Frankincense essential oil prepared from hydrodistillation of Boswellia sacra gum resins induces human pancreatic cancer cell death in cultures and in a xenograft murine model.

BACKGROUND: Regardless of the availability of therapeutic options, the overall 5-year survival for patients diagnosed with pancreatic cancer remains less than 5%. Gum resins from Boswellia species, also known as frankincense, have been used as a major ingredient in Ayurvedic and Chinese medicine to treat a variety of health-related conditions. Both frankincense chemical extracts and essential oil prepared from Boswellia species gum resins exhibit anti-neoplastic activity, and have been investigated as potential anti-cancer agents. The goals of this study are to identify optimal condition for preparing frankincense essential oil that possesses potent anti-tumor activity, and to evaluate the activity in both cultured human pancreatic cancer cells and a xenograft mouse cancer model. METHODS: Boswellia sacra gum resins were hydrodistilled at 78°C; and essential oil distillate fractions were collected at different durations (Fraction I at 0-2 h, Fraction II at 8-10 h, and Fraction III at 11-12 h). Hydrodistillation of the second half of gum resins was performed at 100°C; and distillate was collected at 11-12 h (Fraction IV). Chemical compositions were identified by gas chromatography-mass spectrometry (GC-MS); and total boswellic acids contents were quantified by high-performance liquid chromatography (HPLC). Frankincense essential oil-modulated pancreatic tumor cell viability and cytotoxicity were determined by colorimetric assays. Levels of apoptotic markers, signaling molecules, and cell cycle regulators expression were characterized by Western blot analysis. A heterotopic (subcutaneous) human pancreatic cancer xenograft nude mouse model was used to evaluate anti-tumor capability of Fraction IV frankincense essential oil in vivo. Frankincense essential oil-induced tumor cytostatic and cytotoxic activities in animals were assessed by immunohistochemistry. RESULTS: Longer duration and higher temperature hydrodistillation produced more abundant high molecular weight compounds, including boswellic acids, in frankincense essential oil fraactions. Human pancreatic cancer cells were sensitive to Fractions III and IV (containing higher molecular weight compounds) treatment with suppressed cell viability and increased cell death. Essential oil activated the caspase-dependent apoptotic pathway, induced a rapid and transient activation of Akt and Erk1/2, and suppressed levels of cyclin D1 cdk4 expression in cultured pancreatic cancer cells. In addition, Boswellia sacra essential oil Fraction IV exhibited anti-proliferative and pro-apoptotic activities against pancreatic tumors in the heterotopic xenograft mouse model. CONCLUSION: All fractions of frankincense essential oil from Boswellia sacra are capable of suppressing viability and inducing apoptosis of a panel of human pancreatic cancer cell lines. Potency of essential oil-suppressed tumor cell viability may be associated with the greater abundance of high molecular weight compounds in Fractions III and IV. Although chemical component(s) responsible for tumor cell cytotoxicity remains undefined, crude essential oil prepared from hydrodistillation of Boswellia sacra gum resins might be a useful alternative therapeutic agent for treating patients with pancreatic adenocarcinoma, an aggressive cancer with poor prognosis.

Bladder regeneration in a canine model using hyaluronic acid-poly(lactic-co-glycolic-acid) nanoparticle modified porcine small intestinal submucosa.

OBJECTIVE: • To determine if hyaluronic acid (HA) can be incorporated into porcine small intestinal submucosa (SIS) through poly (lactide-co-glycolide-acid) (PLGA) nanoparticles to improve the consistency of the naturally derived biomaterial and promote bladder tissue regeneration. METHODS: • Beagle dogs were subjected to 40% partial cystectomy followed by bladder augmentation with commercial SIS or HA-PLGA-modified SIS. • Urodynamic testing was performed before and after augmentation to assess bladder volume. • A scoring system was created to evaluate gross and histological presentations of regenerative bladders. RESULTS: • All dogs showed full-thickness bladder regeneration. • Histological assessment showed improved smooth muscle regeneration in the HA-PLGA-modified SIS group. • For both groups of dogs, urodynamics and graft measurements showed an approximate 40% reduction in bladder capacity and graft size from pre-augmentation to post-regeneration measurements. • Application of the scoring system and statistical analysis failed to show a significant difference between the groups. CONCLUSIONS: • SIS can be modified through the addition of HA-PLGA nanoparticles. The modified grafts showed evidence of improved smooth muscle regeneration on histological assessment, although this difference was not evident on a novel grading scale. • The volume loss and graft shrinkage experienced are consistent with previous models of SIS bladder regeneration at the 10-week time point. • Additional research into the delivery of HA and the long-term benefits of HA on bladder regeneration is needed to determine the full benefit of HA-PLGA-modified SIS. In addition, a more objective biochemical characterization will be needed to evaluate the quality of regeneration.

