Nitrogen Mustard-Induced Ex Vivo Human Cornea Injury Model and Therapeutic Intervention by Dexamethasone.

Sulfur mustard (SM), a vesicating agent first used during World War I, remains a potent threat as a chemical weapon to cause intentional/accidental chemical emergencies. Eyes are extremely susceptible to SM toxicity. Nitrogen mustard (NM), a bifunctional alkylating agent and potent analog of SM, is used in laboratories to study mustard vesicant-induced ocular toxicity. Previously, we showed that SM-/NM-induced injuries (in vivo and ex vivo rabbit corneas) are reversed upon treatment with dexamethasone (DEX), a US Food and Drug Administration-approved, steroidal anti-inflammatory drug. Here, we optimized NM injuries in ex vivo human corneas and assessed DEX efficacy. For injury optimization, one cornea (randomly selected from paired eyes) was exposed to NM: 100 nmoles for 2 hours or 4 hours, and 200 nmoles for 2 hours, and the other cornea served as a control. Injuries were assessed 24 hours post NM-exposure. NM 100 nmoles exposure for 2 hours was found to cause optimal corneal injury (epithelial thinning [∼69%]; epithelial-stromal separation [6-fold increase]). In protein arrays studies, 24 proteins displayed ≥40% change in their expression in NM exposed corneas compared with controls. DEX administration initiated 2 hours post NM exposure and every 8 hours thereafter until 24 hours post-exposure reversed NM-induced corneal epithelial-stromal separation [2-fold decrease]). Of the 24 proteins dysregulated upon NM exposure, six proteins (delta-like canonical Notch ligand 1, FGFbasic, CD54, CCL7, endostatin, receptor tyrosine-protein kinase erbB-4) associated with angiogenesis, immune/inflammatory responses, and cell differentiation/proliferation, showed significant reversal upon DEX treatment (Student's t test; P ≤ 0.05). Complementing our animal model studies, DEX was shown to mitigate vesicant-induced toxicities in ex vivo human corneas. SIGNIFICANCE STATEMENT: Nitrogen mustard (NM) exposure-induced injuries were optimized in an ex vivo human cornea culture model and studies were carried out at 24 h post 100 nmoles NM exposure. Dexamethasone (DEX) administration (started 2 h post NM exposure and every 8 h thereafter) reversed NM-induced corneal injuries. Molecular mediators of DEX action were associated with angiogenesis, immune/inflammatory responses, and cell differentiation/proliferation, indicating DEX aids wound healing via reversing vesicant-induced neovascularization (delta-like canonical Notch ligand 1 and FGF basic) and leukocyte infiltration (CD54 and CCL7).

Establishing a Dexamethasone Treatment Regimen To Alleviate Sulfur Mustard-Induced Corneal Injuries in a Rabbit Model.

Sulfur mustard (SM) is an ominous chemical warfare agent. Eyes are extremely susceptible to SM toxicity; injuries include inflammation, fibrosis, neovascularization (NV), and vision impairment/blindness, depending on the exposure dosage. Effective countermeasures against ocular SM toxicity remain elusive and are warranted during conflicts/terrorist activities and accidental exposures. We previously determined that dexamethasone (DEX) effectively counters corneal nitrogen mustard toxicity and that the 2-hour postexposure therapeutic window is most beneficial. Here, the efficacy of two DEX dosing frequencies [i.e., every 8 or 12 hours (initiated, as previously established, 2 hours after exposure)] until 28 days after SM exposure was assessed. Furthermore, sustained effects of DEX treatments were observed up to day 56 after SM exposure. Corneal clinical assessments (thickness, opacity, ulceration, and NV) were performed at the day 14, 28, 42, and 56 post-SM exposure time points. Histopathological assessments of corneal injuries (corneal thickness, epithelial degradation, epithelial-stromal separation, inflammatory cell, and blood vessel counts) using H&E staining and molecular assessments (COX-2, MMP-9, VEGF, and SPARC expressions) were performed at days 28, 42, and 56 after SM exposure. Statistical significance was assessed using two-way ANOVA, with Holm-Sidak post hoc pairwise multiple comparisons; significance was established if P < 0.05 (data represented as the mean ± S.E.M.). DEX administration every 8 hours was more potent than every 12 hours in reversing ocular SM injury, with the most pronounced effects observed at days 28 and 42 after SM exposure. These comprehensive results are novel and provide a comprehensive DEX treatment regimen (therapeutic-window and dosing-frequency) for counteracting SM-induced corneal injuries. SIGNIFICANCE STATEMENT: The study aims to establish a dexamethasone (DEX) treatment regimen by comparing the efficacy of DEX administration at 12 versus 8 hours initiated 2 hours after exposure. DEX administration every 8 hours was more effective in reversing sulfur mustard (SM)-induced corneal injuries. SM injury reversal during DEX administration (initial 28 days after exposure) and sustained [further 28 days after cessation of DEX administration (i.e., up to 56 days after exposure)] effects were assessed using clinical, pathophysiological, and molecular biomarkers.

