To demonstrate lipid-specific imaging of the retina through the use of third harmonic generation (THG), a multiphoton microscopic technique in which tissue contrast is generated from optical inhomogeneities.

Hypoxia is an independent prognostic indicator of poor outcome in several malignancies. However, precise mechanism through which hypoxia promotes disease aggressiveness is still unclear. Here, we report that under hypoxia (1% O2), human prostate cancer (PCA) cells, and extracellular vesicles (EVs) released by these cells, are significantly enriched in triglycerides due to the activation of lipogenesis-related enzymes and signaling molecules. This is likely a survival response to hypoxic stress as accumulated lipids could support growth following reoxygenation. Consistent with this, significantly higher proliferation was observed in hypoxic PCA cells following reoxygenation associated with rapid use of accumulated lipids. Importantly, lipid utilization inhibition by CPT1 inhibitor etomoxir and shRNA-mediated CPT1-knockdown significantly compromised hypoxic PCA cell proliferation following reoxygenation. Furthermore, COX2 inhibitor celecoxib strongly reduced growth and invasiveness following hypoxic PCA cells reoxygenation, and inhibited invasiveness induced by hypoxic PCA EVs. This establishes a role for COX2 enzymatic products in the enhanced PCA growth and invasiveness. Importantly, concentration and loading of EVs secreted by PCA cells were significantly compromised under delipidized serum condition and by lipogenesis inhibitors (fatostatin and silibinin). Overall, present study highlights the biological significance of lipid accumulation in hypoxic PCA cells and its therapeutic relevance in PCA.

In addition to their key role as structural lens proteins, α-crystallins also appear to confer protection against many eye diseases, including cataract, retinitis pigmentosa, and macular degeneration. Exogenous recombinant α-crystallin proteins were examined for their ability to prevent cell death induced by heat or oxidative stress in a human lens epithelial cell line (HLE-B3). Wild type αA- or αB-crystallin (WT-αA and WT-αB) and αA- or αB-crystallins, modified by the addition of a cell penetration peptide (CPP) designed to enhance the uptake of proteins into cells (gC-αB, TAT-αB, gC-αA), were produced by recombinant methods. In vitro chaperone-like assays were used to assay the ability of α-crystallins to protect client proteins from chemical or heat induced aggregation. In vivo viability assays were performed in HLE-B3 to determine whether pre-treatment with α-crystallins reduced death after exposure to oxidative or heat stress. Most of the five recombinant α-crystallin proteins tested conferred some in vitro protection from protein aggregation, with the greatest effect seen with WT-αB and gC-αB. All α-crystallins displayed significant protection to oxidative stress induced cell death, while only the αB-crystallins reduced cell death induced by thermal stress. Our findings indicate that the addition of the gC tag enhanced the protective effect of αB-crystallin against oxidative but not thermally-induced cell death. In conclusion, modifications that increase the uptake of α-crystallin proteins into cells, without destroying their chaperone-like activity and anti-apoptotic functions, create the potential to use these proteins therapeutically.

The role of neo-angiogenesis in prostate cancer (PCA) growth and metastasis is well established, but the development of effective and non-toxic pharmacological inhibitors of angiogenesis remains an unaccomplished goal. In this regard, targeting aberrant angiogenesis through non-toxic phytochemicals could be an attractive angiopreventive strategy against PCA. The rationale of the present study was to compare the anti-angiogenic potential of four pure diastereoisomeric flavonolignans, namely silybin A, silybin B, isosilybin A and isosilybin B, which we established previously as biologically active constituents in Milk Thistle extract. Results showed that oral feeding of these flavonolignans (50 and 100 mg/kg body weight) effectively inhibit the growth of advanced human PCA DU145 xenografts. Immunohistochemical analyses revealed that these flavonolignans inhibit tumor angiogenesis biomarkers (CD31 and nestin) and signaling molecules regulating angiogenesis (VEGF, VEGFR1, VEGFR2, phospho-Akt and HIF-1α) without adversely affecting the vessel-count in normal tissues (liver, lung, and kidney) of tumor bearing mice. These flavonolignans also inhibited the microvessel sprouting from mouse dorsal aortas ex vivo, and the VEGF-induced cell proliferation, capillary-like tube formation and invasiveness of human umbilical vein endothelial cells (HUVEC) in vitro. Further studies in HUVEC showed that these diastereoisomers target cell cycle, apoptosis and VEGF-induced signaling cascade. Three dimensional growth assay as well as co-culture invasion and in vitro angiogenesis studies (with HUVEC and DU145 cells) suggested the differential effectiveness of the diastereoisomers toward PCA and endothelial cells. Overall, these studies elucidated the comparative anti-angiogenic efficacy of pure flavonolignans from Milk Thistle and suggest their usefulness in PCA angioprevention.

