholesterol treatment drugs alone and in combination on prostate tumor xenograft growth

Abstract Background: Epidemiologic data suggest cholesterol-lowering drugs may prevent the progression of prostate cancer (PC), but not the incidence of the disease. This may occur by lowering levels of low-density lipoproteins (LDL). Statins and cholesterol-uptake inhibitors like ezetimibe lower serum LDL and are commonly prescribed for patients with high cholesterol. In this study, we determined the therapeutic role of simvastatin, ezetimibe, or both in vitro using several PC cell lines and in vivo using a LAPC-4 xenograft model. Methods: We treated 4 PC cell lines (LAPC-4, CWR22rv1, PC-3, and DU145) with varying concentrations of simvastatin (0-1000nM) or ezetimibe (0-100μM) for 0-5 days. MTS assays were performed to determine cell proliferation, and the IC50 was calculated using BioDataFit 1.02. For our in vivo study, a total of 90 athymic male nude mice were fed a high-fat, high-cholesterol diet (HFHC; 40% fat, 17% protein, 43% carbohydrates, 1.25% cholesterol), subcutaneously injected with 1 x 10^5 LAPC-4 cells, and randomized 2 weeks later to 1 of 4 treatments: vehicle control, 11mg/kg/day simvastatin (delivered via osmotic pump), 30mg/kg diet ezetimibe (delivered in the food), or simvastatin + ezetimibe. Mice remained on study for 42 days post-randomization, at which point they were harvested and samples analyzed. Results: In vitro, simvastatin directly reduced PC cell proliferation in a dose-dependent, cell line-specific manner, but ezetimibe had no inhibitory effect. In vivo, treatment of low continuous dosing of simvastatin or ezetimibe for 42 days had no effect on tumor growth compared to control. However, relative to control, the combination of simvastatin and ezetimibe accelerated tumor growth (p=0.01). The simvastatin-ezetimibe mice also showed lower fat infiltration in the liver and lower serum cholesterol, but higher tumor cholesterol levels than control. Conclusions: Our results suggest that low continuous dosing of simvastatin or ezetimibe may have no effect on PC growth in a slow-growing LAPC-4 xenograft model, but the combination of simvastatin and ezetimibe may accelerate PC growth, possibly by stimulating cholesterol uptake within the tumor. This novel finding may be important in implementing new treatments for men with slow growing PC and high cholesterol. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3220. doi:1538-7445.AM2012-3220

Abstract 2421: Iron deficiency suppresses EMT through down-regulation of N-cadherin in esophageal cancer.

Abstract [Introduction] Epithelial-Mesenchymal-Transition(EMT) plays a great role in cancer metastasis and invasion. Mechanism of EMT still remains unclear, However E-cadherin and N-cadherin are known to have contribution to EMT. In particular, down-regulation of N-cadherin has been reported to reduce cancer metastasis and invasiveness. Iron metabolism and its relationship with cancer cell were studied for a long time. Iron overload is known to induce some kinds of cancer, and iron deficiency is also known to suppress the tumor growth in vivo study. But the correlation of iron metabolism and tumor progression isn't unclear. Recently, it was reported that iron metabolism was essential for EMT and iron deficiency down-regulated EMT differentiation. In this study, we hypothesized that iron deficiency down-regulated the expression of N-cadherin, and suppressed the ability of metastasis and invasiveness. Here we demonstrate our results. [Materials and Methods] In vitro study, TE-4 and TE-10 esophageal squamous cell carcinoma and OE19 esophageal adenocarcinoma cell line were used in this study. Cell viability was determined by XTT assay. Iron chelator deferasirox was used to simulate iron deficient condition. To determine that iron deficiency suppresses EMT and invasiveness of cancer cells, we investigated western blotting analysis, three-dimensional spheroid model analysis and migration assay. Moreover, to determine the expression N-cadherin in mRNA level, PCR assay was performed. In vivo study, iron depletion condition was made by iron deficient diet. Normal and iron deficient diet were fed for 3 weeks before implantation of TE-4 cells in athymic male nude mice(n=8/group). After implantation, each diet was fed continuously for a month. [Results] In vivo study, iron deficient diet suppressed the tumor growth(tumor volume: normal diet vs iron deficient diet = 30.7±1.1 mm3 vs 10.9±2.2 mm3). In vitro study, deferasirox reduces proliferation of all cell lines in a concentration manner. According to western blotting analysis, deferasirox induces reduction of N-cadherin protein in TE-4 and TE-10 cell lines. N-cadherin down-regulation declines invasiveness of TE-4 and TE-10 cell lines in the three-dimensional spheroid model analysis and migration assay. Real time PCR assay shows that down-regulation of N-cadherin is dependent on reduction of N-cadherin mRNA. [Conclusion] Iron controlled treatment can be a novel therapy for invasiveness of cancer under EMT. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2421. doi:1538-7445.AM2012-2421

Osteogenic potential of human umbilical cord-derived mesenchymal stromal cells cultured with umbilical cord blood-derived autoserum