Dermal Exposure to Vesicating Nettle Agent Phosgene Oxime: Clinically Relevant Biomarkers and Skin Injury Progression in Murine Models.

Phosgene oxime (CX), categorized as a vesicating chemical threat agent, causes effects that resemble an urticant or nettle agent. CX is an emerging potential threat agent that can be deployed alone or with other chemical threat agents to enhance their toxic effects. Studies on CX-induced skin toxicity, injury progression, and related biomarkers are largely unknown. To study the physiologic changes, skin clinical lesions and their progression, skin exposure of SKH-1 and C57BL/6 mice was carried out with vapor from 10 µl CX for 0.5-minute or 1.0-minute durations using a designed exposure system for consistent CX vapor exposure. One-minute exposure caused sharp (SKH-1) or sustained (C57BL/6) decrease in respiratory and heart rate, leading to mortality in both mouse strains. Both exposures caused immediate blanching, erythema with erythematous ring (wheel) and edema, and an increase in skin bifold thickness. Necrosis was also observed in the 0.5-minute CX exposure group. Both mouse strains showed comparative skin clinical lesions upon CX exposure; however, skin bifold thickness and erythema remained elevated up to 14 days postexposure in SKH-1 mice but not in C57BL/6 mice. Our data suggest that CX causes immediate changes in the physiologic parameters and gross skin lesions resembling urticaria, which could involve mast cell activation and intense systemic toxicity. This novel study recorded and compared the progression of skin injury to establish clinical biomarkers of CX dermal exposure in both the sexes of two murine strains relevant for skin and systemic injury studies and therapeutic target identification. SIGNIFICANCE STATEMENT: Phosgene oxime (CX), categorized as a vesicating agent, is considered as a potent chemical weapon and is of high military and terrorist threat interest since it produces rapid onset of severe injury as an urticant. However, biomarkers of clinical relevance related to its toxicity and injury progression are not studied. Data from this study provide useful clinical markers of CX skin toxicity in mouse models using a reliable CX exposure system for future mechanistic and efficacy studies.

Effect of nonsteroidal anti-inflammatory drugs (aspirin and naproxen) on inflammation-associated proteomic profiles in mouse plasma and prostate during TMPRSS2-ERG (fusion)-driven prostate carcinogenesis.

Recent preclinical studies have shown that the intake of nonsteroidal anti-inflammatory drugs (NSAIDs) aspirin and naproxen could be an effective intervention strategy against TMPRSS2-ERG fusion-driven prostate tumorigenesis. Herein, as a follow-up mechanistic study, employing TMPRSS2-ERG (fusion) positive tumors and plasma from TMPRSS2-ERG. Ptenflox/flox mice, we profiled the stage specific proteomic changes (focused on inflammatory circulating and prostate tissue/tumor-specific cytokines, chemokines, and growth factors/growth signaling-associated molecules) that contribute to prostate cancer (PCa) growth and progression in the TMPRSS2-ERG fusion-driven mouse model of tumorigenesis. In addition, the association of the protective effects of NSAIDs (aspirin 1400 ppm and naproxen 400 ppm) with the modulation of these specific molecular pathways was determined. A sandwich Elisa based membrane array-proteome profiler identifying 111 distinct signaling molecules was employed. Overall, the plasma and prostate tissue sample analyses identified 54 significant and differentially expressed cytokines, chemokines, and growth factors/growth signaling-associated molecules between PCa afflicted mice (TMPRSS2-ERG. Ptenflox/flox, age-matched noncancerous controls, NSAIDs-supplemented and no-drug controls). Bioinformatic analysis of the array outcomes indicated that the protective effect of NSAIDs was associated with reduced expression of (a) tumor promoting inflammatory molecules (M-CSF, IL-33, CCL22, CCL12, CX3CL1, CHI3L1, and CD93), (b) growth factors- growth signaling-associated molecules (Chemerin, FGF acidic, Flt-3 ligand, IGFBP-5, and PEDF), and (c) tumor microenvironment/stromal remodeling proteins MMP2 and MMP9. Overall, our findings corroborate the pathological findings that protective effects of NSAIDs in TMPSS2-ERG fusion-driven prostate tumorigenesis are associated with antiproliferative and anti-inflammatory effects and possible modulation of the immune cell enriched microenvironment.

