Male Nude Mice Research Articles

1455 EFFECT OF POMEGRANATE AND DIET ON PROSTATE CANCER TUMOR GROWTH AND SURVIVAL

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 20111455 EFFECT OF POMEGRANATE AND DIET ON PROSTATE CANCER TUMOR GROWTH AND SURVIVAL Chenwei Wu, Elizabeth M. Masko, Susan H. Poulton, Tammy S. Ho, Alexis R. Gaines, and Stephen J. Freedland Chenwei WuChenwei Wu Durham, NC More articles by this author , Elizabeth M. MaskoElizabeth M. Masko Durham, NC More articles by this author , Susan H. PoultonSusan H. Poulton Durham, NC More articles by this author , Tammy S. HoTammy S. Ho Durham, NC More articles by this author , Alexis R. GainesAlexis R. Gaines Durham, NC More articles by this author , and Stephen J. FreedlandStephen J. Freedland Durham, NC More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1368AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Pomegranate extract (POM) has been shown to slow prostate cancer (PC) growth in xenograft models. However, mice in these studies ate standard mouse chow, which is very low-fat (∼12% kcals) and not representative of the typical diet for men with PC. Whether POM slows PC growth when taken with a Western diet is unknown. As our group showed diet to separately affect PC xenograft growth, with a no-carbohydrate ketogenic diet (NCKD) being protective vs. a Western diet, we tested the effects of POM on PC growth and survival in mice fed a Western or NCK diet. METHODS 120 male athymic nude mice (age 6-8 weeks) were fed an ad lib Western diet and injected subcutaneously with 1x106 LAPC-4 cells. Two weeks later, mice were randomized to remain on Western diet alone or switched to Western diet + POM, NCKD alone, or NCKD + POM treatments (n=30 per arm). Western diet mice were fed ad lib and NCKD mice via a modified pair-feeding protocol. POM arms received gavage with 0.8 mg POMx extract (PomWonderful) Monday-Friday and control mice received PBS (0.2 mL all arms). Mice were sacrificed when tumors exceeded 1,000 mm3. Tumor volumes were compared between arms using Kruskal-Wallis. Survival between arms was tested using Cox proportional hazards. RESULTS The study is currently on-going and results are preliminary for day 53 post-injection. To date, significant differences in tumor volume have been noted across the study arms (p=0.007) with NCKD + POM mice having significantly smaller tumors than mice fed base Western diet (p=0.03). In mice fed NCKD, the addition of POM resulted in a trend toward smaller tumors (p=0.07). In contrast, in mice on Western diet, the addition of POM resulted in a trend toward larger tumors (p=0.13). Diet type alone did not affect tumor volume (p=1.00). Preliminary survival data suggest that treatment group impacted survival (p=0.055). Specifically, survival for the NCKD + POM arm was better than either Western + POM or NCKD alone arms (all p≤0.045). CONCLUSIONS In a xenograft mouse model, the combination of POM and NCKD slows PC tumor growth relative to a Western diet and improves survival vs. either POM or NCKD treatments alone. Interestingly, our data show that POM delays tumor growth in mice fed NCKD but may actually accelerate tumor growth in mice fed a Western diet. Prior pomegranate studies reporting delayed tumor growth used very low-fat diets, which we have also shown to slow tumor growth vs. a Western diet, while our findings suggest that dietary background may profoundly influence the effect of POM on PC growth and survival. This is especially important given that men with PC are more likely to consume a Western diet. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e583 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Chenwei Wu Durham, NC More articles by this author Elizabeth M. Masko Durham, NC More articles by this author Susan H. Poulton Durham, NC More articles by this author Tammy S. Ho Durham, NC More articles by this author Alexis R. Gaines Durham, NC More articles by this author Stephen J. Freedland Durham, NC More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

613 IMMEDIATE, COMPARED TO DEFERRED, COMBINED ANDROGEN BLOCKADE PROLONGS TIME TO CASTRATE-RESISTANT LNCAP PROSTATE CANCER PROGRESSION IN VIVO

