Detection of carcinogen-induced bladder cancer by fluorocoxib A.

BACKGROUND: Conventional cystoscopy can detect advanced stages of bladder cancer; however, it has limitations to detect bladder cancer at the early stages. Fluorocoxib A, a rhodamine-conjugated analog of indomethacin, is a novel fluorescent imaging agent that selectively targets cyclooxygenase-2 (COX-2)-expressing cancers. METHODS: In this study, we have used a carcinogen N-butyl-N-4-hydroxybutyl nitrosamine (BBN)-induced bladder cancer immunocompetent mouse B6D2F1 model that resembles human high-grade invasive urothelial carcinoma. We evaluated the ability of fluorocoxib A to detect the progression of carcinogen-induced bladder cancer in mice. Fluorocoxib A uptake by bladder tumors was detected ex vivo using IVIS optical imaging system and Cox-2 expression was confirmed by immunohistochemistry and western blotting analysis. After ex vivo imaging, the progression of bladder carcinogenesis from normal urothelium to hyperplasia, carcinoma-in-situ and carcinoma with increased Ki67 and decreased uroplakin-1A expression was confirmed by histology and immunohistochemistry analysis. RESULTS: The specific uptake of fluorocoxib A correlated with increased Cox-2 expression in progressing bladder cancer. In conclusion, fluorocoxib A detected the progression of bladder carcinogenesis in a mouse model with selective uptake in Cox-2-expressing bladder hyperplasia, CIS and carcinoma by 4- and 8-fold, respectively, as compared to normal bladder urothelium, where no fluorocoxib A was detected. CONCLUSIONS: Fluorocoxib A is a targeted optical imaging agent that could be applied for the detection of Cox-2 expressing human bladder cancer.

Platelet-rich plasma affects the proliferation of canine bone marrow-derived mesenchymal stromal cells in vitro.

BACKGROUND: Reported efficacy of platelet-rich plasma (PRP) in regenerative medicine is contradictory. We validated the effects of PRP on proliferation of canine bone marrow-derived multipotent mesenchymal stromal cells (K9BMMSCs) in vitro. PRP was extracted from blood of six dogs with osteoarthritis. K9BMMSCs were established from bone marrow and characterized for CD90 and CD19 expression by immunocytochemistry. Effects of PRP concentrations on viability of matching autologous K9BMMSCs were validated using MTS assay. RESULTS: Positive CD90 and negative CD19 expression confirmed MSC origin. PRP at 40% volume/volume concentration increased, while PRP at 80 and 100% v/v concentrations suppressed viability of tested K9BMMSCs. CONCLUSION: PRP concentration plays an important role in K9BMMSCs viability, which could affect tissue repairs in vivo.

Inhibition of the PI3K/AKT Pathway Sensitizes Oral Squamous Cell Carcinoma Cells to Anthracycline-Based Chemotherapy In Vitro.

Anthracycline-based chemotherapy, such as doxorubicin (Dox), while effective against many solid tumors, is not widely used for head and neck cancers. In this study, we evaluated the efficacy of Dox, and its derivative AD198 in human, canine, and feline oral squamous cell carcinomas cells (OSCC) in vitro. Dox and AD198 had significant an anti-proliferative effect on human, canine, and feline OSCC cells in dose-dependent manner. AD198 inhibited cell proliferation more effectively than Dox in tested OSCC cells. In the human oral squamous cell carcinoma SCC25 cells, Dox and AD198 increased the production of reactive oxygen species and subsequently increased apoptosis through activation of caspase signaling pathway. Dox and AD198 increased activation of AKT, ERK1/2, and p38 MAPK signaling pathways in tested OSCC cells by dose-dependent manner. The efficacy of Dox and AD198 treatments in inhibition of cell proliferation was increased in tested OSCC when combined with PI3K/AKT inhibitor, LY294002 treatment. Inhibition of PI3K/AKT reduced Dox- and AD198-induced activation of ERK1/2 and further increased Dox- and AD198-induced phosphorylation of p38 MAPK in OSCC. Our results suggest that the anthracycline therapies, such as Dox or AD198, can be more effective for treatment of OSCC when combined with inhibitors of the PI3K/AKT pathway. J. Cell. Biochem. 118: 2615-2624, 2017. © 2016 Wiley Periodicals, Inc.