Dexamethasone targets actin cytoskeleton signaling and inflammatory mediators to reverse sulfur mustard-induced toxicity in rabbit corneas.

PURPOSE: Sulfur mustard (SM), a bi-functional alkylating agent, was used during World War I and the Iran-Iraq war. SM toxicity is ten times higher in eyes than in other tissues. Cornea is exceptionally susceptible to SM-injuries due to its anterior positioning and mucous-aqueous interphase. Ocular SM exposure induces blepharitis, photosensitivity, dry eye, epithelial defects, limbal ischemia and stem cell deficiency, and mustard gas keratopathy leading to temporary or permanent vision impairments. We demonstrated that dexamethasone (Dex) is a potent therapeutic intervention against SM-induced corneal injuries; however, its mechanism of action is not well known. Investigations employing proteomic profiling (LC-MS/MS) to understand molecular mechanisms behind SM-induced corneal injury and Dex efficacy were performed in the rabbit cornea exposed to SM and then received Dex treatment. PEAKS studio was used to extract, search, and summarize peptide identity. Ingenuity Pathway Analysis was used for pathway identification. Validation was performed using immunofluorescence. One-Way ANOVA (FDR < 0.05; p < 0.005) and Student's t-test (p < 0.05) were utilized for analyzing proteomics and IF data, respectively. Proteomic analysis revealed that SM-exposure upregulated tissue repair pathways, particularly actin cytoskeleton signaling and inflammation. Prominently dysregulated proteins included lipocalin2, coronin1A, actin-related protein2, actin-related protein2/3 complex subunit2, actin-related protein2/3 complex subunit4, cell division cycle42, ezrin, bradykinin/kininogen1, moesin, and profilin. Upregulated actin cytoskeleton signaling increases F-actin formation, dysregulating cell shape and motility. Dex reversed SM-induced increases in the aforementioned proteins levels to near control expression profiles. Dex aids corneal wound healing and improves corneal integrity via actin cytoskeletal signaling and anti-inflammatory effects following SM-induced injuries.

Mouse Model of Nitrogen Mustard Ocular Surface Injury Characterization and Sphingolipid Signaling.

Vesicating chemicals like sulfur mustard (SM) or nitrogen mustard (NM) can cause devastating damage to the eyes, skin, and lungs. Eyes, being the most sensitive, have complicated pathologies that can manifest immediately after exposure (acute) and last for years (chronic). No FDA-approved drug is available to be used as medical counter measures (MCMs) against such injuries. Understanding the pathological mechanisms in acute and chronic response of the eye is essential for developing effective MCMs. Here, we report the clinical and histopathological characterization of a mouse model of NM-induced ocular surface injury (entire surface) developed by treating the eye with 2% (w/v) NM solution for 5 min. Unlike the existing models of specific injury, our model showed severe ocular inflammation, including the eyelids, structural deformity of the corneal epithelium and stroma, and diminished visual and retinal functions. We also observed alterations of the inflammatory markers and their expression at different phases of the injury, along with an activation of acidic sphingomyelinase (aSMase), causing an increase in bioactive sphingolipid ceramide and a reduction in sphingomyelin levels. This novel ocular surface mouse model recapitulated the injuries reported in human, rabbit, and murine SM or NM injury models. NM exposure of the entire ocular surface in mice, which is similar to accidental or deliberate exposure in humans, showed severe ocular inflammation and caused irreversible alterations to the corneal structure and significant vision loss. It also showed an intricate interplay between inflammatory markers over the injury period and alteration in sphingolipid homeostasis in the early acute phase.

Acacetin induces sustained ERK1/2 activation and RIP1-dependent necroptotic death in breast cancer cells.

Acacetin (AC), a naturally occurring flavonoid has shown anticancer potential. Herein, we studied the mechanisms of cell death and growth inhibition by AC in breast carcinoma T-47D and MDA-MB-231 cells. AC (10-40 µM) significantly decreased the levels of G2/M phase cyclins and CDKs, simultaneously increasing the expression of CDK inhibitors including Cip1/p21. A concentration-dependent increase in cell death was noted in both breast cancer cell lines with no such considerable effects on MCF-10A non-tumorigenic breast cells. The cell death-inducing potential of AC was further confirmed using confocal microscopy and flow cytometry analysis. AC resulted in mitochondrial superoxide generation, DNA damage, and ROS generation. N-acetyl cysteine (NAC) pre-treatment inhibited ROS generation and partially reversed ERK1/2 activation as well as cell death by AC. Further, AC enhanced the expression of RIP1 and RIP3, which mediate necroptosis. RIP1-specific inhibitor Necrostatin-1 (NS-1) reversed the AC-induced DNA damage and cell death. Collectively, these findings, for the first time, suggested that AC exerts its antitumor potential through ROS induction and RIP1-dependent necroptosis in breast carcinoma cells.