Boswellia sacra essential oil induces tumor cell-specific apoptosis and suppresses tumor aggressiveness in cultured human breast cancer cells.

BACKGROUND: Gum resins obtained from trees of the Burseraceae family (Boswellia sp.) are important ingredients in incense and perfumes. Extracts prepared from Boswellia sp. gum resins have been shown to possess anti-inflammatory and anti-neoplastic effects. Essential oil prepared by distillation of the gum resin traditionally used for aromatic therapy has also been shown to have tumor cell-specific anti-proliferative and pro-apoptotic activities. The objective of this study was to optimize conditions for preparing Boswellea sacra essential oil with the highest biological activity in inducing tumor cell-specific cytotoxicity and suppressing aggressive tumor phenotypes in human breast cancer cells. METHODS: Boswellia sacra essential oil was prepared from Omani Hougari grade resins through hydrodistillation at 78 or 100 °C for 12 hours. Chemical compositions were identified by gas chromatography-mass spectrometry; and total boswellic acids contents were quantified by high-performance liquid chromatography. Boswellia sacra essential oil-mediated cell viability and death were studied in established human breast cancer cell lines (T47D, MCF7, MDA-MB-231) and an immortalized normal human breast cell line (MCF10-2A). Apoptosis was assayed by genomic DNA fragmentation. Anti-invasive and anti-multicellular tumor properties were evaluated by cellular network and spheroid formation models, respectively. Western blot analysis was performed to study Boswellia sacra essential oil-regulated proteins involved in apoptosis, signaling pathways, and cell cycle regulation. RESULTS: More abundant high molecular weight compounds, including boswellic acids, were present in Boswellia sacra essential oil prepared at 100 °C hydrodistillation. All three human breast cancer cell lines were sensitive to essential oil treatment with reduced cell viability and elevated cell death, whereas the immortalized normal human breast cell line was more resistant to essential oil treatment. Boswellia sacra essential oil hydrodistilled at 100 °C was more potent than the essential oil prepared at 78 °C in inducing cancer cell death, preventing the cellular network formation (MDA-MB-231) cells on Matrigel, causing the breakdown of multicellular tumor spheroids (T47D cells), and regulating molecules involved in apoptosis, signal transduction, and cell cycle progression. CONCLUSIONS: Similar to our previous observations in human bladder cancer cells, Boswellia sacra essential oil induces breast cancer cell-specific cytotoxicity. Suppression of cellular network formation and disruption of spheroid development of breast cancer cells by Boswellia sacra essential oil suggest that the essential oil may be effective for advanced breast cancer. Consistently, the essential oil represses signaling pathways and cell cycle regulators that have been proposed as therapeutic targets for breast cancer. Future pre-clinical and clinical studies are urgently needed to evaluate the safety and efficacy of Boswellia sacra essential oil as a therapeutic agent for treating breast cancer.

Elevated AKR1C3 expression promotes prostate cancer cell survival and prostate cell-mediated endothelial cell tube formation: implications for prostate cancer progression.

BACKGROUND: Aldo-keto reductase (AKR) 1C family member 3 (AKR1C3), one of four identified human AKR1C enzymes, catalyzes steroid, prostaglandin, and xenobiotic metabolism. In the prostate, AKR1C3 is up-regulated in localized and advanced prostate adenocarcinoma, and is associated with prostate cancer (PCa) aggressiveness. Here we propose a novel pathological function of AKR1C3 in tumor angiogenesis and its potential role in promoting PCa progression. METHODS: To recapitulate elevated AKR1C3 expression in cancerous prostate, the human PCa PC-3 cell line was stably transfected with an AKR1C3 expression construct to establish PC3-AKR1C3 transfectants. Microarray and bioinformatics analysis were performed to identify AKR1C3-mediated pathways of activation and their potential biological consequences in PC-3 cells. Western blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and an in vitro Matrigel angiogenesis assays were applied to validate the pro-angiogenic activity of PC3-AKR1C3 transfectants identified by bioinformatics analysis. RESULTS: Microarray and bioinformatics analysis suggested that overexpression of AKR1C3 in PC-3 cells modulates estrogen and androgen metabolism, activates insulin-like growth factor (IGF)-1 and Akt signaling pathways, as well as promotes tumor angiogenesis and aggressiveness. Levels of IGF-1 receptor (IGF-1R) and Akt activation as well as vascular endothelial growth factor (VEGF) expression and secretion were significantly elevated in PC3-AKR1C3 transfectants in comparison to PC3-mock transfectants. PC3-AKR1C3 transfectants also promoted endothelial cell (EC) tube formation on Matrigel as compared to the AKR1C3-negative parental PC-3 cells and PC3-mock transfectants. Pre-treatment of PC3-AKR1C3 transfectants with a selective IGF-1R kinase inhibitor (AG1024) or a non-selective phosphoinositide 3-kinases (PI3K) inhibitor (LY294002) abolished ability of the cells to promote EC tube formation. CONCLUSIONS: Bioinformatics analysis followed by functional genomics demonstrated that AKR1C3 overexpression promotes angiogenesis and aggressiveness of PC-3 cells. These results also suggest that AKR1C3-mediated tumor angiogenesis is regulated by estrogen and androgen metabolism with subsequent IGF-1R and Akt activation followed by VEGF expression in PCa cells.