BCL-2 family protein, BAD is down-regulated in breast cancer and inhibits cell invasion.

We have previously demonstrated that the anti-apoptotic protein BAD is expressed in normal human breast tissue and shown that BAD inhibits expression of cyclin D1 to delay cell-cycle progression in breast cancer cells. Herein, expression of proteins in breast tissues was studied by immunohistochemistry and results were analyzed statistically to obtain semi-quantitative data. Biochemical and functional changes in BAD-overexpressing MCF7 breast cancer cells were evaluated using PCR, reporter assays, western blotting, ELISA and extracellular matrix invasion assays. Compared to normal tissues, Grade II breast cancers expressed low total/phosphorylated forms of BAD in both cytoplasmic and nuclear compartments. BAD overexpression decreased the expression of ß-catenin, Sp1, and phosphorylation of STATs. BAD inhibited Ras/MEK/ERK and JNK signaling pathways, without affecting the p38 signaling pathway. Expression of the metastasis-related proteins, MMP10, VEGF, SNAIL, CXCR4, E-cadherin and TlMP2 was regulated by BAD with concomitant inhibition of extracellular matrix invasion. Inhibition of BAD by siRNA increased invasion and Akt/p-Akt levels. Clinical data and the results herein suggest that in addition to the effect on apoptosis, BAD conveys anti-metastatic effects and is a valuable prognostic marker in breast cancer.

Phosphatidylinositol- 3-kinase inhibitor induces chemosensitivity to a novel derivative of doxorubicin, AD198 chemotherapy in human bladder cancer cells in vitro.

BACKGROUND: Doxorubicin (Dox) is widely used to treat progressed bladder cancer after transurethral resection. The use of Dox-chemotherapy has been limited due to induced drug resistance and cumulative cardiotoxic effects. N-benzyladriamycin-14-valerate (AD198), a novel derivative of Dox, has a potential to become a more effective treatment than Dox by overcoming drug resistance and cardio-toxicity as shown in the rodent model of lymphoma in vivo. The purpose of this study was to compare the efficacy of Dox and AD198 and explore their mechanisms in inhibition on human bladder cancer cells in vitro. METHODS: We evaluated the effects of Dox and AD198 on cell viability of human transitional cell carcinoma (TCC) cell lines T24 and UMUC3 by MTS assay in vitro. The effects of Dox and AD198 on cell apoptosis were determined by caspase 3/7 assay, generation of reactive oxygen species (ROS), and Western Blotting (WB) analysis. RESULTS: AD198 was more effective than Dox in inhibition of cell viability of T24 and UMUC3 cells in vitro. Both Dox and AD198 significantly increased the generation of ROS and induced apoptosis in caspase-dependent and -independent manner in T24 and UMUC3 cells. AD 198 induced significantly higher production of ROS as compared to Dox in human TCC cells. Dox and AD198 activated the pro-apoptotic p38 MAPK pathway; however, on the other hand also increased phosphorylation of AKT, an anti-apoptotic signaling pathway, in T24 and UMUC3 cells. Combined treatment of PI3K inhibitor (LY294002) with Dox or AD198 inhibited cell viability of T24 and UMUC3 cells more effectively than any of drug treatments alone. CONCLUSIONS: These data suggest that AD198 as novel derivative of Dox, could be a used as effective treatment for bladder cancer. Dox and AD198 induced PI3K/AKT signaling pathway that is a one of the indicators of pro-survival and possible drug-resistance mechanisms of chemotherapies in bladder cancer. Combined therapies of Dox or AD198 with inhibitors of PI3K/AKT signaling pathway might lead to more effective treatment outcome for patients diagnosed with bladder cancer based on our in vitro experiments.

Animal model of naturally occurring bladder cancer: characterization of four new canine transitional cell carcinoma cell lines.