Metabolomics for identifying pathways involved in vesicating agent lewisite-induced corneal injury.

Lewisite (LEW) is an arsenical vesicant that can be a potentially dangerous chemical warfare agent (CWA). Eyes are particularly susceptible to vesicant induced injuries and ocular LEW exposure can act swiftly, causing burning of eyes, edema, inflammation, cell death and even blindness. In our previous studies, we developed a LEW exposure-induced corneal injury model in rabbit and showed increased inflammation, neovascularization, cell death, and structural damage to rabbit corneas upon LEW exposure. In the present study, we further assessed the metabolomic changes to delineate the possible mechanisms underlying the LEW-induced corneal injuries. This information is vital and could help in the development of effective targeted therapies against ocular LEW injuries. Thus, the metabolomic changes associated with LEW exposures in rabbit corneas were assessed as a function of time, to delineate pathways from molecular perturbations at the genomic and proteomic levels. New Zealand white rabbit corneas (n = 3-6) were exposed to LEW vapor (0.2 mg/L; flow rate: 300 ml/min) for 2.5 min (short exposure; low dose) or 7.5 min (long-exposure; high dose) and then collected at 1, 3, 7, or 14 days post LEW exposure. Samples were prepared using the automated MicroLab STAR® system, and proteins precipitated to recover the chemically diverse metabolites. Metabolomic analysis was carried out by reverse phase UPLC-MS/MS and gas chromatography (GC)-MS. The data obtained were analyzed using Metabolon's software. The results showed that LEW exposures at high doses were more toxic, particularly at the day 7 post exposure time point. LEW exposure was shown to dysregulate metabolites associated with all the integral functions of the cornea and cause increased inflammation and immune response, as well as generate oxidative stress. Additionally, all important metabolic functions of the cells were also affected: lipid and nucleotide metabolism, and energetics. The high dose LEW exposures were more toxic, particularly at day 7 post LEW exposure (>10-fold increased levels of histamine, quinolinate, N-acetyl-ß-alanine, GMP, and UPM). LEW exposure dysregulated integral functions of the cornea, caused inflammation and heightened immune response, and generated oxidative stress. Lipid and nucleotide metabolism, and energetics were also affected. The novel information about altered metabolic profile of rabbit cornea following LEW exposure could assist in delineating complex molecular events; thus, aid in identifying therapeutic targets to effectively ameliorate ocular trauma.

Chemopreventive efficacy of silibinin against basal cell carcinoma growth and progression in UVB-irradiated Ptch+/- mice.

The factors (environmental and genetic) contributing to basal cell carcinoma (BCC) pathogenesis are well-established; however, effective agents for BCC prevention are marred by toxic side-effects. Herein, we assessed the efficacy of flavonolignan silibinin against ultraviolet B (UVB)-induced BCC in Ptch+/- (heterozygous patched homolog 1 gene) mouse model. Both male and female Ptch+/- mice were irradiated with a 240 mJ/cm2 UVB dose 3 times/week for 26 or 46 weeks, with or without topical application of silibinin (9 mg/200 µl in acetone, applied 30 min before or after UVB exposure). Results indicated that silibinin application either pre- or post-UVB exposure for 26 weeks significantly decreased the number of BCC lesions by 65% and 39% (P < 0.001 for both) and the area covered by BCCs (72% and 45%, P < 0.001 for both), respectively, compared to UVB alone. Furthermore, continuous UVB exposure for 46 weeks increased the BCC lesion number and the BCC area covered by ~6 and ~3.4 folds (P < 0.001), respectively. Notably, even in this 46 week prolonged UVB exposure, silibinin (irrespective of pre- or post-UVB treatment) significantly halted the growth of BCCs by 81-94% (P < 0.001) as well as other epidermal lesions; specifically, silibinin treated tissues had less epidermal dysplasia, fibrosarcoma, and squamous cell carcinoma. Immunohistochemistry and immunofluorescence studies revealed that silibinin significantly decreased basal cell proliferation (Ki-67) and the expression of cytokeratins (14 and 15), and Hedgehog signaling mediators Smo and Gli1 in the BCC lesions. Together, our findings demonstrate strong potential of silibinin to be efficacious in preventing the growth and progression of UVB-induced BCC.