Regional variations in small intestinal submucosa evoke differences in inflammation with subsequent impact on tissue regeneration in the rat bladder augmentation model.

OBJECTIVE: To examine the histological differences in the inflammatory response and regenerative outcomes of distal vs proximal porcine small intestinal submucosa (SIS) grafts in the rat bladder, as SIS from distal small intestine yields reliable and reproducible bladder regeneration, while SIS from proximal portions of small intestine does not provide similar results. MATERIALS AND METHODS: In all, 30 Sprague-Dawley rats underwent hemi-cystectomy followed by anastomosis of a bladder patch of SIS prepared from either distal or proximal small intestine. After bladder harvest, immunohistochemistry was used to quantify mast cells, eosinophils, macrophages, and neutrophils (PMNs). Total cell count per unit area was compared across the time course in univariate and logistic regression modelling. RESULTS: There were more eosinophils and mast cells in proximal SIS grafts, while there were more macrophages and PMNs in distal SIS grafts (all P < 0.05). Trichrome analysis showed increased collagen deposition in proximal SIS grafts and little smooth muscle regeneration. There was also significant graft contracture in proximal SIS grafts compared with distal SIS grafts (P < 0.05). CONCLUSIONS: We conclude that the location of SIS origin may evoke different inflammatory responses, which results in altered bladder tissue regeneration.

Protective essential oil attenuates influenza virus infection: an in vitro study in MDCK cells.

BACKGROUND: Influenza is a significant cause of morbidity and mortality. The recent pandemic of a novel H1N1 influenza virus has stressed the importance of the search for effective treatments for this disease. Essential oils from aromatic plants have been used for a wide variety of applications, such as personal hygiene, therapeutic massage and even medical practice. In this paper, we investigate the potential role of an essential oil in antiviral activity. METHODS: We studied a commercial essential oil blend, On Guard™, and evaluated its ability in modulating influenza virus, A/PR8/34 (PR8), infection in Madin-Darby canine kidney (MDCK) cells. Influenza virus was first incubated with the essential oil and infectivity in MDCK cells was quantified by fluorescent focus assay (FFA). In order to determine the mechanism of effects of essential oil in viral infection inhibition, we measured hemagglutination (HA) activity, binding and internalization of untreated and oil-treated virus in MDCK cells by flow cytometry and immunofluorescence microscopy. In addition, the effect of oil treatment on viral transcription and translation were assayed by relative end-point RT-PCR and western blot analysis. RESULTS: Influenza virus infectivity was suppressed by essential oil treatment in a dose-dependent manner; the number of nascent viral particles released from MDCK cells was reduced by 90% and by 40% when virus was treated with 1:4,000 and 1:6,000 dilutions of the oil, respectively. Oil treatment of the virus also decreased direct infection of the cells as the number of infected MDCK cells decreased by 90% and 45% when virus was treated with 1:2,000 and 1:3,000 dilutions of the oil, respectively. This was not due to a decrease in HA activity, as HA was preserved despite oil treatment. In addition, oil treatment did not affect virus binding or internalization in MDCK cells. These effects did not appear to be due to cytotoxicity of the oil as MDCK cell viability was only seen with concentrations of oil that were 2 to 6 times greater than the doses that inhibited viral infectivity. RT-PCR and western blotting demonstrated that oil treatment of the virus inhibited viral NP and NS1 protein, but not mRNA expression. CONCLUSIONS: An essential oil blend significantly attenuates influenza virus PR8 infectivity in vitro without affecting viral binding or cellular internalization in MDCK cells. Oil treated virus continued to express viral mRNAs but had minimal expression of viral proteins, suggesting that the antiviral effect may be due to inhibition of viral protein translation.

Unique patterns of molecular profiling between human prostate cancer LNCaP and PC-3 cells.