ObjectiveOsteogenesis in the bone defect at the site of an alveolar cleft is important to enable patients with cleft lip and palate to acquire dental articulation. The presence of umbilical cord-derived mesenchymal stem cells has been reported. In this study, we used autoserum derived from the umbilical cord blood (UCB) of neonates in an attempt to examine the osteoblastic differentiation potential of umbilical cord-derived mesenchymal stromal cells (UC-MSCs) in nude mice. Materials and methodsUCB, hydroxyapatite, and rhBMP were used as the supply source of autoserum, scaffold, and osteoinductive growth factor, respectively. MSCs, obtained from Wharton's jelly and cultured for 3–4weeks to induce their differentiation into osteoblasts, were implanted subcutaneously into the dorsum of male nude mice for 6weeks before the assessment by real-time reverse transcriptase chain reaction of osteoblast marker expression. ResultsUCB-derived autoserum was a viable source for the culture and implantation of UC-MSCs. The osteoblastic differentiation potential of UC-MSCs was demonstrated in nude mice by performing immunohistochemical staining and by the presence of osteoblast marker expression. ConclusionsOur results confirm the osteogenic potential of UC-MSCs and provide basic evidence for the realization of regenerative medicine using autologous tissues.

Targeting Free Prostate-Specific Antigen forIn VivoImaging of Prostate Cancer Using a Monoclonal Antibody Specific for Unique Epitopes Accessible on Free Prostate-Specific Antigen Alone

This study investigated the feasibility of targeting the free, unbound forms of prostate-specific antigen (fPSA) for in vivo imaging of prostate adenocarcinomas (PCa), as PSA is produced and secreted at abundance during every clinical stage and grade of PCa, including castration-resistant disease. We injected (125)I-labeled monoclonal antibody PSA30 (specific for an epitope uniquely accessible on fPSA alone) intravenously in male nude mice carrying subcutaneous xenografts of LNCaP tumors (n=36). Mice were sacrificed over a time course from 4 hours to 13 days after injecting (125)I-labeled PSA30. Tissue uptake of (125)I-PSA30 at 48 and 168 hours after intravenous injection was compared with two clinically used positron emission tomography radiopharmaceuticals, (18)F-fluoro-deoxy-glucose ((18)F-FDG) or (18)F-choline, in cryosections using Digital AutoRadiography (DAR) and also compared with immunohistochemical staining of PSA and histopathology. On DAR, the areas with high (125)I-PSA30 uptake corresponded mainly to morphologically intact and PSA-producing LNCaP cells, but did not associate with the areas of high uptake of either (18)F-FDG or (18)F-choline. Biodistribution of (125)I-PSA30 measured in dissected organs ex vivo during 4 to 312 hours after intravenous injection demonstrated maximum selective tumor uptake 24-48 hours after antibody injection. Our data showed selective uptake in vivo of a monoclonal antibody highly specific for fPSA in LNCaP cells. Hence, in vivo imaging of fPSA may be feasible with putative usefulness in disseminated PCa.

1322 THE EFFECT OF POMEGRANATE EXTRACT DOSE ON PROSTATE CANCER GROWTH IN A CARBOHYDRATE RESTRICTED MOUSE XENOGRAFT MODEL

You have accessJournal of UrologyProstate Cancer: Basic Research II1 Apr 20121322 THE EFFECT OF POMEGRANATE EXTRACT DOSE ON PROSTATE CANCER GROWTH IN A CARBOHYDRATE RESTRICTED MOUSE XENOGRAFT MODEL Michael Abern, Elizabeth Masko, Chenwei Wu, Alexis Gaines, Salvatore Pizzo, and Stephen Freedland Michael AbernMichael Abern Durham, NC More articles by this author , Elizabeth MaskoElizabeth Masko Durham, NC More articles by this author , Chenwei WuChenwei Wu Durham, NC More articles by this author , Alexis GainesAlexis Gaines Durham, NC More articles by this author , Salvatore PizzoSalvatore Pizzo Durham, NC More articles by this author , and Stephen FreedlandStephen Freedland Durham, NC More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.1669AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Preclinical studies have shown that dietary carbohydrate restriction slows prostate tumor growth in multiple xenograft models in part due to a reduction insulin-like growth factor (IGF) axis signaling. Pomegranate extract (PE) has been shown to slow prostate tumor growth in-vitro and in vivo similarly through this pathway, as well as decrease markers of angiogenesis and inflammation. In this study, we determined the dose-response of PE on prostate tumor growth in the setting of dietary carbohydrate restriction in a slow-growing mouse xenograft model. For this study, we chose a standardized PE (POMx, Pom Wonderful, Los Angeles, CA) at doses which are easily translatable to human equivalents. METHODS Male athymic nude mice housed 5 per cage (n = 130) were subcutaneously injected with 1 × 105 LAPC-4 cells after a 10 day acclimation period. 14 days post-injection, all mice were all placed on an ad-libitum no-carbohydrate ketogenic diet (NCKD: 83% fat, 0% carbohydrate, 17% protein) (day 0). Each cage was randomly assigned to 1 of 4 doses of PE (placebo = 0 mg, low dose = 2.62 mg, intermediate dose = 5.25mg, or high dose = 7.87mg) suspended in 0.2 mL of PBS delivered via oral gavage 5 days per week. These doses are equivalent to 0, 1, 2, or 3 POMx capsules/day consumed by a 70 kg human. Mice will be sacrificed when tumors reach ≥1,000 mm3. RESULTS At the time of randomization, there were no differences in median body weight between the groups (p = 0.623). Currently, at day 32, overall survival is decreased in the placebo group (hazard ratio 2.26, 95% confidence interval, 0.83 - 6.10, p = 0.109) relative to the low dose PE group, and there are no survival differences between PE groups. Median tumor volumes for the placebo group are 83.3mm3, compared to 74.5mm3, 76.3mm3, and 70.1mm3 for the low, intermediate, and high PE doses respectively which are not statistically different (p = 0.98). CONCLUSIONS Preliminary data show a trend toward improved survival with the combination of an NCKD and PE at the selected doses. Currently, tumor volumes are not significantly different, however completed results will be presented. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e536 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Michael Abern Durham, NC More articles by this author Elizabeth Masko Durham, NC More articles by this author Chenwei Wu Durham, NC More articles by this author Alexis Gaines Durham, NC More articles by this author Salvatore Pizzo Durham, NC More articles by this author Stephen Freedland Durham, NC More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