Stage-specific differential expression of zinc transporter SLC30A and SLC39A family proteins during prostate tumorigenesis.

Prostate cancer (PCa) initiation and progression uniquely modify the prostate milieu to aid unrestrained cell proliferation. One salient modification is the loss of the ability of prostate epithelial cells to accumulate high concentrations of zinc; however, molecular alterations associated with loss of zinc accumulating capability in malignant prostate cells remain poorly understood. Herein, we assessed the stage-specific expression of zinc transporters (ZNTs) belonging to the ZNT (SLC30A) and Zrt- and Irt-like protein (ZIP) (SLC39A) solute-carrier family in the prostate tissues of different genetically engineered mouse models (GEMM) of PCa (TMPRSS2-ERG.Ptenflox/flox , Hi-Myc+/- , and transgenic adenocarcinoma of mouse prostate), their age-matched wild-type controls, and 104 prostate core biopsies from human patients with different pathological lesions. Employing immunohistochemistry, differences in the levels of protein expression and spatial distribution of ZNT were evaluated as a function of the tumor stage. Results indicated that the expression of zinc importers (ZIP1, ZIP2, and ZIP3), which function to sequester zinc from circulation and prostatic fluid, was low to negligible in the membranes of the malignant prostate cells in both GEMM and human prostate tissues. Regarding zinc exporters (ZNT1, ZNT2, ZNT9, and ZNT10) that export excess zinc into the extracellular spaces or intracellular organelles, their expression was low in normal prostate glands of mice and humans; however, it was significantly upregulated in prostate adenocarcinoma lesions in GEMM and PCa patients. Together, our findings provide new insights into altered expression of ZNTs during the progression of PCa and indicate that changes in zinc homeostasis could possibly be an early-initiation event during prostate tumorigenesis and a likely prevention/intervention target.

Characterization of stage-specific tumor progression in TMPRSS2-ERG (fusion)-driven and non-fusion-driven prostate cancer in GEM models.

In the present study, we performed a comparative stage-specific pathological and molecular marker evaluation of TMPRSS2-ERG fusion and PTEN loss-driven (TMPRSS2-ERG. Ptenflox/flox ) versus non-fusion-driven prostate tumorigenesis (Hi-Myc) in mice. Anterior, ventral, and dorsolateral prostates were collected from mice at different ages (or time points post-Cre induction). Results indicated that growth and progression of prostatic intraepithelial lesions to adenocarcinoma stages occurred in both mice models albeit at different rates. In the TMPRSS2-ERG. Ptenflox/flox mice, the initiation of tumorigenesis was slow, but subsequent progression through different stages became increasingly faster. Adenocarcinoma stage was reached early on; however, no high-grade undifferentiated tumors were observed. Conversely, in the Hi-Myc+/- mice, tumorigenesis initiation was rapid; however, progression through different stages was relatively slower and it took a while to reach the more aggressive phenotype stage. Nevertheless, at the advanced stages in the Hi-Myc+/- mice, high-grade undifferentiated tumors were observed compared to the later stage tumors observed in the fusion-driven TMPRSS2-ERG. Ptenflox/flox mice. These results were corroborated by the stage specific-pattern in the molecular expression of proliferation markers (PCNA and c-Myc); androgen receptor (AR); fusion-resultant overexpression of ERG; Prostein (SLC45-A3); and angiogenesis marker (CD-31). Importantly, there was a significant increase in immune cell infiltrations, which increased with the stage of tumorigenesis, in the TMPRSS2-ERG fusion-positive tumors relative to fusion negative tumors. Together, these findings are both novel and highly significant in establishing a working preclinical model for evaluating the efficacy of interventions during different stages of tumorigenesis in TMPRSS2-ERG fusion-driven PCa.

Gender-based effect of absence of gut microbiota on the protective efficacy of Bifidobacterium longum-fermented rice bran diet against inflammation-associated colon tumorigenesis.