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 2011613 IMMEDIATE, COMPARED TO DEFERRED, COMBINED ANDROGEN BLOCKADE PROLONGS TIME TO CASTRATE-RESISTANT LNCAP PROSTATE CANCER PROGRESSION IN VIVO Hidetoshi Kuruma, Kilian Gust, Hiroaki Matsumoto, Amina Zoubeidi, Chirstian Thomas, Francois Lamoureux, and Martin Gleave Hidetoshi KurumaHidetoshi Kuruma Vancouver, Canada More articles by this author , Kilian GustKilian Gust Vancouver, Canada More articles by this author , Hiroaki MatsumotoHiroaki Matsumoto Vancouver, Canada More articles by this author , Amina ZoubeidiAmina Zoubeidi Vancouver, Canada More articles by this author , Chirstian ThomasChirstian Thomas Vancouver, Canada More articles by this author , Francois LamoureuxFrancois Lamoureux Vancouver, Canada More articles by this author , and Martin GleaveMartin Gleave Vancouver, Canada More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1466AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Androgen deprivation therapy is standard for advanced prostate cancer (PCa). Combined androgen blockade (CAB) refers to surgical or medical castration plus an anti-androgen is considered maximum androgen blockade therapy. However, there is no consensus about optimal timing of anti-androgen induction after castration. In this study, we compared effects of immediate vs. delayed administration of bicalutamide after castration on time to castrate-resistant LNCaP tumor growth. METHODS Androgen dependent human prostate cancer LNCaP cells were inoculated into male athymic nude mice subcutaneously. Serum PSA level and tumour volumes were followed weekly. When serum PSA values reached 50ng/ml, mice were castrated and divided into 3 groups including immediate CAB, CAB after progression to castration resistance (deferred CAB), or vehicle-only dosing. 50mg/kg of bicalutamide was used as an anti- androgen. RESULTS Forty one mice entered this study. Averages of PSA and tumour volume were similar prior to castration in all three groups. In the immediate CAB group, PSA declined to −91.2% from baseline 2 weeks after CAB, compared to −64.6% for vehicle. In the deferred CAB group, PSA declined −50.4% by 1W after castration, and returned to pre-castration levels at 4 weeks. By 12 weeks post-castration, PSA levels were −29.0%, +197.0% and +118.1% from baseline for the immediate CAB, deferred CAB and vehicle groups, respectively. PSA levels in the immediate CAB group were significantly lower than deferred CAB (p=0.04 at 12 weeks). Tumor volume in the immediate CAB group decreased −10.2% at 2 weeks after CAB, while tumor volume in the deferred CAB and vehicle-only groups continued to increase post-castration. By 12 weeks after castration, tumor volume increased by +98.2%, +307.4% and +546.9% from base line for immediate CAB, deferred CAB and vehicle groups, respectively. CONCLUSIONS Immediate CAB, at time of first-line castration, prolonged time to recurrence compared deferred CAB in the androgen-sensitive LNCaP models, and has ongoing implications to the clinical disease as more potent anti-androgens are under development. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e247 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hidetoshi Kuruma Vancouver, Canada More articles by this author Kilian Gust Vancouver, Canada More articles by this author Hiroaki Matsumoto Vancouver, Canada More articles by this author Amina Zoubeidi Vancouver, Canada More articles by this author Chirstian Thomas Vancouver, Canada More articles by this author Francois Lamoureux Vancouver, Canada More articles by this author Martin Gleave Vancouver, Canada More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

1456 CENTRAL ROLE FOR PELP1-ANDROGEN RECEPTOR COMPLEX IN PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 20111456 CENTRAL ROLE FOR PELP1-ANDROGEN RECEPTOR COMPLEX IN PROSTATE CANCER Lin Yang, Preethi Ravindranath, and Ganesh Raj Lin YangLin Yang Dallas, TX More articles by this author , Preethi RavindranathPreethi Ravindranath Dallas, TX More articles by this author , and Ganesh RajGanesh Raj Dallas, TX More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1369AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Previously, we have shown that Estrogen Receptor beta (ER)β, PELP1 and Androgen Receptor (AR) can form a heterotrmeric protein complex in Prostate Cancer cells (PCa) in tissue culture. We had also shown that this protein complex plays a central role in the non-androgenic activation of AR in PCa. In this study, we examined whether ERβ, PELP1 and AR also formed a complex in vivo in both PCa cell lines, xenografts and human PCa tumors. METHODS Extracts from a large variety of PCa cell lines were prepared and evaluated by immunohistochemistry (IHC) and by co-immunoprecipitations (CO-IPs) for the presence of the heterotrimeric complex following treatment with estradiol (E2) or dihydrotestosterone (DHT). Then, following subcutaneously injection of Androgen-independent C4-2 Pca cells into 12 male nude mice and establishment of tumors, six mice were subject to surgical castration. Tumor samples from these mice were then harvested and extracts prepared to examine the formation of the trimeric complex before and after castration by IHC and CO-IP. In addition, fifty-eight samples from androgen dependent (ADPCa) and androgen independent human PCa (AIPCa) were screened for ERβ, PELP1 and AR expression by IHC and complex formation by CO-IP. RESULTS The complex between ERβ, PELP1 and AR was consistently detected in all PCa cell lines with AR. Treatment with DHT or E2 increased the detectability of the complex. In those PCa cell lines without AR, introduction of AR by transient or stable transfection restored the trimeric complex. The heterotrimeric complex between ERβ, PELP1 and AR was consistently detected PCa tumor samples from xenograft mice. Castration did not appear to significantly affect the detection of this trimeric complex in xenograft mice. In the human PCa tissue, PELP1 expression was increased significantly in AIPCa, while AR and ERβ expression was slightly decreased. Interestingly, while the complex between AR and PELP1 was detectable in both ADPCa and AIPCa samples, the level of the complex was significantly upregulated in AIPCa samples. CONCLUSIONS These data suggest that ERβ, PELP1 and AR form a heterotrimeric protein complex in PCa cells in vivo. This complex is present in all PCa cells with an intact AR. The complex appears to be upregulated up on androgenic and non-androgenic activation and in AIPCa Coupled with previous observations about the functional importance of this complex, these data appear to validate our approach to design agents targeting the formation of this complex. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e584 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Lin Yang Dallas, TX More articles by this author Preethi Ravindranath Dallas, TX More articles by this author Ganesh Raj Dallas, TX More articles by this author Expand All Advertisement Advertisement Loading ...