969 PI3K/AKT DUAL INHIBITOR RESTORES DOCETAXEL SENSITIVITY IN CASTRATION RESISTANT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research V1 Apr 2012969 PI3K/AKT DUAL INHIBITOR RESTORES DOCETAXEL SENSITIVITY IN CASTRATION RESISTANT PROSTATE CANCER Yota Yasumizu, Akira Miyajima, Takeo Kosaka, Suguru Shirotake, Eiji Kikuchi, and Mototsugu Oya Yota YasumizuYota Yasumizu Tokyo, Japan More articles by this author , Akira MiyajimaAkira Miyajima Tokyo, Japan More articles by this author , Takeo KosakaTakeo Kosaka Tokyo, Japan More articles by this author , Suguru ShirotakeSuguru Shirotake Tokyo, Japan More articles by this author , Eiji KikuchiEiji Kikuchi Tokyo, Japan More articles by this author , and Mototsugu OyaMototsugu Oya Tokyo, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.1068AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The specific aim of this study was to establish a new treatment strategy for docetaxel resistant CRPC, with a special focus on dual regulation of the phosphoinositide3-kinase (PI3K) and Akt signaling pathways. METHODS The efficacy of PI3K/Akt signaling pathway inhibition by the PI3K/Akt dual inhibitor BEZ235 in combination with docetaxel was examined in C4-2 and C4-2AT6 cells in vivo and in vitro. C4-2AT6 cells are a new CRPC cell line established by our laboratory under androgen ablation conditions lasting for 6 months. RESULTS We compared the cytotoxic effect of docetaxel on C4-2 and C4-2AT6 cells. The cytotoxic response was measured by WST assay. In C4-2 cells and C4-2AT6 cells, the mean cell viability at 24 h of treatment with 2nM docetaxel was 43.0% ± 5.2 % and 69.8% ± 6.3 %, respectively. These results indicated that C4-2AT6 cells showed significantly higher resistance to docetaxel than C4-2 cells. We investigated the expression of phosphorylation of Akt (pAkt) in C4-2 and C4-2AT6 cells by Western blotting and immunohistochemistry. C4-2AT6 cells showed significantly higher pAkt expression than C4-2 cells, suggesting the possibility of a treatment strategy targeting PI3K/Akt. In C4-2AT6 cells WST assay showed that cell viability was 44.8 ± 4.2 % with 10nM BEZ235 and 20.9 ± 3.9 % with combined 2nM docetaxel and 10nM BEZ235. Combine administration of BEZ235 and docetaxel showed significantly higher cytotoxicity in C4-2AT6 than monotherapy with either BEZ235 or docetaxel alone (p<0.01). Combination index was 0.31, suggesting the synergic effect of this combination therapy. Western blotting showed 50nM BEZ235 inhibited the expression of pAKT in a time-dependent manner. In vivo, we assigned castrated male nude mice to 4 groups; control, BEZ235 monotherapy (40mg/kg/day), docetaxel monotherapy (4mg/kg), and BEZ235 combined with docetaxel. We examined the time course changes in C4-2AT6 tumor growth with BEZ235 and docetaxel. In the control group tumor size grew up to 298% on day 15, compared with day 1. In contrast, the ratio of tumor size on day 15 to day 0 was 103% and 140% in the BEZ235 and docetaxel monotherapy groups, respectively. In the combination group, tumor volume was significantly decreased to 43.9% (p<0.01). These results indicate that the combination of BEZ235 and docetaxel has a significant antitumor effect in C4-2AT6 in vivo, as well as in vitro. CONCLUSIONS The combination of BEZ235 and docetaxel showed significant anti-tumor effects in CRPC, suggesting that PI3K/Akt dual inhibitor may be a promising modality for docetaxel resistant CRPC. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e394-e395 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yota Yasumizu Tokyo, Japan More articles by this author Akira Miyajima Tokyo, Japan More articles by this author Takeo Kosaka Tokyo, Japan More articles by this author Suguru Shirotake Tokyo, Japan More articles by this author Eiji Kikuchi Tokyo, Japan More articles by this author Mototsugu Oya Tokyo, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