BACKGROUND: Development and further characterization of animal models for human cancers is important for the improvement of cancer detection and therapy. Canine bladder cancer closely resembles human bladder cancer in many aspects. In this study, we isolated and characterized four primary transitional cell carcinoma (K9TCC) cell lines to be used for future in vitro validation of novel therapeutic agents for bladder cancer. METHODS: Four K9TCC cell lines were established from naturally-occurring canine bladder cancers obtained from four dogs. Cell proliferation rates of K9TCC cells in vitro were characterized by doubling time. The expression profile of cell-cycle proteins, cytokeratin, E-cadherin, COX-2, PDGFR, VEGFR, and EGFR were evaluated by immunocytochemistry (ICC) and Western blotting (WB) analysis and compared with established human bladder TCC cell lines, T24 and UMUC-3. All tested K9TCC cell lines were assessed for tumorigenic behavior using athymic mice in vivo. RESULTS: Four established K9TCC cell lines: K9TCC#1Lillie, K9TCC#2Dakota, K9TCC#4Molly, and K9TCC#5Lilly were confirmed to have an epithelial-cell origin by morphology analysis, cytokeratin, and E-cadherin expressions. The tested K9TCC cells expressed UPIa (a specific marker of the urothelial cells), COX-2, PDGFR, and EGFR; however they lacked the expression of VEGFR. All tested K9TCC cell lines confirmed a tumorigenic behavior in athymic mice with 100% tumor incidence. CONCLUSIONS: The established K9TCC cell lines (K9TCC#1Lillie, K9TCC#2Dakota, K9TCC#4Molly, and K9TCC#5Lilly) can be further utilized to assist in development of new target-specific imaging and therapeutic agents for canine and human bladder cancer.

A machine learning decision criterion for reducing scan time for hyperspectral neutron computed tomography systems.

We present the first machine learning-based autonomous hyperspectral neutron computed tomography experiment performed at the Spallation Neutron Source. Hyperspectral neutron computed tomography allows the characterization of samples by enabling the reconstruction of crystallographic information and elemental/isotopic composition of objects relevant to materials science. High quality reconstructions using traditional algorithms such as the filtered back projection require a high signal-to-noise ratio across a wide wavelength range combined with a large number of projections. This results in scan times of several days to acquire hundreds of hyperspectral projections, during which end users have minimal feedback. To address these challenges, a golden ratio scanning protocol combined with model-based image reconstruction algorithms have been proposed. This novel approach enables high quality real-time reconstructions from streaming experimental data, thus providing feedback to users, while requiring fewer yet a fixed number of projections compared to the filtered back projection method. In this paper, we propose a novel machine learning criterion that can terminate a streaming neutron tomography scan once sufficient information is obtained based on the current set of measurements. Our decision criterion uses a quality score which combines a reference-free image quality metric computed using a pre-trained deep neural network with a metric that measures differences between consecutive reconstructions. The results show that our method can reduce the measurement time by approximately a factor of five compared to a baseline method based on filtered back projection for the samples we studied while automatically terminating the scans.

The Complex, Unique, and Powerful Imaging Instrument for Dynamics (CUPI2D) at the Spallation Neutron Source (invited).

The Oak Ridge National Laboratory is planning to build the Second Target Station (STS) at the Spallation Neutron Source (SNS). STS will host a suite of novel instruments that complement the First Target Station's beamline capabilities by offering an increased flux for cold neutrons and a broader wavelength bandwidth. A novel neutron imaging beamline, named the Complex, Unique, and Powerful Imaging Instrument for Dynamics (CUPI2D), is among the first eight instruments that will be commissioned at STS as part of the construction project. CUPI2D is designed for a broad range of neutron imaging scientific applications, such as energy storage and conversion (batteries and fuel cells), materials science and engineering (additive manufacturing, superalloys, and archaeometry), nuclear materials (novel cladding materials, nuclear fuel, and moderators), cementitious materials, biology/medical/dental applications (regenerative medicine and cancer), and life sciences (plant-soil interactions and nutrient dynamics). The innovation of this instrument lies in the utilization of a high flux of wavelength-separated cold neutrons to perform real time in situ neutron grating interferometry and Bragg edge imaging-with a wavelength resolution of δλ/λ ≈ 0.3%-simultaneously when required, across a broad range of length and time scales. This manuscript briefly describes the science enabled at CUPI2D based on its unique capabilities. The preliminary beamline performance, a design concept, and future development requirements are also presented.