BACKGROUND: Human prostate cancer LNCaP and PC-3 cell lines have been extensively used to study prostate cancer progression and to develop therapeutic agents. Although LNCaP and PC-3 cells are generally assumed to represent early and late stages of prostate cancer, respectively, there is limited information regarding gene expression patterns between these two cell lines and its relationship to prostate cancer. METHODS: Comprehensive gene expression analysis was performed. Total RNA was isolated from cultured cells and hybridized to Illumina human BeadChips representing 24,526 transcripts. Bioinformatics analysis was applied to identify cell line specific genes as well as biological mechanisms, pathways, and functions related to the genes. RESULTS: A total of 2,198 genes were differentially expressed between LNCaP and PC-3 cells. Using a robust statistical analysis and high significance criteria, 115 and 188 genes were identified to be unique to LNCaP and PC-3 cells, respectively. LNCaP cells maintained various metabolic pathways including a gene cluster that encodes UDP-glucuronosyltransferases. Several transcription factors including Tal alpha/beta, GATA-1, and c-Myc/Max may be responsible for regulating LNCaP cell specific genes. By contrast, PC-3 cells were characterized by their unique expression of cytoskeleton-related genes and other genes including VEGFC, IL8, and TGF beta 2. CONCLUSIONS: This study showed that LNCaP and PC-3 cells represent two distinct prostate cancer cell lineages. LNCaP cells retain many prostate cell specific properties, whereas PC-3 cells have acquired a more aggressive phenotype. Future studies for prostate cancer research need to consider similarities and differences between these two cells and their relationship to prostate cancer.

Temporal differentiation and maturation of regenerated rat urothelium.

OBJECTIVE: To determine if porcine small intestinal submucosa (SIS)-regenerated urothelium expresses markers of urothelial differentiation, uroplakin and zona occludens-1 (ZO-1), and whether their expression correlates with the histological appearance of the urothelium. MATERIALS AND METHODS: In all, 15 rats underwent partial cystectomy and bladder replacement with SIS. Regenerated bladders were harvested at either 2, 7, 14, 28, or 56 days after SIS grafting. Histological examination with haematoxylin and eosin staining was conducted to assess tissue regeneration. Immunohistochemistry was performed with uroplakin and ZO-1 antibodies. RESULTS: By 14 days after SIS grafting, the urothelial layer was completely confluent over the SIS. Expression of uroplakin and ZO-1, evident at 2 days after SIS grafting, progressed from a cytoplasmic pattern of expression to a mature pattern of cytoplasmic and membrane expression by 56 days after SIS grafting. CONCLUSION: In vivo tissue regeneration produces histologically and phenotypically mature urothelium within 2 weeks of SIS implantation. Regeneration of functional urothelium is probably essential for the subsequent development of the remaining bladder.

Frankincense oil derived from Boswellia carteri induces tumor cell specific cytotoxicity.

BACKGROUND: Originating from Africa, India, and the Middle East, frankincense oil has been important both socially and economically as an ingredient in incense and perfumes for thousands of years. Frankincense oil is prepared from aromatic hardened gum resins obtained by tapping Boswellia trees. One of the main components of frankincense oil is boswellic acid, a component known to have anti-neoplastic properties. The goal of this study was to evaluate frankincense oil for its anti-tumor activity and signaling pathways in bladder cancer cells. METHODS: Frankincense oil-induced cell viability was investigated in human bladder cancer J82 cells and immortalized normal bladder urothelial UROtsa cells. Temporal regulation of frankincense oil-activated gene expression in bladder cancer cells was identified by microarray and bioinformatics analysis. RESULTS: Within a range of concentration, frankincense oil suppressed cell viability in bladder transitional carcinoma J82 cells but not in UROtsa cells. Comprehensive gene expression analysis confirmed that frankincense oil activates genes that are responsible for cell cycle arrest, cell growth suppression, and apoptosis in J82 cells. However, frankincense oil-induced cell death in J82 cells did not result in DNA fragmentation, a hallmark of apoptosis. CONCLUSION: Frankincense oil appears to distinguish cancerous from normal bladder cells and suppress cancer cell viability. Microarray and bioinformatics analysis proposed multiple pathways that can be activated by frankincense oil to induce bladder cancer cell death. Frankincense oil might represent an alternative intravesical agent for bladder cancer treatment.

Immunomodulatory response of layered small intestinal submucosa in a rat bladder regeneration model.

Based on the hypothesis that bioscaffold permeability is a major factor in determining the outcome of histologically complete and functional bladder regeneration, we evaluated regeneration processes of four-layer porcine small intestinal submucosa (SIS) construct; and compared results between rat bladders augmented with single-layer SIS bioscaffolds. Sprague-Dawley female rats were subjected to hemi-cystectomy followed by anastomosis of a patch of either single- or four-layer porcine SIS. Permeability was analyzed in situ using magnetic resonance imaging (MRI) at post-operative days 7 and 14. Bladder sections excised at days 7, 14, 28, and 56 post-operation Samples were assessed by H&E and Masson's trichrome stains. Urothelial differentiation was analyzed using cytokeratin AE1/AE3, and uroplakin III (UPIII). In addition, quantitative and qualitative evaluations of neutrophils, mast cells, eosinophils, and macrophages were performed using anti-myeloperoxidase, Alcian blue, Giemsa stain, and anti-CD68 staining methods, respectively. Four-layer SIS was consistently impermeable as evidenced by the absence of intravesical administered gadolinium with diethylenetriaminepentacetate (Gd-DTPA) contrast signal in peripheral regions of augmented bladders compared with single-layer SIS bioscaffold. Elevated and sustained eosinophil and neutrophil infiltrations were prominent in four-layered SIS-augmented bladders compared with single-layer SIS with comparable impermeability. Delayed but consistent urothelial regeneration and differentiation were observed in four-layer SIS-augmented bladders; and urothelial differentiation was observed at day 56 post-augmentation. In conclusion, four-layer SIS enacts an elevated inflammatory response along with extended urothelial regeneration. Four-layer SIS may activate a different but yet to be identified mechanism for inflammatory responses. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1960-1969, 2019.