EXPRESSION OF VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR 2 CORRELATES WITH METASTATIC POTENTIAL IN PROSTATE TUMOR CELLS

Studies in recent years have suggested that VEGFR2 could be expressed in non‐endothelial cell populations. This study explores the expression and function of VEGFR2 on non‐endothelial prostate tumor cells of differing metastatic potential. PC3M and PC3M‐LN4 human prostate carcinoma cell lines, low and high metastatic respectively, were analyzed using RT‐PCR, western blot, ELISA, and in vitro growth assays. Additionally, PC3M and PC3M‐LN4 cells were injected orthotopically into the prostate of male nude mice for in vivo tumor growth analysis. PC3M‐LN4 cells showed expression of VEGFR2 mRNA in RT‐PCR and VEGFR2 protein in western blot analysis whereas PC3M cells did not. ELISAs of PC3M and PC3M‐LN4 cells showed that both cells lines produce high levels of VEGF. Culturing cells in serum free media containing only VEGF‐A resulted in the proliferation and growth of PC3M‐LN4 cells but not PC3M. Tumors resected from nude mice injected with PC3M tumor cells were small and non‐metastatic whereas PC3M‐LN4 tumors were large, highly vascularized, and with metastases to the para‐aortic lymph nodes. These results pose an autocrine growth function for VEGFR2 in highly metastatic prostate tumor cells as well as a correlation between VEGFR2 expression and metastatic potential in these cells. Clinically, these data suggest that VEGFR2 inhibition may target both the tumor and stromal compartments to inhibit disease progression.

Combination of calcitriol and dietary soy exhibits enhanced anticancer activity and increased hypercalcemic toxicity in a mouse xenograft model of prostate cancer

The potential role of vitamin D and soy in prostate cancer (PCa) prevention/treatment has gained much attention in recent years. In this study, we evaluated the anticancer activity of calcitriol, the active form of vitamin D, dietary soy, and their combinations in a mouse model of PCa. Athymic male nude mice bearing PC-3 human PCa xenografts received diets containing 10 or 20 kcal% soy, calcitriol injections, or a combination of dietary soy and calcitriol. Changes in tumor growth, serum levels of 1,25(OH)(2)D and calcium, and regulation of tumor gene expression were examined. The combination treatments resulted in substantially greater inhibition of tumor growth than either agent alone. Soy diets alone caused a modest elevation in serum 1,25(OH)(2)D, whereas the calcitriol-soy combinations led to substantially elevated serum 1,25(OH)(2) D, hypercalcemia, and in some cases lethal toxicity. The combinations enhanced calcitriol activity in regulating target gene expression, including greater up-regulation of anti-proliferative (p21, IGFBP-3) and pro-apoptotic (Bax) genes, increased inhibition of anti-apoptotic (Bcl-2) and cell cycle promoting (cyclin D1) genes, and suppression of prostaglandin (PG) synthesis and signaling (COX-2, 15-PGDH, PG receptors). Increases in serum calcium were accompanied by elevated expression of intestinal calcium absorption genes (TRPV6, calbindin-9k). Soy increases the bioavailability of endogenous and administered calcitriol, thereby enhancing its anticancer effects and risk of hypercalcemia. Since both agents are easily available as dietary supplements, the increased potential for hypercalcemic toxicity becomes an important factor when considering the combined use of vitamin D and soy in PCa therapy.

Carbohydrate restriction and lactate transporter inhibition in a mouse xenograft model of human prostate cancer