Neutron Radiography and Computed Tomography of Biological Systems at the Oak Ridge National Laboratory's High Flux Isotope Reactor.

Neutrons have historically been used for a broad range of biological applications employing techniques such as small-angle neutron scattering, neutron spin echo, diffraction, and inelastic scattering. Unlike neutron scattering techniques that obtain information in reciprocal space, attenuation-based neutron imaging measures a signal in real space that is resolved on the order of tens of micrometers. The principle of neutron imaging follows the Beer-Lambert law and is based on the measurement of the bulk neutron attenuation through a sample. Greater attenuation is exhibited by some light elements (most notably, hydrogen), which are major components of biological samples. Contrast agents such as deuterium, gadolinium, or lithium compounds can be used to enhance contrast in a similar fashion as it is done in medical imaging, including techniques such as optical imaging, magnetic resonance imaging, X-ray, and positron emission tomography. For biological systems, neutron radiography and computed tomography have increasingly been used to investigate the complexity of the underground plant root network, its interaction with soils, and the dynamics of water flux in situ. Moreover, efforts to understand contrast details in animal samples, such as soft tissues and bones, have been explored. This manuscript focuses on the advances in neutron bioimaging such as sample preparation, instrumentation, data acquisition strategy, and data analysis using the High Flux Isotope Reactor CG-1D neutron imaging beamline. The aforementioned capabilities will be illustrated using a selection of examples in plant physiology (herbaceous plant/root/soil system) and biomedical applications (rat femur and mouse lung).

Pharmacokinetic characterization of fluorocoxib D, a cyclooxygenase-2-targeted optical imaging agent for detection of cancer.

SIGNIFICANCE: Fluorocoxib D, N-[(rhodamin-X-yl)but-4-yl]-2-[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetamide, is a water-soluble optical imaging agent to detect cyclooxygenase-2 (COX-2)-expressing cancer cells. AIM: We evaluated the pharmacokinetic and safety properties of fluorocoxib D and its ability to detect cancer cells in vitro and in vivo. APPROACH: Pharmacokinetic parameters of fluorocoxib D were assessed from plasma collected at designated time points after intravenous administration of 1 mg / kg fluorocoxib D in six research dogs using a high-performance liquid chromatography analysis. Safety of fluorocoxib D was assessed for 3 days after its administration using physical assessment, complete blood count, serum chemistry profile, and complete urinalysis in six research dogs. The ability of fluorocoxib D to detect COX-2-expressing cancer cells was performed using human 5637 cells in vitro and during rhinoscopy evaluation of specific fluorocoxib D uptake by canine cancer cells in vivo. RESULTS: No evidence of toxicity and no clinically relevant adverse events were noted in dogs. Peak concentration of fluorocoxib D (114.8 ± 50.5 ng / ml) was detected in plasma collected at 0.5 h after its administration. Pretreatment of celecoxib blocked specific uptake of fluorocoxib D in COX-2-expressing human 5637 cancer cells. Fluorocoxib D uptake was detected in histology-confirmed COX-2-expressing head and neck cancer during rhinoscopy in a client-owned dog in vivo. Specific tumor-to-normal tissue ratio of detected fluorocoxib D signal was in an average of 3.7 ± 0.9 using Image J analysis. CONCLUSIONS: Our results suggest that fluorocoxib D is a safe optical imaging agent used for detection of COX-2-expressing cancers and their margins during image-guided minimally invasive biopsy and surgical procedures.

Peroxisome proliferator-activated receptor ligand MCC-555 suppresses intestinal polyps in ApcMin/+ mice via extracellular signal-regulated kinase and peroxisome proliferator-activated receptor-dependent pathways.