Curcumin is a bioactive natural compound with a wide range of pharmacological properties, including antitumor activity; however, its clinical application has been limited because of its low solubility, stability, and bioavailability. In this study, a solid phase approach was proposed for the combinatorial synthesis of a mini library of the mimics of curcumin in good purity and yield. The non-effective findings in pancreatic cancer cells switched to strong growth inhibition and cell death efficacy for PC3 prostate cancer cells, and mimic 9, in which tyrosol (TYR) and homovanillyl alcohol (HVA) units were linked by a phosphodiester bond, was quite effective not only in cell growth inhibition but also in causing strong cell death under the study conditions and treatments that were not effective in PANC1 cells. The results got more exciting when we also consider the findings in SW480 human colorectal carcinoma cell line, where the growth inhibitor effects were more in line with that of the PC3 cells, but the lack of cell death effect was more in line with the PANC1 cells.

Sulfur mustard (SM) is a cytotoxic, vesicating, chemical warfare agent, first used in 1917; corneas are particularly vulnerable to SM exposure. They may develop inflammation, ulceration, neovascularization (NV), impaired vision, and partial/complete blindness depending upon the concentration of SM, exposure duration, and bio-physiological conditions of the eyes. Comprehensive in vivo studies have established ocular structural alterations, opacity, NV, and inflammation upon short durations (<4 min) of SM exposure. In this study, detailed analyses of histopathological alterations in corneal structure, keratocytes, inflammatory cells, blood vessels, and expressions of cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)-9, vascular endothelial growth factor (VEGF), and cytokines were performed in New Zealand white rabbits, in a time-dependent manner till 28 days, post longer durations (5 and 7 min) of ocular SM exposure to establish quantifiable endpoints of injury and healing. Results indicated that SM exposure led to duration-dependent increases in corneal thickness, opacity, ulceration, epithelial-stromal separation, and epithelial degradation. Significant increases in NV, keratocyte death, blood vessels, and inflammatory markers (COX-2, MMP-9, VEGF, and interleukin-8) were also observed for both exposure durations compared to the controls. Collectively, these findings would benefit in temporal delineation of mechanisms underlying SM-induced corneal toxicity and provide models for testing therapeutic interventions.

To demonstrate the ability of multiphoton microscopy to obtain full three-dimensional high-resolution images of the intact mouse eye anterior chamber without need for enucleation.

To visualize and quantify the three-dimensional (3D) spatial relationships of the structures of the aqueous outflow system (AOS) within intact enucleated mouse eyes using spectral two-photon microscopy (TPM) techniques.

Prostate cancer (PCa) is the leading malignancy among men. Importantly, this disease is mostly diagnosed at early stages offering a unique chemoprevention opportunity. Therefore, there is an urgent need to identify and target signaling molecules with higher expression/activity in prostate tumors and play critical role in PCa growth and progression. Here we report that NADPH oxidase (NOX) expression is directly associated with PCa progression in TRAMP mice, suggesting NOX as a potential chemoprevention target in controlling PCa. Accordingly, we assessed whether NOX activity in PCa cells could be inhibited by Graviola pulp extract (GPE) that contains unique acetogenins with strong anti-cancer effects. GPE (1-5 μg/ml) treatment strongly inhibited the hypoxia-induced NOX activity in PCa cells (LNCaP, 22Rv1 and PC3) associated with a decrease in the expression of NOX catalytic and regulatory sub-units (NOX1, NOX2 and p47(phox)). Furthermore, GPE-mediated NOX inhibition was associated with a strong decrease in nuclear HIF-1α levels as well as reduction in the proliferative and clonogenic potential of PCa cells. More importantly, GPE treatment neither inhibited NOX activity nor showed any cytotoxicity against non-neoplastic prostate epithelial PWR-1E cells. Overall, these results suggest that GPE could be useful in the prevention of PCa progression via inhibiting NOX activity.