What's known on the subject? and What does the study add? It is known that both lactate inhibition and carbohydrate restriction inhibit tumour growth. What is unknown is whether the two work synergistically together. This study adds that though the combination of lactate inhibition and carbohydrate restriction did not synergistically slow tumour growth in our model, we confirmed that carbohydrate restriction started after tumour inoculation slowed tumour growth. Moreover, lactate inhibition resulted in changes in the tumour microenvironment that may have implications for future metabolic targeting of prostate cancer growth. To determine if a no-carbohydrate ketogenic diet (NCKD) and lactate transporter inhibition can exert a synergistic effect on delaying prostate tumour growth in a xenograft mouse model of human prostate cancer. 120 nude athymic male mice (aged 6-8 weeks) were injected s.c. in the flank with 1.0 × 10(5) LAPC-4 prostate cancer cells. • Mice were randomized to one of four treatment groups: Western diet (WD, 35% fat, 16% protein, 49% carbohydrate) and vehicle (Veh) treatment; WD and mono-carboxylate transporter-1 (MCT1) inhibition via α-cyano-4-hydroxycinnamate (CHC) delivered through a mini osmotic pump; NCKD (84% fat, 16% protein, 0% carbohydrate) plus Veh; or NCKD and MCT1 inhibition. • Mice were fed and weighed three times per week and feed was adjusted to maintain similar body weights. • Tumour size was measured twice weekly and the combined effect of treatment was tested via Kruskal-Wallis analysis of all four groups. Independent effects of treatment (NCKD vs WD and CHC vs Veh) on tumour volume were tested using linear regression analysis. • All mice were killed on Day 53 (conclusion of pump ejection), and serum and tumour sections were analysed for various markers. Again, combined and independent effects of treatment were tested using Kruskal-Wallis and linear regression analysis, respectively. There were no significant differences in tumour volumes among the four groups (P= 0.09). • When testing the independent effects of treatment, NCKD was significantly associated with lower tumour volumes at the end of the experiment (P= 0.026), while CHC administration was not (P= 0.981). However, CHC was associated with increased necrotic fraction (P < 0.001). Differences in tumour volumes were observed only in comparisons between mice fed a NCKD and mice fed a WD. • MCT1 inhibition did not have a significant effect on tumour volume, although it was associated with increased necrotic fraction.

Digoxin Inhibits Blood Vessel Density and HIF‐1a Expression in Castration‐Resistant C4‐2 Xenograft Prostate Tumors

Recent studies suggest a potential application for digoxin in the prevention and/or treatment of prostate cancer. However, the effect of digoxin on androgen receptor (AR)-positive prostate tumor in vivo is not clear. This study is designed to determine if digoxin can inhibit AR-positive xenograft prostate tumors. Athymic male nude mice were utilized to establish subcutaneous C4-2 castration-resistant prostate tumors. The animals were castrated and then treated with daily intraperitoneal (i.p.) injection of digoxin at 2 mg/kg along with vehicle controls for 7 consecutive days. Tumor growth was determined by measuring tumor volume changes, blood vessel density by immunostaining of CD31, and cell proliferation by BrdU labeling. The expression of HIF-1α in C4-2 tumors was measured by Western blot and real-time RT-PCR. Digoxin inhibited blood vessel density about fourfold and down-regulated HIF-1α expression at both mRNA and protein levels. However, digoxin did not inhibit C4-2 tumor growth. Digoxin is a potent inhibitor of HIF-1α signaling pathway and blood vessel formation in C4-2 castration-resistant prostate tumors.

“Combi-targeting” mitozolomide: Conferring novel signaling inhibitory properties to an abandoned DNA alkylating agent in the treatment of advanced prostate cancer

At the preclinical stage, mitozolomide (MTZ) showed exciting preclinical activity but failed later in clinical trial due to toxic side effects. We surmised that by targeting MTZ to epidermal growth factor receptor (EGFR), we may not only alter its toxicity profile, but also enhance its potency in EGFR-overexpressing tumors. To test this hypothesis, we designed JDF12, studied its mechanism of action in human prostate cancer (PCa) cells and determined its potency in vivo. To analyze its mixed EGFR-DNA targeting potential, we performed an enzyme linked immunosorbent assay (ELISA) and western blotting analysis of EGFR phosphorylation in cells stimulated with EGF. DNA damage was analyzed using the comet assay, and apoptosis quantitated by annexin V binding assay. Growth inhibition in vitro was determined by the sulforhodamine B (SRB) assay and in vivo efficacy analyzed in male CD-1 nude mice. The results showed that: Under physiological conditions, JDF12 was hydrolyzed to JDF04R and both agents were capable of inhibiting isolated EGFR tyrosine kinase (TK) and EGFR phosphorylation in EGF-stimulated cells. JDF12 significantly damaged DNA, induced apoptosis in DU145 cells and was up to 2-10-fold more potent than equieffective combinations of MTZ and JDF04R or Iressa in a panel that also included LNCaP and its EGFR and ErbB2 transfectants. In vivo, it induced significant antitumor activity in a DU145 xenograft model. The results suggest that the superior cytotoxicity of JDF12 when compared with MTZ and JDF04R may be imputed to its potent EGFR-DNA targeting properties and confirm the ability of this novel strategy to confer EGFR targeting properties to a classical alkylator.