A large body of studies has suggested that peroxisome proliferator-activated receptor gamma (PPARgamma) ligands, such as thiazolidinedione, are potent candidates for chemopreventive agents. MCC-555 is a PPARgamma/alpha dual agonist and has been shown previously to induce apoptosis in vitro; however, the molecular mechanisms by which MCC-555 affects antitumorigenesis in vivo are poorly understood. In this study, we explored the antitumorigenic effects of MCC-555 both in cell culture and in Apc-deficient mice, an animal model for human familial adenomatous polyposis. MCC-555 increased MUC2 expression in colorectal and lung cancer cells, and treatment with the PPARgamma antagonist GW9662 revealed that MUC2 induction by MCC-555 was mediated in a PPARgamma-dependent manner. Moreover, MCC-555 increased transcriptional activity of human and mouse MUC2 promoters. Subsequently, treatment with MCC-555 (30 mg/kg/d) for 4 weeks reduced the number of small intestinal polyps to 54.8% of that in control mice. In agreement with in vitro studies, enhanced Muc2 expression was observed in the small intestinal tumors of Min mice treated with MCC-555, suggesting that MUC2 expression may be associated at least in part with the antitumorigenic action of MCC-555. In addition, highly phosphorylated extracellular signal-regulated kinase (ERK) was found in the intestinal tumors of MCC-555-treated Min mice, and inhibition of the ERK pathway by a specific inhibitor markedly suppressed MCC-555-induced Muc2 expression in vitro. Overall, these results indicate that MCC-555 has a potent tumor suppressor activity in intestinal tumorigenesis, likely involving MUC2 up-regulation by ERK and PPARgamma pathways.

Detection of tyrosine kinase inhibitors-induced COX-2 expression in bladder cancer by fluorocoxib A.

Among challenges of targeted therapies is the activation of alternative pro-survival signaling pathways in cancer cells, resulting in an acquired drug resistance. Cyclooxygenase-2 (COX-2) is overexpressed in bladder cancer cells, making it an attractive molecular target for the detection and treatment of cancer. Fluorocoxib A is an optical imaging agent that selectively targets COX-2. In this study, we evaluated the ability of fluorocoxib A to monitor the responses of bladder cancer to targeted therapies in vivo. The effects of several tyrosine kinase inhibitors (TKIs: axitinib, AB1010, toceranib, imatinib, erlotinib, gefitinib, imatinib, sorafenib, vandetanib, SP600125, UO126, and AZD 5438) on COX-2 expression were validated in ten human and canine bladder cancer cell lines (J82, RT4, T24, UM-UC-3, 5637, SW780, TCCSUP, K9TCC#1Lillie, K9TCC#2Dakota, K9TCC#5Lilly) in vitro. The effects of TKIs on bladder cancer in vivo were evaluated using the COX-2-expressing K9TCC#5Lilly xenograft mouse model and detected by fluorocoxib A. The increased COX-2 expression was detected by all tested TKIs in at least one of the tested COX-2-expressing bladder cancer cell lines (5637, SW780, TCCSUP, K9TCC#1Lillie, K9TCC#2Dakota, and K9TCC#5Lilly) in vitro. In addition, fluorocoxib A uptake correlated with the AB1010- and imatinib-induced COX-2 expression in the K9TCC#5Lilly xenografts in vivo. In conclusion, these results indicate that fluorocoxib A could be used for the monitoring the early responses to targeted therapies in COX-2-expressing bladder cancer.

Multiple mechanisms are involved in 6-gingerol-induced cell growth arrest and apoptosis in human colorectal cancer cells.