5alpha-androstane-3alpha,17beta-diol supports human prostate cancer cell survival and proliferation through androgen receptor-independent signaling pathways: implication of androgen-independent prostate cancer progression.

Androgen and androgen receptor (AR) are involved in growth of normal prostate and development of prostatic diseases including prostate cancer. Androgen deprivation therapy is used for treating advanced prostate cancer. This therapeutic approach focuses on suppressing the accumulation of potent androgens, testosterone and 5alpha-dihydrotestosterone (5alpha-DHT), or inactivating the AR. Unfortunately, the majority of patients with prostate cancer eventually advance to androgen-independent states and no longer respond to the therapy. In addition to the potent androgens, 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), reduced from 5alpha-DHT through 3alpha-hydroxysteroid dehydrogenases (3alpha-HSDs), activated signaling may represent a novel pathway responsible for the progression to androgen-independent prostate cancer. Androgen sensitive human prostate cancer LNCaP cells were used to compare 5alpha-DHT and 3alpha-diol activated androgenic effects. In contrast to 5alpha-DHT, 3alpha-diol regulated unique patterns of beta-catenin and Akt expression as well as Akt phosphorylation in parental and in AR-silenced LNCaP cells. More significantly, 3alpha-diol, but not 5alpha-DHT, supported AR-silenced LNCaP cells and AR negative prostate cancer PC-3 cell proliferation. 3alpha-diol-activated androgenic effects in prostate cells cannot be attributed to the accumulation of 5alpha-DHT, since 5alpha-DHT formation was not detected following 3alpha-diol administration. Potential accumulation of 3alpha-diol, as a result of elevated 3alpha-HSD expression in cancerous prostate, may continue to support prostate cancer growth in the presence of androgen deprivation. Future therapeutic strategies for treating advanced prostate cancer might need to target reductive 3alpha-HSD to block intraprostatic 3alpha-diol accumulation.

Tissue distribution of human AKR1C3 and rat homolog in the adult genitourinary system.

Human aldo-keto reductase (AKR) 1C3 (type 2 3alpha-hydroxysteroid dehydrogenase/type 5 17beta-hydroxysteroid dehydrogenase) catalyzes androgen, estrogen, and prostaglandin metabolism. AKR1C3 is therefore implicated in regulating ligand access to the androgen receptor, estrogen receptor, and peroxisome proliferator activating receptor gamma in hormone target tissues. Recent reports on close relationships between ARK1C3 and various cancers including breast and prostate cancers implicate the involvement of AKR1C3 in cancer development or progression. We previously described the characterization of an isoform-specific monoclonal antibody against AKR1C3 that does not cross-react with related, >86% sequence identity, human AKR1C1, AKR1C2, or AKR1C4, human aldehyde reductase AKR1A1, or rat 3alpha-hydroxysteroid dehydrogenase (AKR1C9). In this study, a clone of murine monoclonal antibody raised against AKR1C3 was identified and characterized for its recognition of rat homolog. Tissue distribution of human AKR1C3 and its rat homolog in adult genitourinary systems including kidney, bladder, prostate, and testis was studied by IHC. A strong immunoreactivity was detected not only in classically hormone-associated tissues such as prostate and testis but also in non-hormone-associated tissues such as kidney and bladder in humans and rats. The distribution of these two enzymes was comparable but not identical between the two species. These features warrant future studies of AKR1C3 in both hormone- and non-hormone-associated tissues and identification of the rodent homolog for establishing animal models.

AKR1C2 and AKR1C3 mediated prostaglandin D2 metabolism augments the PI3K/Akt proliferative signaling pathway in human prostate cancer cells.