Adipose Tissue Engineering Using Injectable, Oxidized Alginate Hydrogels

Current treatment modalities for soft tissue augmentation which use autologous grafting and commercially available fillers present a number of challenges and limitations, such as donor site morbidity and volume loss over time. Adipose tissue engineering technology may provide an attractive alternative. This study investigated the feasibility of a degradable alginate hydrogel system with commercially available cryopreserved human adipose stem cells (hADSCs) to engineer adipose tissue. hADSCs were differentiated into adipogenic cells, and encapsulated in alginate hydrogels made susceptible to hydrolysis by partial periodate oxidation of the polymer chains. Cell laden gels were subcutaneously injected into the chest wall of male nude mice, and a cell suspension without alginate served as control. After 10 weeks, specimens were harvested and analyzed morphologically, histologically, and with immunoblotting of tissue extractions. Newly generated tissues were semitransparent and soft in all experimental mice, grossly resembling adipose tissue. Analysis using confocal live imaging, immunohistochemisty and western blot analysis revealed that the newly generated tissue was adipose tissue. This study demonstrates that degradable, injectable alginate hydrogels provide a suitable delivery vehicle for preconditioned cryopreserved hADSCs to engineer adipose tissue.

Comparison of animal studies between interstitial magnetic resonance lymphography and radiocolloid SPECT/CT lymphoscintigraphy in the head and neck region

To comparatively assess two techniques, radiocolloid SPECT/CT lymphoscintigraphy and interstitial MR lymphography using SPIO and gadoxetate disodium, in animal models. We used twenty one 8-week-old male nude mice of strain BALB/c Slc-nu/nu, weighing 23-27g. The 4.7-T MRI equipment was used to detect the SNs. T2*WI of gradient-echo sequences was acquired sequentially up to 24h after administering SPIO, ferucarbotran. T1WI was acquired sequentially up to 80min after administering gadoxetate disodium. (99m)Tc-phytate SPECT/CT lymphoscintigraphy was taken at 30min after the injection to detect the SNs using animal-dedicated whole-body SPECT/CT hybrid scanner. The injection was submucosally performed in the right tongue margin of each mouse. Reading performances concerning SN visualization and its quality on interstitial MR lymphogram and SPECT/CT lymphoscintigram were performed by 3 radiologists. The SN intensities were 0.43 for the right, 0.61 for the left at 30min after ferucarbotran injection, with gradual decrease in intensity, and 1.43 for the right, 1.33 for the left at 10min after gadoxetate disodium injection with a fast decrease in intensity. The base value of 1.0 was at pre-examination. The mean numbers of lymph nodes visualized were 4.00 nodes for on SPECT/CT lymphoscintigram and 2.0 for interstitial MR lymphogram. There was a statistically significant difference in the mean scores between SPECT/CT lymphoscintigraphy and interstitial MR lymphography (two factor mixed design with repeated measures on one factor: p<0.0002). In our comparative study using mice, the results of radiocolloid SPECT/CT lymphoscintigraphy were superior to those of interstitial MR lymphography, while both SPIO and gadoxetate disodium have a potential of being employed for sentinel node navigation surgery by interstitial MR lymphography in the head and neck region.

Do phthalates affect steroidogenesis by the human fetal testis? Exposure of human fetal testis xenografts to di-n-butyl phthalate.

Phthalates are ubiquitous environmental chemicals. Fetal exposure to certain phthalates [e.g. di-n-butyl phthalate (DBP)] causes masculinization disorders in rats, raising concern for similar effects in humans. We investigated whether DBP exposure impairs steroidogenesis by the human fetal testis. The aim of the study was to determine effects of DBP exposure on testosterone production by normally growing human fetal testis xenografts. Human fetal testes (14-20 wk gestation; n=12) were xenografted into castrate male nude mice that were treated for 4-21 d with vehicle, or 500 mg/kg·d DBP, or monobutyl phthalate (active metabolite of DBP); all mice were treated with human chorionic gonadotropin to mimic normal human pregnancy. Rat fetal testis xenografts were exposed for 4 d to DBP as a positive control. Testosterone production was assessed by measuring host serum testosterone and seminal vesicle (SV) weights at termination, plus testis gene expression (rats). Human fetal testis xenografts showed similar survival (∼80%) and total graft weight (8.6 vs. 10.1 mg) in vehicle and DBP-exposed hosts, respectively. Serum testosterone (0.56 vs. 0.64 ng/ml; P>0.05) and SV weight (67.2 vs. 81.9 mg; P>0.05) also did not differ. Exposure to monobutyl phthalate gave similar results. In contrast, exposure of rat fetal xenografts to DBP significantly reduced SV weight and testis Cyp11a1/StAR mRNA expression and lowered testosterone levels, confirming that DBP exposure can inhibit steroidogenesis in xenografts, further validating the negative findings on testosterone production in the human. Exposure of human fetal testes to DBP is unlikely to impair testosterone production as it does in rats. This has important safety and regulatory implications.