6-Gingerol, a natural product of ginger, has been known to possess anti-tumorigenic and pro-apoptotic activities. However, the mechanisms by which it prevents cancer are not well understood in human colorectal cancer. Cyclin D1 is a proto-oncogene that is overexpressed in many cancers and plays a role in cell proliferation through activation by beta-catenin signaling. Nonsteroidal anti-inflammatory drug (NSAID)-activated gene-1 (NAG-1) is a cytokine associated with pro-apoptotic and anti-tumorigenic properties. In the present study, we examined whether 6-gingerol influences cyclin D1 and NAG-1 expression and determined the mechanisms by which 6-gingerol affects the growth of human colorectal cancer cells in vitro. 6-Gingerol treatment suppressed cell proliferation and induced apoptosis and G(1) cell cycle arrest. Subsequently, 6-gingerol suppressed cyclin D1 expression and induced NAG-1 expression. Cyclin D1 suppression was related to inhibition of beta-catenin translocation and cyclin D1 proteolysis. Furthermore, experiments using inhibitors and siRNA transfection confirm the involvement of the PKCepsilon and glycogen synthase kinase (GSK)-3beta pathways in 6-gingerol-induced NAG-1 expression. The results suggest that 6-gingerol stimulates apoptosis through upregulation of NAG-1 and G(1) cell cycle arrest through downregulation of cyclin D1. Multiple mechanisms appear to be involved in 6-gingerol action, including protein degradation as well as beta-catenin, PKCepsilon, and GSK-3beta pathways.

Gene alterations by peroxisome proliferator-activated receptor gamma agonists in human colorectal cancer cells.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear transcription factor that controls the genes involved in metabolism and carcinogenesis. In the present study, we examined the alteration of gene expression in HCT-116 human colorectal cancer cells by PPARgamma agonists: MCC-555 (5 microM), rosiglitazone (5 microM), and 15-deoxy-Delta12,14-prostaglandin J2 (1 microM). The long-oligo microarray data revealed a list of target genes commonly induced (307 genes) and repressed (32 genes) by tested PPARgamma agonists. These genes were analyzed by Onto-Express software and KEGG pathway analysis and revealed that PPARgamma agonists are involved in cell proliferation, focal adhesion, and several signaling pathways. Eight genes were selected to confirm the microarray data by RT-PCR and real-time PCR, from which CSTA, DAP13, TAF12, RIS1, CDKN3 and MAGOH were up-regulated, and KLHL11 and NCOA2 were down-regulated. This study elucidates the commonly induced genes modulated by tested PPARgamma ligands involved in the different signaling pathways and metabolisms, probably mediated in a PPARgamma-dependent manner in colorectal cancer cells and helps to better understand the pleiotropic actions of PPARgamma ligands.

Cyclooxygenase inhibitors potentiate receptor tyrosine kinase therapies in bladder cancer cells in vitro.

PURPOSE: Receptor tyrosine kinase inhibitors (RTKIs) are used as targeted therapies for patients diagnosed with cancer with highly expressed receptor tyrosine kinases (RTKs), including the platelet-derived growth factor receptor (PDGFR) and c-Kit receptor. Resistance to targeted therapies is partially due to the activation of alternative pro-survival signaling pathways, including cyclooxygenase (COX)-2. In this study, we validated the effects of two RTKIs, axitinib and AB1010, in combination with COX inhibitors on the V-akt murine thymoma oncogene homolog 1 (Akt) and COX-2 signaling pathways in bladder cancer cells. METHODS: The expression of several RTKs and their downstream signaling targets was analyzed by Western blot (WB) analysis in human and canine bladder transitional cell carcinoma (TCC) cell lines. The effects of RTKIs and COX inhibitors in bladder TCC cells were assessed by MTS for cell viability, by Caspase-3/7 and Annexin V assay for apoptosis, by WB analysis for detection of COX-2 and Akt signaling pathways, and by enzyme-linked immunosorbent assay for detection of prostaglandin E2 (PGE2) levels. RESULTS: All tested TCC cells expressed the c-Kit and PDGFRα receptors, except human 5637 cells that had low RTKs expression. In addition, all tested cells expressed COX-1, COX-2, Akt, extracellular signal regulated kinases 1/2, and nuclear factor kappa-light-chain-enhance of activated B cells proteins, except human UM-UC-3 cells, where no COX-2 expression was detected by WB analysis. Both RTKIs inhibited cell viability and increased apoptosis in a dose-dependent manner in tested bladder TCC cells, which positively correlated with their expression levels of the PDGFRα and c-Kit receptors. RTKIs increased the expression of COX-2 in h-5637 and K9TCC#1Lillie cells. Co-treatment of indomethacin inhibited AB1010-induced COX-2 expression leading to an additive effect in inhibition of cell viability and PGE2 production in tested TCC cells. CONCLUSION: Co-treatment of RTKIs with indomethacin inhibited cell viability and AB1010-induced COX-2 expression resulting in decreased PGE2 production in tested TCC cells. Thus, COX inhibition may further potentiate RTKIs therapies in bladder cancer.

