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.

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.

Vesicating agent, Sulfur mustard (SM), causes devastating eye injury; however, there are no effective antidotes available. Using nitrogen mustard (NM), a bi-functional analog of SM, we have earlier reported that NM-induced corneal injury in ex vivo rabbit cornea organ culture model parallels corneal injury reported with SM. Using this model, we have demonstrated the therapeutic efficacy of dexamethasone (DEX), doxycycline (DOX) and silibinin (SB) in reversing NM (2h exposure)-induced corneal injuries when added immediately after washing NM. In the present study, we further examined the efficacy of similar/higher doses of these agents when added immediately, 2, or 4h after washing NM following its 2h exposure. All three treatment agents caused a reversal in established NM-induced injury biomarkers when added immediately or 2h after washing NM following its 2h exposure; however, when treatments were carried out 4h after washing NM, there was no significant effect. Together, our results further show the beneficial effect of these agents in reversing NM-induced corneal injury and indicate the time window for effective treatment. This could be useful towards future development of targeted therapeutics against vesicant-induced ocular injury.

Understanding the dynamics of autophagy and apoptosis crosstalk in cancer progression remains a challenging task. Here, we reported how the autophagy protein ATG14 induces lipophagy-mediated mitochondrial apoptosis. The overexpression of ATG14 in HeLa cells inhibited cell viability and increased mitochondrial apoptosis and endoplasmic reticulum (ER) stress. Furthermore, inhibition of this ATG14-induced autophagy promoted apoptosis. ATG14 overexpression resulted in the accumulation of free fatty acids (FFA), with a concomitant decrease in the number of lipid droplets. Our data showed that ER stress induced by ATG14 was due to the lipophagy-mediated FFA accumulation, which resulted in ROS-dependent mitochondrial stress leading to apoptosis. Inhibition of lipophagy in HeLa-ATG14 cells enhanced the cellular viability and rescued them from lipotoxicity. Mechanistically, we found that ATG14 interacted with Ulk1 and LC3, and knock down of Ulk1 prevented the lipidation of LC3 and autophagy in HeLa-ATG14 cells. We also identified a phosphatidylethanolamine (PE) binding region in ATG14, and the addition of Ulk1 to Hela-ATG14 cells decreased the ATG14-PE interaction. Lastly, confocal microscopy studies showed that the decrease in ATG14-PE binding was concomitant with the increase in LC3 lipidation over time, confirming the importance of Ulk1 to sort PE to LC3 during ATG14 mediated lipophagy induction. In conclusion, ATG14 and Ulk1 interact to induce lipophagy resulting in FFA accumulation leading to ER stress-mediated apoptosis.

Prostate cancer (PCa) deaths are typically the result of metastatic castration-resistant PCa (mCRPC). Recently, enzalutamide (Enz), an oral androgen receptor inhibitor, was approved for treating patients with mCRPC. Invariably, all PCa patients eventually develop resistance against Enz. Therefore, novel strategies aimed at overcoming Enz resistance are needed to improve the survival of PCa patients. The role of exosomes in drug resistance has not been fully elucidated in PCa. Therefore, we set out to better understand the exosome's role in the mechanism underlying Enz-resistant PCa. Results showed that Enz-resistant PCa cells (C4-2B, CWR-R1, and LNCaP) secreted significantly higher amounts of exosomes (2-4 folds) compared to Enz-sensitive counterparts. Inhibition of exosome biogenesis in resistant cells by GW4869 and dimethyl amiloride strongly decreased their cell viability. Mechanistic studies revealed upregulation of syntaxin 6 as well as its increased colocalization with CD63 in Enz-resistant PCa cells compared to Enz-sensitive cells. Syntaxin 6 knockdown by specific small interfering RNAs in Enz-resistant PCa cells (C4-2B and CWR-R1) resulted in reduced cell number and increased cell death in the presence of Enz. Furthermore, syntaxin 6 knockdown significantly reduced the exosome secretion in both Enz-resistant C4-2B and CWR-R1 cells. The Cancer Genome Atlas analysis showed increased syntaxin 6 expressions associated with higher Gleason score and decreased progression-free survival in PCa patients. Importantly, IHC analysis showed higher syntaxin 6 expression in cancer tissues from Enz-treated patients compared to Enz naïve patients. Overall, syntaxin 6 plays an important role in the secretion of exosomes and increased survival of Enz-resistant PCa cells.

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.

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.

Prostate cancer (PCa) is the most common cancer in men, and the global burden of the disease is rising. The majority of PCa deaths are due to metastasis that are highly resistant to current hormonal treatments; this state is called castration-resistant prostate cancer (CRPC). In this study, we focused on the role of the lipid catabolism enzyme CPT1A in supporting CRPC growth in an androgen-dependent manner. We found that androgen withdrawal promoted the growth of CPT1A over-expressing (OE) tumors while it decreased the growth of CPT1A under-expressing (KD) tumors, increasing their sensitivity to enzalutamide. Mechanistically, we found that CPT1A-OE cells burned more lipid and showed increased histone acetylation changes that were partially reversed with a p300 specific inhibitor. Conversely, CPT1A-KD cells showed less histone acetylation when grown in androgen-deprived conditions. Our results suggest that CPT1A supports CRPC by supplying acetyl groups for histone acetylation, promoting growth and antiandrogen resistance.

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.