The Effect of Finasteride and Dutasteride on the Growth of WPE1-NA22 Prostate Cancer Xenografts in Nude Mice

Background5α-reductase 1 (5αR1) and 5α-reductase 2 (5αR2) convert testosterone into the more potent androgen dihydrotestosterone. 5αR2 is the main isoenzyme in normal prostate tissue; however, most prostate tumors have increased 5αR1 and decreased 5αR2 expression. Previously, finasteride (5αR2 inhibitor) treatment begun 3 weeks post-tumor implantation had no effect on Dunning R3327-H rat prostate tumor growth. We believe the tumor compensated for finasteride treatment by increasing tumor 5αR1 expression or activity. We hypothesize that finasteride treatment would not significantly alter tumor growth even if begun before tumor implantation, whereas dutasteride (5αR1 and 5αR2 inhibitor) treatment would decrease tumor growth regardless of whether treatment was initiated before or after tumor implantation.Methodology/Principal FindingsSixty 8-week-old male nude mice were randomized to Control, Pre- and Post-Finasteride, and Pre- and Post-Dutasteride (83.3 mg drug/kg diet) diet groups. Pre- and post-groups began their treatment diets 1–2 weeks prior to or 3 weeks after subcutaneous injection of 1×105 WPE1-NA22 human prostate cancer cells, respectively. Tumors were allowed to grow for 22 weeks; tumor areas, body weights, and food intakes were measured weekly. At study's conclusion, prostate and seminal vesicle weights were significantly decreased in all treatment groups versus the control; dutasteride intake significantly decreased seminal vesicle weights compared to finasteride intake. No differences were measured in final tumor areas or tumor weights between groups, likely due to poor tumor growth. In follow-up studies, proliferation of WPE1-NA22 prostate cancer cells and parent line RWPE-1 prostate epithelial cells were unaltered by treatment with testosterone, dihydrotestosterone, or mibolerone, suggesting that these cell lines are not androgen-sensitive.ConclusionThe lack of response of WPE1-NA22 prostate cancer cells to androgen treatment may explain the inadequate tumor growth observed. Additional studies are needed to determine whether finasteride and dutasteride are effective in decreasing prostate cancer development/growth.

MR-guided pulsed high intensity focused ultrasound enhancement of docetaxel combined with radiotherapy for prostate cancer treatment

The purpose of this study is to evaluate the efficacy of the enhancement of docetaxel by pulsed focused ultrasound (pFUS) in combination with radiotherapy (RT) for treatment of prostate cancer in vivo. LNCaP cells were grown in the prostates of male nude mice. When the tumors reached a designated volume by MRI, tumor bearing mice were randomly divided into seven groups (n = 5): (1) pFUS alone; (2) RT alone; (3) docetaxel alone; (4) docetaxel + pFUS; (5) docetaxel + RT; (6) docetaxel + pFUS + RT, and (7) control. MR-guided pFUS treatment was performed using a focused ultrasound treatment system (InSightec ExAblate 2000) with a 1.5T GE MR scanner. Animals were treated once with pFUS, docetaxel, RT or their combinations. Docetaxel was given by i.v. injection at 5 mg kg−1 before pFUS. RT was given 2 Gy after pFUS. Animals were euthanized 4 weeks after treatment. Tumor volumes were measured on MRI at 1 and 4 weeks post-treatment. Results showed that triple combination therapies of docetaxel, pFUS and RT provided the most significant tumor growth inhibition among all groups, which may have potential for the treatment of prostate cancer due to an improved therapeutic ratio.

Sexual dimorphism of STGC3 tumor suppressor function in nasopharyngeal carcinoma CNE2 cells

STGC3 is a potential tumor suppressor in nasopharyngeal carcinoma. We previously found that CNE2 cells that re-expressed STGC3 formed smaller tumors in female mice than in male mice. Here, we investigated the sexual dimorphism of STGC3 as a tumor-suppressor in female and male nude mice injected subcutaneously with pcDNA3.1(+)-STGC3/CNE2 cells. ER-α was positively expressed in vitro in the CNE2 cells. The pcDNA3.1(+)-STGC3/CNE2 cell growth rate decreased after treatment with β-estradiol in vitro. There were significant differences in tumor size or mass between pcDNA3.1(+)-STGC3/CNE2 and control cases (P < 0.05), but there were significant differences in tumor size between female and male nude mice in the STGC3 transfection groups, and the pcDNA3.1(+)-STGC3/CNE2 tumor growth rate in the female nude mice was the lowest in all cases (P < 0.05). There were no significant differences between female and male nude mice in control groups. Furthermore, a greater number of cells were blocked in the G(0)/G(1) phase in pcDNA3.1(+)-STGC3/ CNE2 tumor xenografts in the female mice. Protemic analysis found 9 differentially expressed proteins in the pcDNA3.1-STGC3/CNE2 xenograft tissues in females and males. A heat shock 70 protein 8 isoform 2 variant was identified as a down-regulated protein associated with cell cycle control and its downstream factor cyclin D1 was also decreased in STGC3-repressed xenografts in female mice. The data above suggest that STGC3 and its associated proteins play an important role in nasopharyngeal carcinoma gender differences.