Antitumor effects of AR-42, a novel histone deacetylase inhibitor, in embryonal carcinoma.

232 Background: Histone deacetylase inhibitors (HDACIs) modulate gene expression and induce cellular differentiation, growth inhibition and apoptotic cell death by chromatin hyperacetylation. Developmental arrest of germ cell differentiation earlier in the life is responsible for the pathogenesis of germ cell tumors (GCT). With current treatment nearly 95% of patients with GCT can be cured. Yet, effective agents with less toxicity are desired. In addition, those with relapsed/refractory disease have a dismal prognosis, indicating a clear need for new, more effective agents. Here we assess the antitumor effects of AR-42, a novel HDACI in in vitro and in vivo models of embryonal carcinoma. Methods: In vitro effects of AR-42 and suberoylanilide hydroxamic acid (SAHA) were evaluated in NTERA-2, an embryonal carcinoma (EC) cell line derived from a human testicular cancer. Cell viability (MTS assay), apoptosis (caspase 3/7 activity and PARP cleavage), cell cycle analysis (flow cytometry) and HDAC inhibition (immunoblotting) were assessed. The in vivo efficacy of AR-42 was assessed in a NTERA-2 xenograft tumor model in male athymic nude mice. Mice were fed control diet and diet containing AR-42 at an average dose of 25 mg/kg/day. Tumor volumes and weights were used as in vivo endpoints. Results: Treatment of NTERA-2 cells with both agents at 0.1-10 μM concentrations showed a time- and dose-dependent reduction in cell viability. Both agents significantly induced apoptosis, cell cycle inhibition and hyperacetylation of histones H-3 and H-4 in a dose-dependent manner. In vitro studies showed that AR-42 was more potent than SAHA. In our rodent model, AR-42-containing diet resulted in a significant reduction in tumor volumes and weights (50% and 56%, respectively). The results for intratumoral changes of proliferation and apoptosis are pending. There were no significant toxicities associated with AR-42, except for testicular atrophy, known to be reversible. Conclusions: AR-42 appears to be a potent inhibitor of EC through different mechanisms, orally bioavailable and well tolerated in our rodent model. Our data indicates that AR- 42 may have clinical value in the treatment of GCT and requires further investigation in clinical trials. No significant financial relationships to disclose.

Blockade of transforming growth factor‐beta (TGFβ) signaling inhibits osteoblastic tumorigenesis by a novel human prostate cancer cell line

The skeleton is the most common site of prostate cancer metastasis, which often results in osteoblastic lesions. The role of transforming growth factor-beta (TGFβ) signaling in prostate cancer-induced osteoblastic metastasis is not clear. We investigated the role of TGFβ signaling in prostate cancer-induced bone metastasis using a novel human prostate cancer cell line, PacMetUT1. We injected PacMetUT1/Luc-GFP cells in male nude mice by intracardiac and intratibia injections and then investigated the effect of TGFβ signaling abrogation on osteoblastic tumor growth and incidence in vivo by using fluorescence and bioluminescence imaging analysis and quantifying bone and tumor volume by histomorphometry analysis. Osteoclasts were counted using TRAP assay. Osteoblastic bone metastasis in skull, rib, and femur was detected after 10-16 weeks of intracardiac injection of the PacMetUT1 cells. Stable knockdown of TGFβ1 with an shRNA resulted in decreased tumor incidence and bone formation when the cells were directly injected into the tibiae. Systemic administration of either a small inhibitor of TGFβ type I receptor kinase or a pan TGFβ binding protein (BG(E) RII) also decreased bone tumor growth and osteoblastic bone formation in vivo after 7 weeks of treatment. Our results for the first time indicate that blockade of TGFβ signaling in the PacMetUT1 model significantly inhibits osteoblastic bone formation and tumor incidence. Thus, TGFβ signaling pathway may be a viable target for the prevention and treatment of prostate cancer-induced bone metastasis.