Mutations of p53 decrease sensitivity to the anthracycline treatments in bladder cancer cells.

Due to doxorubicin (Dox) cardiotoxicity, the next generation of novel non-cardiotoxic anthracyclines, including AD 312 and AD 198, were synthesized and validated. In this study, we assessed the efficacy and mechanisms of anthracyclines-induced apoptosis and inhibition of cell viability in human bladder cancer cells expressing wild-type (wt) p53 (RT4 and SW780) and mutated (mt) p53 (UM-UC-3, 5637, T-24, J82, and TCCSUP) protein. Anthracyclines inhibited cell viability in tested TCC cells, but were less effective in mt-p53 TCC cells, especially in the drug-resistant J82 and TCCSUP cells. Anthracyclines upregulated the expression of wt p53 protein in RT4 and SW780 cells, but had no effect on expression of mt p53 protein in UM-UC-3, 5637, T-24, J82, and TCCSUP cells. The anthracyclines activated caspase 3/7 and cleavage of PARP in wt-p53 RT4 and SW780 cells, and mt-p53 5637, UM-UC-3, and T-24, but not in mt-p53 J82 and TCCSUP cells. The anthracyclines-induced cleavage of PARP was blocked by p53 siRNA in wt-p53 RT4 cells. Co-treatment of AD 198 with PRIMA-1 significantly inhibited cell viability of mt-p53 J82 cells, but had no effect in wt-p53 RT4 cells. AD 198 blocked c-myc expression in mt-p53 UM-UC-3, 5637, T-24, and J82 cells, however no expression of c-myc was detected in wt-p53 RT4 and SW780 cells. In conclusion, our results demonstrated that the anthracycline-induced resistance in bladder cancer cells positively correlated with TP53 mutations in the tetramerization domain in J82 and TCCSUP cells. Further, AD 312 and AD 198 are promising chemotherapeutic drugs for bladder cancer, especially in combination with PRIMA-1.

Aligned nanofiber material supports cell growth and increases osteogenesis in canine adipose-derived mesenchymal stem cells in vitro.

Tissue engineering shows great promise for the treatment of degenerative diseases, including bone repair. Polymer nanofibers provide a three-dimensional (3-D) scaffold for attachment and growth of mesenchymal stem cells. Increasing evidence supports that fiber alignment on scaffolds plays a major role in the viability and differentiation of stem cells. We compared the cell viability of canine adipose tissue-derived mesenchymal stem cells (cADMSCs) cultured in the aligned- (NanoAligned™) and random- (NanoECM™) oriented polycaprolactone (PCL) nanofiber-coated plates to control polystyrene tissue culture plates using a proliferation assay. Ability of the plates to induce differentiation of cADMSCs into osteocytes, adipocytes, and neurons was evaluated based on expression of the osteocyte markers, COL1A1 and osterix; adipocyte markers PPARγ2 and LPL; and neuronal marker nestin using RT-PCR. Proliferation results demonstrated that aligned-oriented PCL nanofiber-coated plates were more suitable substrate for cADMSCs after 7 days in culture compared to random-oriented PCL nanofiber-coated or control plates. Additionally, we demonstrated that both 3-D PCL nanofiber-coated plates were a better scaffold for cADMSCs differentiation into osteocytes compared to control plates. In conclusion, our results confirm that PCL nanofiber is a suitable tissue engineering material for use in regenerative medicine for canine patients in vivo. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1780-1788, 2018.