Altered Lung Motion is a Sensitive Indicator of Regional Lung Disease

Since lung diseases adversely affect airflow during breathing, they must also alter normal lung motion, which can be exploited to detect these diseases. However, standard imaging techniques such as CT and MRI imaging during breath-holds provide little or no information on lung motion and cannot detect diseases that cause subtle changes in lung structure. Phase-contrast X-ray imaging provides images of high contrast and spatial resolution with temporal resolutions that allow multiple images to be acquired throughout the respiratory cycle. Using X-ray phase-contrast imaging, coupled with velocimetry, we have measured lung tissue movement and determined velocity fields that define speed and direction of regional lung motion throughout a breath in normal Balb/c nude male mice and mice exposed to bleomycin. Regional maps of lung tissue motion reveal both the heterogeneity of normal lung motion, as well as abnormal motion induced by bleomycin treatment. Analysed histologically, bleomycin treatment caused pathological changes in lung structure that were heterogenous, occupying less than 12% of the lung at 6 days after treatment. Moreover, plethysmography failed to detect significant changes in compliance at either 36 h or 6 days after treatment. Detailed analysis of the vector fields demonstrated major differences (p < 0.001) in regional lung motion between control and bleomycin-treated mice at both 36 h and 6 days after treatment. The results of this study demonstrate that X-ray phase-contrast imaging, coupled with velocimetry, can detect early stage, subtle and non-uniform lung disease.

MR Molecular Imaging of Prostate Cancer with a Peptide-Targeted Contrast Agent in a Mouse Orthotopic Prostate Cancer Model

To study the effectiveness of a peptide targeted nanoglobular Gd-DOTA complexes for MR molecular imaging of prostate cancer in a mouse orthotopic PC-3 prostate cancer model. A CLT1 (CGLIIQKNEC) peptide-targeted generation 2 nanoglobular Gd-DOTA monoamide conjugate [CLT1-G2-(Gd-DOTA)] was used for imaging fibrin-fibronectin complexes in prostate tumor using a non-specific peptide KAREC modified conjugate, KAREC-G2-(Gd-DOTA) as a control. Cy5 conjugates of CLT1 and KAREC were synthesized for binding studies. Orthotopic PC-3 prostate tumors were established in the prostate of athymic male nude mice. MRI study was performed on a Bruker 7T small animal MRI system. CLT1 peptide showed specific binding in the prostate tumor with no binding in normal tissues. The control peptide had little binding in normal and tumor tissues. CLT1-G2-(Gd-DOTA) resulted in stronger contrast enhancement in tumor tissue than KAREC-G2-(Gd-DOTA). CLT1-G2-(Gd-DOTA) generated ~100% increase in contrast-to-noise ratio (CNR) in the tumor compared to precontrast CNR at 1min post-injection, while KAREC-G2-(Gd-DOTA) resulted in 8% increase. CLT1-G2-(Gd-DOTA) is a promising molecular MRI contrast agent for fibrin-fibronectin complexes in tumor stroma. It has potential for diagnosis and assessing prognosis of malignant tumors with MRI.

Dynamic Ultrasound Assessment of the Effects of Knee and Ankle Position on Achilles Tendon Apposition Following Acute Rupture

Previous reports have suggested that knee flexion improves tendon edge apposition following acute rupture of the Achilles tendon. The aim of the present study was to determine, with use of ultrasonography, the effects of knee and ankle position on the Achilles tendon gap distance in patients with an acute rupture. Twenty-six patients with a unilateral acute complete Achilles tendon rupture that had been confirmed with ultrasonography were recruited within a week after the injury. The mean age at the time of presentation was forty-one years. Ultrasound measurements included the location of the rupture and the gap distance between the superficial tendon edges with the ankle in neutral and the knee extended. The gap distance was sequentially measured with the foot in maximum equinus at the ankle and with 0°, 30°, 60°, and 90° of knee flexion. The mean distance of the rupture from the calcaneal enthesis was 52 mm (range, 40 to 76 mm). The mean gap distance with the ankle in neutral and the knee extended was 12 mm (95% confidence interval, 10 to 13 mm). This distance decreased to 5 mm (95% confidence interval, 4 to 7 mm) when the foot was placed in maximum ankle equinus with 0° of knee flexion and to 4 mm (95% confidence interval, 3 to 5 mm) with 30° of knee flexion, 3 mm (95% confidence interval, 2 to 4 mm) with 60° of knee flexion, and 2 mm (95% confidence interval, 1 to 2 mm) with 90° of knee flexion. Expressing the reduction in gap distance with each successive position as a percentage of the gap distance with the knee extended and the ankle in neutral revealed a mean reduction of 55.7%, 64.4%, 75.4%, and 84.8% with maximum ankle equinus and 0°, 30°, 60°, and 90° of knee flexion, respectively. The difference in gap distance between each of these positions was significant (p < 0.05). Maximum ankle equinus alone significantly reduces the gap distance after acute Achilles tendon rupture. Increasing knee flexion further reduces the gap distance by small increments that, although significant, may not be clinically important.