Phosphorylated Akt up‐regulates angiotensin II type‐1 receptor expression in castration resistant prostate cancer

Accumulating evidences has suggested that the renin-angiotensin system (RAS) participates in the regulation of tumor angiogenesis. We previously demonstrated that castration-resistant prostate cancer (CRPC) showed significantly higher angiotensin II (Ang II) type-1 receptor (AT1R) expression, and that AT1R blockade (ARB) exerted protective effects by inhibiting angiogenesis. However, the detailed molecular mechanisms for the increase of AT1R expression in CRPC has not been fully elucidated yet. In this study we used C4-2 and C4-2AT6 cells, which were PTEN-null, androgen receptor (AR) positive, PSA-producing CRPC cell lines. We investigated the association between phosphorylated Akt (pAkt) and AT1R expression, and used LY294002 as a PI3K/Akt inhibitor. Western blot analysis revealed C4-2AT6 cells showed significantly higher pAkt expression than C4-2 cells, although there were no significant differences in total Akt (tAkt) expression. Immunohistochemical (IHC) analysis also revealed significant higher pAkt expression in C4-2AT6 tumors obtained from castrated male nude mice. These results indicated that C4-2AT6 cells acquired elevated pAkt status under androgen-ablated treatment in vitro. Treatment with LY294002 at the same dose reduced the viability of C4-2AT6 more effectively than that of C4-2, reflecting the dependency of cancer cells on PI3K/Akt pathway. The up-regulated AT1R expression in C4-2AT6 cells was reduced by LY294002 in a dose-dependent manner. On the other hand, in C4-2 cells, serum starvation induced pAkt up-regulation, which led to an increase of AT1R expression. These findings indicated that up-regulation of pAkt contributed to elevated AT1R expression in CRPC.

Poly(oligo-D-arginine) With Internal Disulfide Linkages as a Cytoplasm-sensitive Carrier for siRNA Delivery

Small interfering RNA (siRNA) has emerged as a therapeutic strategy for various diseases due to its target-specific gene silencing; however, its relatively high molecular weight, negative charge, and low stability hamper in vitro and in vivo applications. Approaches to overcome those drawbacks have relied on nonviral siRNA carriers based on cationic polymers or peptides. Nevertheless, cationic polymer-based siRNA carriers have yet to resolve intrinsic problems such as cytotoxicity and immunogenicity. An environment-sensitive carrier was recently proposed to enhance siRNA bioactivity and to reduce the carrier safety issues. Only a few studies, however, have shown cytoplasm-sensitive dissociation of the polyplex. In the present study, we clearly demonstrated decondensation of siRNA/poly(oligo-D-arginine) polyplex in the cytoplasm in response to intracellular glutathione (GSH) and the enhanced bioactivity of siRNA against VEGF (siVEGF) used as a model both in vitro and in an animal model. Reducible poly(oligo-D-arginine) (rPOA) rapidly dissociated in the cytoplasm, resulting in fast siRNA release to its target location while maintaining siRNA bioactivity both in vitro and in vivo.

Enhancement of blood porphyrin emission intensity with aminolevulinic acid administration: A new concept for photodynamic diagnosis of early prostate cancer

The objective of this paper was to verify if the oral administration of δ-aminolevulinic acid (ALA) in animals with prostate tumor can increase the sensitivity of cancer diagnosis by protoporphyrin IX blood autofluorescence. In this study, the autofluorescence of blood porphyrin was analyzed using fluorescence spectroscopy on healthy male NUDE mice and in those with prostate cancer induced by the inoculation of DU145 cells. A total of 18 male NUDE mice, ∼8 weeks old on arrival were divided into 3 groups: Control, Tumor and ALA Tumor. The autofluorescence of blood porphyrin of the groups was analyzed using fluorescence spectroscopy at different days after tumor induction, to monitor the tumor progression. Emission spectra were obtained by exciting the samples at 405 nm. The animals inoculated had their blood collected with and without oral ALA solution administration to compare PPIX endogenous (Tumor group) and exogenous (ALA Tumor group) signal intensity and to establish a method to diagnosis early prostate cancer. Significant differences were observed in autofluorescence intensities measured in the 575-725 nm spectral regions for the studied groups. The results showed an enhancement of almost 100% in blood PPIX fluorescence, using the oral administration of δ-aminolevulinic acid on male NUDE mice with prostate cancer, making fluorescence measurements more accurate and sensitive since the first week after tumor induction.