Members of the aldo-keto reductase (AKR) superfamily have been implicated in prostaglandin (PG) metabolism and prostate cancer. AKR1C3 possesses 11-ketoprostaglandin reductase activity and is capable of converting PGD2 to 9alpha, 11beta-PGF2alpha, whereas AKR1C2-mediated PG metabolism remains unclear. The accumulation of PGF2alpha may generate proliferative signals to promote prostate cell growth. Levels of AKR1C2 and AKR1C3 expression are elevated in localized and advanced prostate cancer. To study the significance of AKR1C2- and AKR1C3-mediated PGD2 conversion in human prostate cell proliferation, we stably transfected androgen insensitive human prostate cancer PC-3 cells with AKR1C2 or AKR1C3 cDNA. PC-3 cells overexpressing AKR1C2 and AKR1C3 had elevated cell proliferation in response to PGD2 stimulation as compared to mock transfectants. Overexpression of AKR1C2 or AKR1C3 did not alter levels of PGF receptor (FP) expression. Inclusion of an FP antagonist (AL8810) significantly suppressed PGD2-stimulated PC-3 cell proliferation in these stable transfectants. In addition, PGD2 significantly elevated levels of total Akt protein expression and Akt Ser473 phosphorylation in AKR1C2 and AKR1C3 stable transfectants; and inclusion of a phosphatidylinositol 3-kinase (PI3K) chemical inhibitor (LY294002) attenuated PGD2-stimulated cell proliferation in these transfectants. Our results suggested that both AKR1C2 and AKR1C3 mediate similar PGD2 conversion toward the accumulation of proliferative signals through FP and PI3K/Akt signaling pathways to promote prostate cell proliferation.

The incorporation of poly(lactic-co-glycolic) acid nanoparticles into porcine small intestinal submucosa biomaterials.

Small intestinal submucosa (SIS) derived from porcine small intestine has been intensively studied for its capacity in repairing and regenerating wounded and dysfunctional tissues. However, SIS suffers from a large spectrum of heterogeneity in microarchitecture leading to inconsistent results. In this study, we introduced nanoparticles (NPs) to SIS with an intention of decreasing the heterogeneity and improving the consistency of this biomaterial. As determined by scanning electron microscopy and urea permeability, the optimum NP size was estimated to be between 200 nm and 500 nm using commercial monodisperse latex spheres. The concentration of NPs that is required to alter pore sizes of SIS as determined by urea permeability was estimated to be 1 mg/ml 260 nm poly(lactic-co-glycolic) acid (PLGA) NPs. The 1mg/ml PLGA NPs loaded in the SIS did not change the tensile properties of the unmodified SIS or even alter pH values in a cell culture environment. More importantly, PLGA NP modified SIS did not affect human mammary endothelial cells (HMEC-1) morphology or adhesion, but actually enhanced HEMC-1 cell growth.

Reduced urothelial regeneration in rat bladders augmented with permeable porcine small intestinal submucosa assessed by magnetic resonance imaging.

Augmentation enterocystoplasty remains the gold standard surgical bladder reconstruction procedure to increase the capacity and compliance of dysfunctional bladders. Since the use of the patient's intestine has severe risks of complications, alternative biodegradable matrices have been explored. Porcine small intestinal submucosa (SIS) has gained immense interests in bladder reconstruction due to its favorable properties. However, trials have shown inconsistent regeneration with SIS, attributed to the heterogeneity in microstructures and mechanical properties. We hypothesize that uneven SIS permeability to urine is a factor responsible for the inconsistency. We measured permeability to urine in situ using a contrast enhanced-magnetic resonance imaging (MRI), and evaluated urothelium regeneration using immunohistochemical staining of urothelial cell markers in SIS-augmented rat bladders. Results showed significant differences in permeability among SIS-augmented rat bladders. Commercial SIS scaffolds were then categorized into nonleaky and leaky groups based on MRI results. Hematoxylin and eosin staining showed higher numbers of inflammatory cells in leaky SIS on day 14 relative to nonleaky SIS. In addition, trichrome staining showed major changes in the distribution of collagen on day 28 between SIS-augmented bladder groups. Furthermore, expressions of urothelium-associated markers (cytokeratins AE1/AE3, claudin 4, and uroplakin III) were completed in bladders augmented with nonleaky SIS, whereas limited urothelial differentiation was noticed in leaky SIS-augmented bladders at post-augmentative day 14. These results show that scaffold permeability to urine may be responsible for variations in regenerative capacity of porcine SIS. Applications of MRI technique will be helpful to understand a relationship between biomaterial property and regenerative capacity. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1778-1787, 2018.

Transition from androgenic to neurosteroidal action of 5α-androstane-3α, 17ß-diol through the type A γ-aminobutyric acid receptor in prostate cancer progression.