Dietary rice bran (RB) has shown capacity to influence metabolism by modulation of gut microbiota in individuals at risk for colorectal cancer (CRC), which warranted attention for delineating mechanisms for bidirectional influences and cross-feeding between the host and RB-modified gut microbiota to reduce CRC. Accordingly, in the present study, fermented rice bran (FRB, fermented with a RB responsive microbe Bifidobacterium longum), and non-fermented RB were fed as 10% w/w (diet) to gut microbiota-intactspf or germ-free micegf to investigate comparative efficacy against inflammation-associated azoxymethane/dextran sodium sulfate (AOM/DSS)-induced CRC. Results indicated both microbiota-dependent and independent mechanisms for RB meditated protective efficacy against CRC that was associated with reduced neoplastic lesion size and local-mucosal/systemic inflammation, and restoration of colonic epithelial integrity. Enrichment of beneficial commensals (such as, Clostridiales, Blautia, Roseburia), phenolic metabolites (benzoate and catechol metabolism), and dietary components (ferulic acid-4 sulfate, trigonelline, and salicylate) were correlated with anti-CRC efficacy. Germ-free studies revealed gender-specific physiological variables could differentially impact CRC growth and progression. In the germ-free females, the RB dietary treatment showed a ∼72% reduction in the incidence of colonic epithelial erosion when compared to the ∼40% reduction in FRB-fed micegf . Ex vivo fermentation of RB did not parallel the localized-protective benefits of gut microbial metabolism by RB in damaged colonic tissues. Findings from this study suggest potential needs for safety considerations of fermented fiber rich foods as dietary strategies against severe inflammation-associated colon tumorigenesis (particularly with severe damage to the colonic epithelium).

Effect of dexamethasone treatment at variable therapeutic windows in reversing nitrogen mustard-induced corneal injuries in rabbit ocular in vivo model.

Nitrogen mustard (NM) is an analogue of the potent vesicating agent sulfur mustard, with well-established ocular injury models in rabbit eyes to study vesicant-induced ocular toxicity. The effects of NM-exposure to eyes may include irritation, redness, inflammation, fibrosis, epithelial degradation, blurred vision, partial/complete blindness, which may be temporary or permanent, depending on the route, duration, and dosage of exposure. Effective countermeasures against vesicant exposure are presently not available and are warranted in case of any terrorist activity or accidental leakage from stockpiles. Herein, our focus was to evaluate whether dexamethasone (DEX), an FDA approved potent corticosteroid with documented anti-inflammatory activities, could be an effective treatment modality. Accordingly, utilizing NM-induced corneal injuries in rabbit ocular in vivo model, we examined and compared the efficacy of DEX treatments when administration was started at early (2 h), intermediate (4 h), and late (6 h) therapeutic windows of intervention after NM-exposure and administered every 8 h thereafter. The effects of NM-exposure and DEX treatments were evaluated on clinical (corneal opacity, ulceration, and neovascularization), biological (epithelial thickness, epithelial-stromal separation, blood vessels density, and inflammatory cell and keratocyte counts) and molecular (COX-2 and VEGF expression) parameters, at day 1, 3, 7 and 14. Results indicated that DEX treatment markedly and effectively reversed the NM-induced injury markers in rabbit corneas. Early administration of DEX at 2 h was found to be most effective in reversing NM-induced corneal injuries, followed by DEX 4 h and DEX 6 h administration initiation, indicating that DEX has best efficacy at the early therapeutic window in our study model.

Research models of sulfur mustard- and nitrogen mustard-induced ocular injuries and potential therapeutics.

Sulfur mustard (SM) is a notorious, bifunctional alkylating vesicant that was first used in warfare during World War I in 1917 and since then has been deployed in numerous skirmishes with its most recent documented use being during the Middle Eastern conflicts. Apart from its use in combat and terrorist activities, continual threat of accidental exposure from old stockpiles and improperly discarded munitions is ever present, especially to the innocent and unassuming civilian populations. SM can cause devastating injuries, depending on the dosage of SM exposure, route of exposure, as well as the physiological conditions of the individuals exposed. The most common routes of exposure are ocular, dermal, and exposure to the lungs and respiratory tissues through inhalation. Eyes are the most susceptible organ to SM-induced toxicities owing to their high moisture content and rapidly dividing cells. Additionally, ocular injury causes the most expeditious disablement of individuals even upon whole-body exposures. Therefore, it is imperative to understand the mechanisms underlying SM-induced ocular toxicity and design therapeutic interventions to prevent/mitigate ocular injuries. Ocular SM exposure may cause a wide range of symptoms such as inflammation, lacrimation, itching, dryness, photophobia, edema of the cornea/sclera/retina/iris, conjunctivitis, degradation of the corneal layer, fusion of two or more ocular layers, neovascularization, fibrosis, and temporary or permanent structural damage to one or more ocular layers. These symptoms may lead to vision impairments, resulting in partial or complete blindness that may be permanent. The highly toxic and exceedingly notorious nature of SM makes it a highly regulated chemical, requiring very expensive licensing, security, and safety requirements; thus, the more easily accessible analogue, nitrogen mustard (NM) that mimics SM-induced toxicity and injuries is employed in plethora of studies conducted in different animal models and culture systems. This review provides a comprehensive account of the injuries and symptoms that occur upon ocular SM exposures in human patients as well as studies in animal (in vivo, ex vivo) and cell (in vitro) models of SM and NM ocular exposures. Special emphasis has been laid on highlighting the strengths and lacunae in the research as well as the possible unexplored avenues of mechanisms underlying mustard-induced ocular injury that can be explored in future research endeavors. Furthermore, development of therapeutic interventions and targets of interest in the ocular system exposed to SM and NM, based on studies in human patients as well as in vivo, ex vivo, and in vitro models has been discussed in great depth, providing a valuable knowledge database to delineate pathways associated with vesicant-induced toxicity, and strategies/diagnostic tools against SM-induced toxicity.