Xenografting of Human Fetal Testis Tissue: A New Approach to Study Fetal Testis Development and Germ Cell Differentiation

background: Abnormal fetal testis development can result in disorders of sex development (DSDs) and predispose to later testicular dysgenesis syndrome (TDS) disorders such as testicular germ cell tumours. Studies of human fetal testis development are hampered by the lack of appropriate model, and intervention systems. We hypothesized that human fetal testis xenografts can recapitulate normal development. methods: Human fetal testes (at 9 weeks, n ¼ 4 and 14 –18 weeks gestation, n ¼ 6) were xenografted into male nude mice for 6 weeks, with or without hCG treatment of the host, and evaluated for normal cellular development and function using immunohistochemistry, triple immunofluorescence and testosterone assay. The differentiation and proliferation status of germ cells within xenografts was quantified and compared with age-matched controls. results: Xenografts showed .75% survival with normal morphology. In the first-trimester xenografts seminiferous cord formation was initiated and in first- and second-trimester grafts normal functional development of Sertoli, Leydig and peritubular myoid cells was demonstrated using cell-specific protein markers. Grafts produced testosterone when hosts were treated with hCG (P ¼ 0.004 versus control). Proliferation of germ cells and differentiation from gonocytes (OCT4 + ) into pre-spermatogonia (VASA + ) occurred in grafts and quantification showed this progressed comparably with age-matched ungrafted controls. conclusions: Human fetal testis tissue xenografts demonstrate normal structure, function and development after xenografting, including normal germ cell differentiation. This provides an in vivo system to study normal human fetal testis development and its susceptibility to disruption by exogenous factors (e.g. environmental chemicals). This should provide mechanistic insight into the fetal origins of DSDs and TDS disorders.

Vitamin D deficiency promotes prostate cancer growth in bone

Vitamin D is considered as an important determinant of bone turnover as well as cancer growth. Using a murine model of bone metastasis, we investigated the effect of vitamin D deficiency on prostate cancer cell growth in bone. Three-week-old male nude mice were fed either normal chow (control) or a diet deficient in vitamin D. The latter diet resulted in severe hypovitaminosis D within 6 weeks. At this point of time, 5 × 10(4) cells of the prostate cancer cell line, PC-3, were injected either into the bone marrow (tibia) or subcutaneously into soft tissues. Osteoprotegerin (OPG) was co-administered in subgroups of mice to suppress bone remodeling. Osteolytic lesions were monitored by serial X-ray, while soft tissue tumor growth was measured by caliper. All tissues were analyzed by micro-CT and histology at endpoint. Bone turnover was significantly accelerated in vitamin D deficient compared to vitamin D sufficient mice from week 6 onwards. Intra-tibially implanted PC-3 cells resulted in mixed osteolytic and osteosclerotic lesion. At endpoint, osteolytic and osteosclerotic lesion areas, total tumor area, and tumor mitotic activity were all significantly increased in vitamin D deficient mice compared to controls. Regardless of diet, OPG reduced bone turnover, total tumor, and osteosclerotic area as well as tumor mitotic activity, while promoting cell apoptosis. In contrast, vitamin D deficiency did not alter tumor growth in soft tissues. Vitamin D deficiency stimulates prostate cancer growth in bone through modulating the bone microenvironment.

MR-guided focused ultrasound: enhancement of intratumoral uptake of [3H]-docetaxel in vivo

The purpose of this study is to quantify the enhancement of [3H]-docetaxel in implanted prostate tumors treated with MR-guided pulsed focused ultrasound (MRgFUS). Human prostate cancer, LNCaP cells in 25 µl, were implanted into the prostates of male nude mice. The tumor growth was directly monitored on MRI. When the tumor reached a designated size, MRgFUS treatment was performed using a focused ultrasound treatment system (InSightec ExAblate 2000) with a 1.5 T GE MR scanner. The tumor-bearing animals were randomly divided into three groups: group 1, MRgFUS treatment + [3H]-docetaxel; group 2, [3H]-docetaxel only and group 3, as a control. Animals in group 1 were treated with MRgFUS non-invasively. Immediately after the treatment, the animals received a single dose of tail vein injection of docetaxel at 15 mg kg−1 mixed with [3H]-docetaxel at 50 uCi kg−1 in a total volume of 150 µl. Animals in group 2 were treated the same as in group one, however without MRgFUS treatment. Animals in group 3 were treated as a control. Animals were sacrificed 30 min after i.v. injections regardless of whether or not they received focused ultrasound. Tumors were removed and processed. The radioactivity of [3H]-docetaxel in the tumor tissue was quantitatively measured by a liquid scintillation counter. Our study showed that all animals tolerated the MRgFUS treatment well. Our data showed increased 3H-docetaxel concentration in the tumor in the MRgFUS-treated group (1079 ± 132 cmp/75 mg) versus those without MRgFUS treatment (524 ± 201 cmp/75 mg) with P = 0.037.