Given the high rates of incidence and mortality associated with pancreatic cancer (PanC), there is a need to develop alternative strategies to target PanC. Recent studies have demonstrated that fruits of bitter melon (Momordica charantia) exhibit strong anticancer efficacy against PanC. However, the comparative effects of different bitter melon varieties have not been investigated. This has important implications, given that several bitter melon cultivars are geographically available but their differential effects are not known; and that on a global level, individuals could consume different bitter melon varieties sourced from different cultivars for anti-PanC benefits. Considering these shortcomings, in the present study, comparative pre-clinical anti-PanC studies have been conducted using lyophilized-juice and aqueous-methanolic extracts of the two most widely consumed but geographically diverse bitter melon varieties (Chinese [bitter melon juice; BMJ] and Indian [bitter melon extract; BME] variants). We observed that both BMJ and BME possess comparable efficacy against PanC growth and progression; specifically, these preparations have the potential to (a) inhibit PanC cell proliferation and induce cell death; (b) suppress PanC tumor growth, proliferation, and induce apoptosis; (c) restrict capillary tube formation by human umbilical vein endothelial cells, and decrease angiogenesis in PanC tumor xenografts. Thus, given the comparable pre-clinical anti-PanC efficacy of bitter melon cultivars, the geographical non-availability of a certain cultivar should not be a limiting factor in selecting a variant for moving forward for future clinical use/clinical trials either as a preventive or a therapeutic alternative for targeting PanC.

Herein, we assessed the stage-specific efficacy of inositol hexaphosphate (IP6, phytic acid), a bioactive food component, on prostate cancer (PCa) growth and progression in a transgenic mouse model of prostate cancer (TRAMP). Starting at 4, 12, 20, and 30 weeks of age, male TRAMP mice were fed either regular drinking water or 2% IP6 in water for ~8-15 weeks. Pathological assessments at study endpoint indicated that tumor grade is arrested at earlier stages by IP6 treatment; IP6 also prevented progression to more advanced forms of the disease (~55-70% decrease in moderately and poorly differentiated adenocarcinoma incidence was observed in advanced stage TRAMP cohorts). Next, we determined whether the protective effects of IP6 are mediated via its effect on the expansion of the cancer stem cells (CSCs) pool; results indicated that the anti-PCa effects of IP6 are associated with its potential to eradicate the PCa CSC pool in TRAMP prostate tumors. Furthermore, in vitro assays corroborated the above findings as IP6 decreased the % of floating PC-3 prostaspheres (self-renewal of CSCs) by ~90%. Together, these findings suggest the multifaceted chemopreventive-translational potential of IP6 intervention in suppressing the growth and progression of PCa and controlling this malignancy at an early stage.

With a possibility for the use of chemical weapons in battlefield or in terrorist activities, effective therapies against the devastating ocular injuries, from their exposure, are needed. Oxygen plays a vital role in ocular tissue preservation and wound repair. We tested the efficacy of supersaturated oxygen emulsion (SSOE) in reducing ex vivo corneal and keratocyte injury from chloropicrin (CP). CP, currently used as a pesticide, is a chemical threat agent like the vesicating mustard agents and causes severe corneal injury. Since our previous study in human corneal epithelial cells showed the treatment potential of SSOE (55 %), we further tested its efficacy in an ex vivo CP-induced rabbit corneal injury model. Corneas were exposed to CP (700 nmol) for 2 h, washed and cultured with or without SSOE for 24 h or 96 h. At 96 h post CP exposure, SSOE treatment presented a healing tendency of the corneal epithelial layer, and abrogated the CP-induced epithelial apoptotic cell death. SSOE treatment also reduced the CP induced DNA damage (H2A.X phosphorylation) and inflammatory markers (e.g. MMP9, IL-21, MIP-1β, TNFα). Further examination of the treatment efficacy of SSOE alone or in combination with other therapies in in vivo cornea injury models for CP and vesicants, is warranted.