Molecular targets in urothelial cancer: detection, treatment, and animal models of bladder cancer.

Bladder cancer remains one of the most expensive cancers to treat in the United States due to the length of required treatment and degree of recurrence. In order to treat bladder cancer more effectively, targeted therapies are being investigated. In order to use targeted therapy in a patient, it is important to provide a genetic background of the patient. Recent advances in genome sequencing, as well as transcriptome analysis, have identified major pathway components altered in bladder cancer. The purpose of this review is to provide a broad background on bladder cancer, including its causes, diagnosis, stages, treatments, animal models, as well as signaling pathways in bladder cancer. The major focus is given to the PI3K/AKT pathway, p53/pRb signaling pathways, and the histone modification machinery. Because several promising immunological therapies are also emerging in the treatment of bladder cancer, focus is also given on general activation of the immune system for the treatment of bladder cancer.

Evaluation of the role of the cyclooxygenase signaling pathway during inflammation in skin and muscle tissues of ball pythons (Python regius).

OBJECTIVE To determine degrees of production of cyclooxygenase (COX)-1 and -2 and other mediators of inflammation in noninflamed and inflamed skin and muscle tissues in ball pythons (Python regius). ANIMALS 6 healthy adult male ball pythons. PROCEDURES Biopsy specimens of noninflamed skin and muscle tissue were collected from anesthetized snakes on day 0. A 2-cm skin and muscle incision was then made 5 cm distal to the biopsy sites with a CO2 laser to induce inflammation. On day 7, biopsy specimens of skin and muscle tissues were collected from the incision sites. Inflamed and noninflamed tissue specimens were evaluated for production of COX-1, COX-2, phosphorylated protein kinase B (AKT), total AKT, nuclear factor κ-light-chain-enhancer of activated B cells, phosphorylated extracellular receptor kinases (ERKs) 1 and 2, and total ERK proteins by western blot analysis. Histologic evaluation was performed on H&E-stained tissue sections. RESULTS All biopsy specimens of inflamed skin and muscle tissues had higher histologic inflammation scores than did specimens of noninflamed tissue. Inflamed skin specimens had significantly greater production of COX-1 and phosphorylated ERK than did noninflamed skin specimens. Inflamed muscle specimens had significantly greater production of phosphorylated ERK and phosphorylated AKT, significantly lower production of COX-1, and no difference in production of COX-2, compared with production in noninflamed muscle specimens. CONCLUSIONS AND CLINICAL RELEVANCE Production of COX-1, but not COX-2, was significantly greater in inflamed versus noninflamed skin specimens from ball pythons. Additional research into the reptilian COX signaling pathway is warranted.

NNK-induced hamster lung adenocarcinomas over-express beta2-adrenergic and EGFR signaling pathways.

Pulmonary adenocarcinoma (PAC) is the most common type of human lung cancer. A diagnosis of PAC, history of non-smoking and presence of mutations in the EGFR are predictive factors for responsiveness of lung cancer to EGFR-specific tyrosine kinase inhibitors. Unfortunately, less than 50% of PAC cases demonstrate this mutation-based responsiveness. Our immunohistochemical analysis of NNK-induced PAC in hamsters demonstrates the simultaneous over-expression of a beta2-adrenergic receptor pathway, including PKA, cAMP, CREB and phosphorylated CREB and of an EGFR pathway, including over-expression of EGFR-specific phosphorylated tyrosine kinase, Raf-1 and ERK1/2 and their phosphorylated forms. These findings implicate, for the first time, PKA/CREB-mediated signaling in the development and regulation of any type of lung cancer. In light of reports that NNK acts as a beta-adrenergic agonist and that beta-blockers inhibit the growth of PAC of Clara cell lineage in the NNK hamster model and in human cancer cell lines from smokers, our current data suggest transactivation of the EGFR pathway via beta-adrenergic signaling as a novel regulatory mechanism in a subpopulation of PACs in smokers. Taken together, these data point to PKA/CREB as novel targets for the development of cancer therapeutics for PAC patients non-responsive to EGFR-specific tyrosine kinase inhibitors.