Alginate Microencapsulation Technology for the Percutaneous Delivery of Adipose-Derived Stem Cells

Autologous fat is the ideal soft-tissue filler; however, its widespread application is limited because of variable clinical results and poor survival. Engineered fillers have the potential to maximize survival. Alginate is a hydrogel copolymer that can be engineered into spheres of <200 μm, thus facilitating mass transfer, allowing for subcutaneous injection, and protecting cells from shearing forces. Alginate powder was dissolved in saline, and adipose-derived stem cells (ADSCs) were encapsulated (1 million cells/mL) in alginate using an electrostatic bead generator. To assess effects of injection on cell viability, microspheres containing ADSCs were separated into 2 groups: the control group was decanted into culture wells and the injection group was mixed with basal media and injected through a 21-gauge needle into culture wells. Microbeads were cultured for 3 weeks, and cell number and viability were measured weekly using electron and confocal microscopy. To assess effects of percutaneous injection in vivo, twenty-four male nude mice were randomly separated into 2 groups and injected with either empty microcapsules or ADSC-laden microcapsules. Mice were harvested at 1 and 3 months, and the implants were examined microscopically to assess bead and cell viability. A flow rate of 5 mL/h and an electrostatic potential of 7 kV produced viable ADSC-laden microbeads of <200 μm. There were no differences in bead morphology and ADSC viability between microcapsules placed versus injected into tissue culture plates for up to 3 weeks. Microspheres implanted in a nude mouse model show durability up to 3 months with a host response around each individual sphere. ADSCs remained viable and showed signs of mitosis. ADSCs can be readily cultured, encapsulated, and injected in alginate microspheres. Stem cells suspended in alginate microspheres survive in vivo and are seen to replicate in vitro.

구강암 세포주를 이종 이식한 누드마우스에서 apigenin의 경구투여에 따른 항암효능에 관한 연구

Apigenin은 과일과 야채에 들어있는 플라보노이드로 다양한 악성 세포에 항증식효과를 보여준다. 세포성장 저해효과를 확인하기 위하여 KB 구강암세포주를 96 well plate에 <TEX>$6{\times}10^3$</TEX> cells/well로 분주하고 24시간 후에 apigenin을 24시간 동안 처치하여 MTT assay를 수행하였다. Apigenin은 배양 후 용량 의존적으로 세포사를 유도하였다. Apigenin 100 <TEX>${\mu}M$</TEX>을 24시간 동안 처치하고 대조군과 세포성장을 비교하였을 때 유의적인 감소를 확인하였다. KB 구강암세포주에서의 apoptosis를 확인하기 위해 DAPI 염색을 수행하였다. Apigenin을 처치한 세포에서 핵의 응축이 존재함을 형광현미경으로 확인하였다. 우리는 누드마우스에 KB 구강암세포주를 이식하여 세포 성장 억제 효과를 알아보았다. Apigenin을 마우스에 25, 50 mg/kg을 0.2 ml의 PBS에 녹여 경구투여 하였다. 종양 사이즈는 대조군과 25, 50 mg/kg apigenin 투여군을 비교하였을 때 유의적으로 감소하였다. Apoptosis 분석을 위해 TUNEL염색을 수행하였다. 25 mg/kg apigenin 투여군과 대조군을 비교하였을 때 apoptosis의 유의적인 증가를 확인하였다. 육안적 소견을 위한 H&E 염색은 이상이 없었다. 본 연구는 apigenin이 구강암세포주 성장 억제를 apoptosis의 유도를 통하여 확인하였다. Apigenin (4', 5, 7-trihydroxyflavone), a common dietary flavonoid abundantly present in fruits and vegetables, has shown remarkable anti-proliferative effects against various malignant cell lines. To observe the anti-proliferative effects, oral cavity cancer cell lines, <TEX>$6{\times}10^3$</TEX> cells/well (96 well plate) of KB oral cavity tumor cells were plated and 24 hr later treated with apigenin for one day, after which MTT assay was performed. Apigenin induced cell death in a dose-dependent manner after incubation. Cell viability was significantly decreased in the group treated with 100 <TEX>${\mu}M$</TEX> apigenin for 24 hr (p<0.05) compared to the control group. To assess apoptosis, the nuclei of KB cells were stained with DAPI. The presence of chromatin condensation in the apigenin treated cells was detected on a fluorescent microscope (<TEX>${\times}200$</TEX>). We investigated the in vivo growth inhibitory effects of apigenin on oral cavity cancer KB tumor xenograft subcutaneously implanted in male nude mice. Apigenin was administered to mice by gavage at doses of 25 and 50 mg/kg/day in 0.2ml of PBS. Tumor volume was significantly decreased in 25 and 50 mg/kg apigenin-administration groups compared to the control group. For apoptosis analysis, TUNEL staining was performed. A significant increase in TUNEL positive cells was found in the 25 mg/kg apigenin administration group compared to the non- apigenin administration group. Histopathological changes were not observed. These results indicate that apigenin inhibits oral cavity cancer cell growth through the induction of apoptosis.