Solid-phase synthesis of curcumin mimics and their anticancer activity against human pancreatic, prostate, and colorectal cancer cell lines.

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.

Exosomes secreted by prostate cancer cells under hypoxia promote matrix metalloproteinases activity at pre-metastatic niches.

Approximately, 30 000 men die from prostate cancer (PCa) every year in the United States, mainly due to the metastasis. Thus, the key events associated with PCa metastasis are under rigorous investigation, with recent studies showing that preparation of pre-metastatic niches (PMN) in distant organs is an important step. However, the molecular basis for PMN preparation is still unclear. Hypoxia in primary tumors promotes aggressiveness; however, its precise role in metastasis is not clear. We recently reported that exosomes secreted by PCa cells under hypoxia promote stemness and invasiveness in naïve PCa cells; however, whether these extracellular vesicles also influence PMN remains unknown. In the present study, we isolated exosomes from human PCa PC3 cells under normoxic (21% O2 , exosomes secreted under normoxic condition [ExoNormoxic ]) and hypoxic (1% O2 , exosomes secreted under hypoxic condition [ExoHypoxic ]) conditions, and characterized their effect (10 µg exosomes, intraperitoneal (IP) treatment every 48 hours for 4 weeks) on key biomarkers associated with PMN in nude mice. Whole animal fluorescence imaging showed that ExoHypoxic treatment promotes matrix metalloproteinases (MMPs) activity in several putative metastatic sites. Histological studies confirmed that ExoHypoxic treatment enhanced the level of MMP2, MMP9, and extracellular matrix proteins (fibronectin and collagen) as well as increased the number of CD11b+ cells at selective PMN sites. Furthermore, proteomic profiling of exosomes by liquid chromatography/mass spectrometry identified cargo proteins in ExoNormoxic and ExoHypoxic as well as distinct canonical pathways targeted by them. These results suggest that exosomes secreted by PCa cells under hypoxia plausibly remodel distant PMN, and thus, could be a potential target to control metastatic PCa.

Bitter melon juice intake with gemcitabine intervention circumvents resistance to gemcitabine in pancreatic patient-derived xenograft tumors.

Chemoresistance to gemcitabine (GEM)-a frontline chemotherapeutic, resulting from its dysfunctional uptake and metabolism in cancer cells, is a major contributing factor for failed therapy in pancreatic cancer (PanC) patients. Therefore, there is an urgent need for agents that could reverse GEM resistance and allow continued chemosensitivity to the drug. We employed natural nontoxic agent (with anti-PanC potential) bitter melon juice (BMJ) and GEM to examine their combinatorial benefits against tumorigenesis of PanC patient-derived xenograft (PDX)-pancreatic ductal adenocarcinomas explants PDX272 (wild-type KRAS), PDX271 (mutant KRAS and SMAD4), and PDX266 (mutant KRAS). Anti-PanC efficacy of single agents vs combination in the three tumor explants, both at the end of active dosing regimen and following a drug-washout phase were compared. In animal studies, GEM alone treatment significantly inhibited PDX tumor growth, but effects were not sustained, as GEM-treated tumors exhibited regrowth posttreatment termination. However, combination-regimen displayed enhanced and sustained efficacy. Mechanistic assessments revealed that overcoming GEM resistance by coadministration with BMJ was possibly due to modulation of GEM transport/metabolism pathway molecules (ribonucleotide reductase regulatory subunit M1, human equilibrative nucleoside transporter 1, and deoxycytidine kinase). Study outcomes, highlighting significantly higher and sustained efficacy of GEM in combination with BMJ, make a compelling case for a clinical trial in PanC patients, wherein BMJ could be combined with GEM to target and overcome GEM resistance. In addition, given their specific effectiveness against KRAS-mutant tumors, this combination could be potentially beneficial to a broader PanC patient population.