Androgen ablation is the standard of care prescribed to patients with advanced or metastatic prostate cancer (PCa) to slow down disease progression. Unfortunately, a majority of PCa patients under androgen ablation progress to castration-resistant prostate cancer (CRPC). Several mechanisms including alternative intra-prostatic androgen production and androgen-independent androgen receptor (AR) activation have been proposed for CRPC progression. Aldo-keto reductase family 1 member C3 (AKR1C3), a multi-functional steroid metabolizing enzyme, is specifically expressed in the cytoplasm of PCa cells; and positive immunoreactivity of the type A γ-aminobutyric acid receptor (GABAAR), an ionotropic receptor and ligand-gated ion channel, is detected on the membrane of PCa cells. We studied a total of 72 radical prostatectomy cases by immunohistochemistry, and identified that 21 cases exhibited positive immunoreactivities for both AKR1C3 and GABAAR. In the dual positive cancer cases, AKR1C3 and GABAAR subunit α1 were either expressed in the same cells or in neighboring cells. Among several possible substrates, AKR1C3 reduces 5α-dihydrotesterone (DHT) to form 5α-androstane-3α, 17ß-diol (3α-diol). 3α-diol is a neurosteroid that acts as a positive allosteric modulator of the GABAAR in the central nervous system (CNS). We examined the hypothesis that 3α-diol-regulated pathological effects in the prostate are GABAAR-dependent, but are independent of the AR. In GABAAR-positive, AR-negative human PCa PC-3 cells, 3α-diol significantly stimulated cell growth in culture and the in ovo chorioallantoic membrane (CAM) xenograft model. 3α-diol also up-regulated expression of the epidermal growth factor (EGF) family of growth factors and activation of EGF receptor (EGFR) and Src as measured by quantitative polymerase chain reaction and immunoblotting, respectively. Inclusion of GABAAR antagonists reversed 3α-diol-stimulated tumor cell growth, expression of EGF family members, and activation of EGFR and Src to the level observed in untreated cells. Results from the present study suggest that 3α-diol may act as an alternative intra-prostatic neurosteroid that activates AR-independent PCa progression. The involvement of AKR1C3-mediated steroid metabolisms in modulating GABAAR activation and promoting PCa progression requires continued studies.

Differentially expressed gene networks in cultured smooth muscle cells from normal and neuropathic bladder.

Neuropathic bladder dysfunction results from abnormal development of the spine, spinal cord injuries, or diseases such as diabetics. Patients with neuropathic bladders often require surgical intervention such as bladder reconstruction to improve incontinence and prevent renal damage. Tissue engineering with ex-vivo cultured bladder cells has been suggested as one means for improving bladder function. However, we previously demonstrated that cultured bladder smooth muscle cells (SMCs) derived from neuropathic bladder exhibit and maintain altered pathologic phenotypes in culture. To identify genes that are responsible for the abnormal neuropathic phenotypes specifically elevated cell proliferation, the expression levels of 1,185 genes were compared between cultured SMCs derived from normal and neuropathic bladders using a cDNA array consisting of well-annotated genes. The expression data were analyzed using several methods to identify differentially expressed genes. The resulting sets of differentially expressed genes were examined by pathway analysis to identify the networks that remain abnormal in the culture-stable phenotype of neuropathic SMCs. A total of 18 genes that are differentially expressed between cultured normal and neuropathic bladder SMCs were identified. Of these 17 were up-regulated greater than 2-fold in neuropathic bladder SMCs, six of them along with one gene that was not up-regulated greater than 2-fold in cultured neuropathic bladder SMCs were confirmed and identified by more stringent analysis methods including significance analysis of microarrays, class comparison, and class prediction analyses. The major dysregulated pathways include fibroblast growth factor signaling, PTEN signaling, and integrin signaling. Our results further suggest that altered neuropathic bladder SMC phenotypes is stable in the culture environments and that SMCs derived from diseased bladders may not be appropriate for tissue engineering purpose without modification of pathologically altered genes expression.

Cancer Chemopreventive Effects of Boswellia sacra Gum Resin Hydrodistillates on Invasive Urothelial Cell Carcinoma: Report of a Case.

A 52-year-old Hispanic male presented with hematuria and was later diagnosed with a large invasive high-grade urothelial cell carcinoma (UCC) of the urinary bladder, but with ambiguous pT1/pT2 staging regarding musclaris propria invasion by UCC. The conventional treatment including radical cystoprostatectomy followed by neoadjuvant chemotherapy with or without radiation therapy was presented. The patient decided to delay the standard therapy until a later stage, but elected to go through transurethral resection of bladder tumor (TURBT) without Bacillus Calmette-Guérin instillation. Following TURBT, the patient started oral Boswellia sacra gum resin (aka frankincense or Ru Xiang in Chinese) hydrodistillates (BSGRH) administration at 3 mL daily with lifestyle changes, and continued this regimen in the last 25 months. Within the first year after diagnosis, the patient experienced 2 recurrences. Recurrent tumors were removed by TURBT alone and both tumors were far smaller than the original one. After the second recurrence, the patient has no detectible cancer in the bladder based on cystoscopy for 14 months and has an intact genitourinary system. His liver and kidney functions are considered to be normal based on blood chemistry tests. This index case suggests that BSGRH may have cancer chemopreventive effects on UCC. The use of Boswellia-derived products in the management of cancer has been well document in other published studies, and boswellic acids have been suggested to be the major component. However, BSGRH contains very little boswellic acids. Demonstration of cancer chemoprevention using BSGRH is one step forward in isolating the key components other than boswellic acids in frankincense. The critical question as to whether these components can simultaneously activate multiple pathways in cancer cells to execute cancer suppression/cytotoxicity or prevention effects remains to be addressed. More studies including identification of key molecules, pharmacokinetics of major compounds, as well as long-term benefits and possible adverse effects will be needed to meet the guidelines of the US Food and Drug Administration for botanical drug development.