Experiment on enhancing antitumor effect of intravenous epirubicin hydrochloride by acoustic cavitation in situ combined with phospholipid-based microbubbles

An in vivo study on enhancing antitumor effect of intravenous epirubicin hydrochloride (EPI) by acoustic cavitation in situ combined with phospholipid-based microbubbles (PMB) was reported. Five-week-old male nude mice were used, and HCT-116 cells were s.c. (subcutaneous injection) inoculated in axilla of these mice. Five groups were designed and five consecutive treatments were applied to investigate the tumor growth, body weight growth, and normal organ growth. Inhibition effects on tumor growth were observed in all groups treated by EPI. However, the potency of tumor growth retardation caused by EPI depended on the application of PMB and US in situ. An effective retardation on tumor growth and improved mice survival status were observed when intravenous EPI combined with sonicated PMB in situ. Short-term lingering sensitive period for chemotherapeutic drugs after acoustic cavitation was also observed in experiment under asynchronous administration of EPI and sonicated PMB. Intravenous EPI combined with PMB-mediated cavitation in situ might provide a potential application for US-mediated chemotherapy.

Exogenous stimulations change nude mouse hair cycle pattern

The pattern of murine hair growth has been seen as an unpredictable and irregular process. In this study, nude mice were used to investigate the hair growth pattern and find the impact of exogenous stimulations on changing the hair growth pattern. We found nude mouse hair appeared in waves from the head to the posterior part of the back for the first time. Amongst all of the six groups, male nude mice had a more regular hair cycle pattern than females: from the head to the posterior part of the back. When there was no hair on the back of a nude mouse, we named this time the ‘no-hair phase’ and the opposite was the ‘hair-existing phase’. Exogenous stimulations significantly elongated the hair-existing time and shortened the no-hair time but did not work on the hair growth pattern. For male mice, topical application of minoxidil created a shorter no-hair phase and a longer hair-existing phase than other treatment methods. For female nude mice, minoxidil had little more effect than a wound in shortening the no-hair phase. A wound was better than minoxidil in elongating the hair-existing phase in female nude mice, and this effect was indistinctive.

Using CD133 positive U251 glioblastoma stem cells to establish nude mice model of transplanted tumor

This study tried to use CD133 positive U251 glioblastoma stem cells to establish nude mice model of transplanted tumor. CD133 positive U251 stem cells were isolated and identified. Twenty-five male nude mice were divided into three groups: U251 cell group, U251 stem cell group and normal control group. U251 cells and stem cells were respectively inoculated into mouse brain to establish model of transplanted tumor. Mice growing condition and behavior were observed. Magnetic resonance imaging (MRI) was performed to detect tumor growth in brain. HE staining assay, immunohistochemical assay and CD34 marked microvascular density (MVD) test were performed. As a result, the successful rates of both model groups were 100%. Growing condition and behavior in U251 stem cell group was more significantly exaggerated and tumor growth and image in magnetic resonance in U251 stem cell group was more significantly malignant than that in U251 cell group. It could be found that models in U251 stem cell group showed stronger pathogolical malignance features than that in U251 cell group. Glial fibrillary acidic protein (GFAP) expression in U251 cell and stem cell group showed the transplanted tumor originated from astrocytes. CD34 in U251 stem cell group was highest and significantly higher than that in the other two groups. In conclusion, use of U251 stem cells to establish transplanted tumor model in nude mice is an excellent method and the model is more likely and malignant than the one established by cancer cells, which showed a new animal model to research glioma.

Pre-conditioning cryosurgery: Cellular and molecular mechanisms and dynamics of TNF-α enhanced cryotherapy in an in vivo prostate cancer model system