Antiangiogenic therapy with Nintedanib affects hypoxia, angiogenesis and apoptosis in the ventral prostate of TRAMP animals.

The antiangiogenic therapy for prostate cancer with Nintedanib, a potent inhibitor of important growth factor receptors, has been proven to delay tumor progression and arrest tumor growth; thus, the aim herein is to evaluate Nintedanib effects on tumor cells, besides angiogenesis and apoptosis processes, metalloproteinases and hypoxia factor in an animal model. Nintedanib promoted growth inhibition and cell death in a dose-dependent manner, showing no tumor selectivity. Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) were treated with Nintedanib (10 mg/kg/day) in different stages of tumor development and the ventral prostate was examined for protein levels by means of immunohistochemistry and Western blotting and apoptosis evaluation. In vitro antiproliferative activity of Nintedanib was also assessed in nine human tumor cell lines. Early Nintedanib treatment has shown decreased levels of FGF-2, VEGFR-1, MMP-9 and HIF-1α and a significantly increased apoptosis of epithelial cells. Furthermore, late Nintedanib treatment decreased FGF-2, VEGFR-1 and FGFR-3 levels. Importantly, even after treatment discontinuation, treated animals displayed a significant decrease in VEGFR-1 as well as MMP-9. Although Nintedanib treatment in late stages of tumor growth has shown some good results, it is noteworthy that the drug presents the best tissue response when administered in the early stages of disease development. Nintedanib treatment has shown to be a promising approach for prostate cancer therapy, especially in the early stages of the disease, interfering in different carcinogenesis progression pathways.

Multilocus sequence typing of Clostridium perfringens strains from neonatal calves, dairy workers and associated environment in India.

Clostridium perfringens is a globally recognized zoonotic pathogen. We report isolation and genotyping of C. perfringens from neonatal calves, dairy workers and their associated environment in India. A total of 103 fecal samples from neonatal calves, 25 stool swabs from the dairy workers and 50 samples from their associated environment were collected from two dairy farms. C. perfringens was detected in 26 out of 103 (25.2%) neonatal calf samples, 7 out of 25 (28%) human stool samples and 17 out of 50 (34%) environmental samples. C. perfringens type A strains were predominant in neonatal calves (24/26; 92.3%) and associated environment (15/17; 88.2%). In contrast, strains from dairy workers mostly belonged to type F (5/7; 71.4%), which also carried the beta2 toxin gene. Seventeen strains were analyzed by multilocus sequence typing (MLST) for studying genotypic relationship along with 188 C. perfringens strains available from public databases. A total of 112 sequence types (STs) were identified from 205 C. perfringens strains analyzed. A Clonal complex (CC) represented by three STs (ST 98, ST 41 and ST 110) representing predominantly type F (18/20 strains) were mostly associated with human illnesses. Among predominant STs, ST 54 was associated with enteritis cases in foals and dogs and ST 58 associated with necrotic enteritis in poultry. Seventeen Indian strains were assigned to 13 STs. Genetic relatedness among strains of calves, dairy worker and associated environments indicate inter-host transfers and zoonotic spreads.

Targeting Fat Oxidation in Mouse Prostate Cancer Decreases Tumor Growth and Stimulates Anti-Cancer Immunity.

Lipid catabolism represents an Achilles heel in prostate cancer (PCa) that can be exploited for therapy. CPT1A regulates the entry of fatty acids into the mitochondria for beta-oxidation and its inhibition has been shown to decrease PCa growth. In this study, we examined the pharmacological blockade of lipid oxidation with ranolazine in TRAMPC1 PCa models. Oral administration of ranolazine (100 mg/Kg for 21 days) resulted in decreased tumor CD8+ T-cells Tim3 content, increased macrophages, and decreased blood myeloid immunosuppressive monocytes. Using multispectral staining, drug treatments increased infiltration of CD8+ T-cells and dendritic cells compared to vehicle. Functional studies with spleen cells of drug-treated tumors co-cultured with TRAMPC1 cells showed increased ex vivo T-cell cytotoxic activity, suggesting an anti-tumoral response. Lastly, a decrease in CD4+ and CD8+ T-cells expressing PD1 was observed when exhausted spleen cells were incubated with TRAMPC1 Cpt1a-KD compared to the control cells. These data indicated that genetically blocking the ability of the tumor cells to oxidize lipid can change the activation status of the neighboring T-cells. This study provides new knowledge of the role of lipid catabolism in the intercommunication of tumor and immune cells, which can be extrapolated to other cancers with high CPT1A expression.