Hypoxia in tumors is associated with resistance towards various therapies including radiotherapy. In this study, we assessed if hypoxia in cancer spheres could be effectively reduced by adding etomoxir (a β-oxidation inhibitor) immediately after cell irradiation.

African American men face a stark prostate cancer (PCa)-related health disparity, with the highest incidence and mortality rates compared to other races. Additional and innovative measures are warranted to reduce this health disparity. Here, we focused on the identification of a novel serum exosome-based "protein signature" for potential use in the early detection and better prognosis of PCa in African American men. Nanoparticle tracking analyses showed that compared to healthy individuals, exosome concentration (number/ml) was increased by ~3.2-fold (P ˂ 0.05) in the sera of African American men with PCa. Mass spectrometry-based proteomic analysis of serum exosomes identified seven unique and fifty-five overlapping proteins (up- or downregulated) in African Americans with PCa compared to healthy African Americans. Furthermore, ingenuity pathway analyses identified the inflammatory acute-phase response signaling as the top pathway associated with proteins loaded in exosomes from African American PCa patients. Interestingly, African American PCa E006AA-hT cells secreted exosomes strongly induced a proinflammatory M2-phenotype in macrophages and showed calcium response on sensory neurons, suggesting a neuroinflammatory response. Additionally, proteomic analyses showed that the protein Isoform 2 of Filamin A has higher loading (2.6-fold) in exosomes from African Americans with PCa, but a lesser loading (0.6-fold) was observed in exosomes from Caucasian men with PCa compared to race-matched healthy individuals. Interestingly, TCGA and Taylor's dataset as well as IHC analyses of PCa tissue showed a lower Filamin A expression in tissues of PCa patients compared with normal subjects. Overall, these results support the usefulness of serum exosomes to noninvasively detect inflammatory phenotype and to discover novel biomarkers associated with PCa in African American men.

Currently, there is no effective antidote to prevent skin injuries by sulfur mustard (SM) and nitrogen mustard (NM), which are vesicating agents with potential relevance to chemical warfare, terrorist attacks, or industrial/laboratory accidents. Our earlier report has demonstrated the therapeutic efficacy of silibinin, a natural flavanone, in reversing monofunctional alkylating SM analog 2-chloroethyl ethyl sulfide-induced toxic effects in mouse skin. To translate this effect to a bifunctional alkylating vesicant, herein, efficacy studies were carried out with NM. Topical application of silibinin (1 or 2mg) 30 min after NM exposure on the dorsal skin of male SKH-1 hairless mice significantly decreased NM-induced toxic lesions at 24, 72 or 120 h post-exposure. Specifically, silibinin treatment resulted in dose-dependent reduction of NM-induced increase in epidermal thickness, dead and denuded epidermis, parakeratosis and microvesication. Higher silibinin dose also caused a 79% and 51%reversal in NM-induced increases in myeloperoxidase activity and COX-2 levels, respectively. Furthermore, silibinin completely prevented NM-induced H2A.X phosphorylation, indicating reversal of DNA damage which could be an oxidative DNA damage as evidenced by high levels of 8-oxodG in NM-exposed mouse skin that was significantly reversed by silibinin. Together, these findings suggest that attenuation of NM-induced skin injury by silibinin is due to its effects on the pathways associated with DNA damage, inflammation, vesication and oxidative stress. In conclusion, results presented here support the optimization of silibinin as an effective treatment of skin injury by vesicants.