Cryosurgery is increasingly being used to treat prostate cancer; however, a major limitation is local recurrence of disease within the previously frozen tissue. We have recently demonstrated that tumor necrosis factor alpha (TNF-α), given 4 h prior to cryosurgery can yield complete destruction of prostate cancer within a cryosurgical iceball. The present work continues the investigation of the cellular and molecular mechanisms and dynamics of TNF-α enhancement on cryosurgery. In vivo prostate tumor (LNCaP Pro 5) was grown in a dorsal skin fold chamber (DSFC) on a male nude mouse. Intravital imaging, thermography, and post-sacrifice histology and immunohistochemistry were used to assess iceball location and the ensuing biological effects after cryosurgery with and without TNF-α pre-treatment. Destruction was specifically measured by vascular stasis and by the size of histologic zones of injury (i.e., inflammatory infiltrate and necrosis). TNF-α induced vascular pre-conditioning events that peaked at 4 h and diminished over several days. Early events (4–24 h) include upregulation of inflammatory markers (nuclear factor-κB (NFκB) and vascular cell adhesion molecule-1 (VCAM)) and caspase activity in the tumor prior to cryosurgery. TNF-α pre-conditioning resulted in recruitment of an augmented inflammatory infiltrate at day 3 post treatment vs. cryosurgery alone. Finally, pre-conditioning yielded enhanced cryosurgical destruction up to the iceball edge at days 1 and 3 vs. cryosurgery alone. Thus, TNF-α pre-conditioning enhances cryosurgical lesions by vascular mechanisms that lead to tumor cell injury via promotion of inflammation and leukocyte (esp. neutrophil) recruitment.

WSS25 Inhibits Growth of Xenografted Hepatocellular Cancer Cells in Nude Mice by Disrupting Angiogenesis via Blocking Bone Morphogenetic Protein (BMP)/Smad/Id1 Signaling

The highly expressed Id1 (inhibitor of DNA binding/differentiation) protein promotes angiogenesis in HCC and is a well established target for anti-angiogenesis therapeutic strategies. Heparan sulfate (HS) mimetics such as PI-88 can abrogate HS-protein interactions to inhibit angiogenesis. Id1 is the direct downstream effector of bone morphogenetic proteins (BMPs), which are angiogenic and HS-binding proteins. Thus, targeting BMPs by HS mimetics may inhibit angiogenesis via attenuating Id1 expression. We report here that a HS mimetic WSS25 potently inhibited the tube formation of HMEC-1 cells on Matrigel and their migration. Meanwhile, WSS25 (25 μg/ml) nearly completely blocked Id1 expression in the HMEC-1 cells as demonstrated by oligo-angiogenesis microarray analysis and further confirmed by RT-PCR and Western blotting. BMP/Smad/Id1 signaling also was blocked by WSS25 treatment in HMEC-1 cells. Importantly, Id1 knockdown in HMEC-1 cells caused the disruption of their tube formation on Matrigel. By employing quartz crystal microbalance analysis, we found that WSS25 strongly bound to BMP2. Moreover, WSS25 impaired BMP2-induced tube formation of HMEC-1 cells on Matrigel and angiogenesis in Matrigel transplanted into C57BL6 mice. Furthermore, WSS25 (100 mg/kg) abrogated the growth of HCC cells xenografted in male nude mice. Immunohistochemical analysis showed that both the expression of Id1 and the endothelial cell marker CD31 were lower in the WSS25-treated tumor tissue than in the control. Therefore, WSS25 is a potential drug candidate for HCC therapy as a tumor angiogenesis inhibitor.

E0009 Multimodality molecular imaging of adipose-derived mesenchymal stem cells with VEGF in hindlimb ischaemia mice

BackgroundPeripheral arterial disease (PAD) is highly prevalent and particularly in elders, smokers, diabetics or patients with systemic atherosclerosis. Apart from the surgery and medication, stem cell transplantation offers promising approaches...

MiR‐125b promotes growth of prostate cancer xenograft tumor through targeting pro‐apoptotic genes

Increasing evidence demonstrates that aberrantly regulated microRNAs (miRNAs) contribute to the initiation and progression of human cancer. We previously have demonstrated that miR-125b stimulated the growth of prostate cancer (CaP) cells. In this study, we further determined the influence of miR-125b on the pathogenesis of CaP. To evaluate the effect of miR-125b on xenograft tumor growth, male athymic mice were subcutaneously injected with PC-346C-miR-125b cells that stably overexpressed miR-125b. Potential direct target transcripts of miR-125b were identified using a bioinformatics approach and three miR-125b targeted molecules were confirmed by means of biochemical analyses. Enforced expression of miR-125b promoted tumor growth in both intact and castrated male nude mice. In an effort to define the molecular mechanism(s) mediating its tumor growth properties, we found that miR-125b directly targets eight transcripts, including three key pro-apoptotic genes: p53, Puma, and Bak1. Increasing the abundance of miR-125b resulted in a dramatic decrease in the levels of these three proteins in CaP cells. A direct repressive effect on each of these was supported by the ability of miR-125b to significantly reduce the activity of luciferase reporters containing their 3'-untranslated regions of each gene encompassing the miR-125b-binding sites. Additionally, we found that repression of miR-125b activity was able to sensitize CaP cells to different therapeutic interventions. Data obtained in this study demonstrate that miR-125b promotes growth of prostatic xenograft tumors by down-regulating three key pro-apoptotic genes. This suggests that miR-125b is oncogenic and makes it an attractive therapeutic target in CaP.