Increased expression of type 2 3alpha-hydroxysteroid dehydrogenase/type 5 17beta-hydroxysteroid dehydrogenase (AKR1C3) and its relationship with androgen receptor in prostate carcinoma.

Type 2 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) is a multi-functional enzyme that possesses 3alpha-, 17beta- and 20alpha-HSD, as well as prostaglandin (PG) F synthase activities and catalyzes androgen, estrogen, progestin and PG metabolism. Type 2 3alpha-HSD was cloned from human prostate, is a member of the aldo-keto reductase (AKR) superfamily and was named AKR1C3. In androgen target tissues such as the prostate, AKR1C3 catalyzes the conversion of Delta(4)-androstene-3,17-dione to testosterone, 5alpha-dihydrotestosterone to 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), and 3alpha-diol to androsterone. Thus AKR1C3 may regulate the balance of androgens and hence trans-activation of the androgen receptor in these tissues. Tissue distribution studies indicate that AKR1C3 transcripts are highly expressed in human prostate. To measure AKR1C3 protein expression and its distribution in the prostate, we raised a monoclonal antibody specifically recognizing AKR1C3. This antibody allowed us to distinguish AKR1C3 from other AKR1C family members in human tissues. Immunoblot analysis showed that this monoclonal antibody binds to one species of protein in primary cultures of prostate epithelial cells and in LNCaP prostate cancer cells. Immunohistochemistry with this antibody on human prostate detected strong nuclear immunoreactivity in normal stromal and smooth muscle cells, perineurial cells, urothelial (transitional) cells, and endothelial cells. Normal prostate epithelial cells were only faintly immunoreactive or negative. Positive immunoreactivity was demonstrated in primary prostatic adenocarcinoma in 9 of 11 cases. Variable increases in immunoreactivity for AKR1C3 was also demonstrated in non-neoplastic changes in the prostate including chronic inflammation, atrophy and urothelial (transitional) cell metaplasia. We conclude that elevated expression of AKR1C3 is highly associated with prostate carcinoma. Although the biological significance of elevated AKR1C3 in prostatic carcinoma is uncertain, AKR1C3 may be responsible for the trophic effects of androgens and/or PGs on prostatic epithelial cells.

Screening and identification of differentially expressed transcripts in circulating cells of prostate cancer patients using suppression subtractive hybridization.

BACKGROUND: Tumor metastasis and changes in host immunosurveillance are important components in cancer development. Tumor cell invasion into the bloodstream is an essential step for systemic metastasis. Currently, the detection of tumor cells in the circulation is mainly dependent upon the utilization of known epithelial cell markers. However, expression of these molecules is not limited to cancer patients; healthy people also have a small number of epithelial cells in their circulation. Utilizing these markers to detect circulating tumor cells (CTCs) cannot adequately explain the mechanisms of tumor cell survival or their development of metastatic potential in peripheral blood. The immune system can also evolve along with the cancer, actually promoting or selecting the outgrowth of tumor variants. Unfortunately, both metastasis and immunosurveillance remain mysterious and are debatable because we have yet to define the molecules that participate in these processes. We are interested in identifying the existence of expressed genes, or mRNA species, that are specifically associated with circulating cells of cancer-bearing patients using prostate cancer (PCa) as a model. RESULTS: We established two comprehensive subtracted cDNA libraries using a molecular technique called suppression subtractive hybridization. This technique selectively amplifies transcripts that are specifically expressed in circulating cells of either PCa patients or healthy men. Following sequencing reaction, we showed that 17 out of 23 (73.9%) sequenced clones did not match any mRNAs in the GenBank database. This result suggests that genes associated with alterations in circulating cells of cancer-bearing patients are largely unknown. Semi-quantitative RT-PCR confirmed that two genes are up-regulated in circulating cells of PCa patients, whereas another two genes are down-regulated in the same patients. CONCLUSION: The comprehensive gene expression analysis is capable of identifying differentially expressed genes in circulating cells of healthy men and PCa patients. We did not attempt to enrich specific cell types in this study because phenotypes of CTCs and subsets of leukocytes participating in immunosurveillance remain largely unknown. Continuous studies of these differentially expressed genes will eventually lead us to understand the mechanisms involved in tumor metastasis and immune modulation during cancer development.