Chemical warfare agent sulfur mustard (HD) inflicts delayed blistering and incapacitating skin injuries. To identify effective countermeasures against HD-induced skin injuries, efficacy studies were carried out employing HD analog 2-chloroethyl ethyl sulfide (CEES)-induced injury biomarkers in skin cells and SKH-1 hairless mouse skin. The data demonstrate strong therapeutic efficacy of silibinin, a natural flavanone, in attenuating CEES-induced skin injury and oxidative stress. In skin cells, silibinin (10 µM) treatment 30 min after 0.35/0.5 mM CEES exposure caused a significant (p<0.05) reversal in CEES-induced decrease in cell viability, apoptotic and necrotic cell death, DNA damage, and an increase in oxidative stress. Silibinin (1 mg) applied topically to mouse skin 30 min post-CEES exposure (2 mg), was effective in reversing CEES-induced increases in skin bi-fold (62%) and epidermal thickness (85%), apoptotic cell death (70%), myeloperoxidase activity (complete reversal), induction of iNOS, COX-2, and MMP-9 protein levels (>90%), and activation of transcription factors NF-κB and AP-1 (complete reversal). Similarly, silibinin treatment was also effective in attenuating CEES-induced oxidative stress measured by 4-hydroxynonenal and 5,5-dimethyl-2-(8-octanoic acid)-1-pyrolline N-oxide protein adduct formation, and 8-oxo-2-deoxyguanosine levels. Since our previous studies implicated oxidative stress, in part, in CEES-induced toxic responses, the reversal of CEES-induced oxidative stress and other toxic effects by silibinin in this study indicate its pleiotropic therapeutic efficacy. Together, these findings support further optimization of silibinin in HD skin toxicity model to develop a novel effective therapy for skin injuries by vesicants.

Silybin or silibinin, a flavonolignan isolated from Milk thistle seeds, is one of the popular dietary supplements and has been extensively studied for its antioxidant, hepatoprotective and anti-cancer properties. We have envisioned that potency of silybin could be further enhanced through suitable modification/s in its chemical structure. Accordingly, here, we synthesized and characterized a series of silybin derivatives namely 2,3-dehydrosilybin (DHS), 7-O-methylsilybin (7OM), 7-O-galloylsilybin (7OG), 7,23-disulphatesilybin (DSS), 7-O-palmitoylsilybin (7OP), and 23-O-palmitoylsilybin (23OP); and compared their anti-cancer efficacy using human bladder cancer HTB9, colon cancer HCT116 and prostate carcinoma PC3 cells. In all the 3 cell lines, DHS, 7OM and 7OG demonstrated better growth inhibitory effects and compared to silybin, while other silybin derivatives showed lesser or no efficacy. Next, we prepared the optical isomers (A and B) of silybin, DHS, 7OM and 7OG, and compared their anti-cancer efficacy. Isomers of these three silybin derivatives also showed better efficacy compared with respective silybin isomers, but in each, there was no clear cut silybin A versus B isomer activity preference. Further studies in HTB cells found that DHS, 7OM and 7OG exert better apoptotic activity than silibinin. Clonogenic assays in HTB9 cells further confirmed that both the racemic mixtures as well as pure optical isomers of DHS, 7OM and 7OG were more effective than silybin. Overall, these results clearly suggest that the anti-cancer efficacy of silybin could be significantly enhanced through structural modifications, and identify strong anti-cancer efficacy of silybin derivatives, namely DHS, 7OM, and 7OG, signifying that their efficacy and toxicity should be evaluated in relevant pre-clinical cancer models in rodents.

Photodynamic therapy (PDT) laser light in conjunction with the benzoporphyrin derivative verteporfin is a current clinical treatment for choroidal vascular diseases such as age-related macular degeneration. The aim of this study was to examine the effects of PDT laser-activated and inactive verteporfin on various cultured ocular cells.

A better molecular understanding of prostate carcinogenesis is warranted to devise novel targeted preventive and therapeutic strategies against prostate cancer (PCA), a major cause of mortality among men. Here, we examined the role of two epithelial-to-mesenchymal transition (EMT) regulators, the adherens junction protein E-cadherin and its transcriptional repressor SNAI1, in regulating the aggressiveness of PCA cells.

To quantify the effects of modified Ahmed glaucoma valves® (AGV) with anti-fibrotic plate coatings or a plate surface micro-pattern on outflow resistance and tissue response.