Dietary rice bran-mediated inhibition of colon carcinogenesis was demonstrated previously for carcinogen-induced rodent models via multiple anti-cancer mechanisms. This study investigated the role of dietary rice bran-mediated changes to fecal microbiota and metabolites over the time course of colon carcinogenesis and compared murine fecal metabolites to human stool metabolic profiles following rice bran consumption by colorectal cancer survivors (NCT01929122). Forty adult male BALB/c mice were subjected to azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colitis-associated colon carcinogenesis and randomized to control AIN93M (n = 20) or diets containing 10% w/w heat-stabilized rice bran (n = 20). Feces were serially collected for 16S rRNA amplicon sequencing and non-targeted metabolomics. Fecal microbiota richness and diversity was increased in mice and humans with dietary rice bran treatment. Key drivers of differential bacterial abundances from rice bran intake in mice included Akkermansia, Lactococcus, Lachnospiraceae, and Eubacterium xylanophilum. Murine fecal metabolomics revealed 592 biochemical identities with notable changes to fatty acids, phenolics, and vitamins. Monoacylglycerols, dihydroferulate, 2-hydroxyhippurate (salicylurate), ferulic acid 4-sulfate, and vitamin B6 and E isomers significantly differed between rice bran- and control-fed mice. The kinetics of murine metabolic changes by the host and gut microbiome following rice bran consumption complemented changes observed in humans for apigenin, N-acetylhistamine, and ethylmalonate in feces. Increased enterolactone abundance is a novel diet-driven microbial metabolite fecal biomarker following rice bran consumption in mice and humans from this study. Dietary rice bran bioactivity via gut microbiome metabolism in mice and humans contributes to protection against colorectal cancer. The findings from this study provide compelling support for rice bran in clinical and public health guidelines for colorectal cancer prevention and control.

Use of the herb milk thistle (Silybum marianum) is widespread, and its chemistry has been studied for over 50 years. However, milk thistle endophytes have not been studied previously for their fungal and chemical diversity. We examined the fungal endophytes inhabiting this medicinal herb to determine: (1) species composition and phylogenetic diversity of fungal endophytes; (2) chemical diversity of secondary metabolites produced by these organisms; and (3) cytotoxicity of the pure compounds against the human prostate carcinoma (PC-3) cell line. Forty-one fungal isolates were identified from milk thistle comprising 25 operational taxonomic units based on BLAST search via GenBank using published authentic sequences from nuclear ribosomal internal transcribed spacer sequence data. Maximum likelihood analyses of partial 28S rRNA gene showed that these endophytes had phylogenetic affinities to four major classes of Ascomycota, the Dothideomycetes, Sordariomycetes, Eurotiomycetes, and Leotiomycetes. Chemical studies of solid-substrate fermentation cultures led to the isolation of four new natural products. In addition, 58 known secondary metabolites, representing diverse biosynthetic classes, were isolated and characterized using a suite of nuclear magnetic resonance and mass spectrometry techniques. Selected pure compounds were tested against the PC-3 cell line, where six compounds displayed cytotoxicity.

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.

Besides its neurotransmitter and vasoconstriction functions, serotonin is an important mediator of numerous biological processes in peripheral tissues including cell proliferation, steatosis, and fibrogenesis. Recent reports indicate that serotonin may promote tumor growth in liver cancer, however, the molecular mechanisms remain elusive. n this study, we investigated the role and molecular signaling mechanisms mediated by serotonin in liver cancer cell survival, drug resistance, and steatosis.

Phosgene Oxime (CX), an urticant or nettle agent categorized as a vesicant, is a potential chemical warfare and terrorist weapon. Its exposure can result in widespread and devastating effects including high mortality due to its fast penetration and ability to cause immediate severe cutaneous injury. It is one of the least studied chemical warfare agents with no effective therapy available. Thus, our goal was to examine the acute effects of CX following its cutaneous exposure in SKH-1 hairless mice to help establish a relevant injury model. Results from our study show that topical cutaneous exposure to CX vapor causes blanching of exposed skin with an erythematous ring, necrosis, edema, mild urticaria and erythema within minutes after exposure out to 8h post-exposure. These clinical skin manifestations were accompanied with increases in skin thickness, apoptotic cell death, mast cell degranulation, myeloperoxidase activity indicating neutrophil infiltration, p53 phosphorylation and accumulation, and an increase in COX-2 and TNFα levels. Topical CX-exposure also resulted in the dilatation of the peripheral vessels with a robust increase in RBCs in vessels of the liver, spleen, kidney, lungs and heart tissues. These events could cause a drop in blood pressure leading to shock, hypoxia and death. Together, this is the first report on effects of CX cutaneous exposure, which could help design further comprehensive studies evaluating the acute and chronic skin injuries from CX topical exposure and elucidate the related mechanism of action to aid in the identification of therapeutic targets and mitigation of injury.

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.

The current paradigm in the development of new cancer therapies is the ability to target tumor cells while avoiding harm to noncancerous cells. Furthermore, there is a need to develop novel therapeutic options against drug-resistant cancer cells. Herein, we characterized the placental-derived stem cell (PLSC) exosomes (PLSCExo) and evaluated their anti-cancer efficacy in prostate cancer (PCa) cell lines. Nanoparticle tracking analyses revealed the size distribution (average size 131.4 ± 0.9 nm) and concentration of exosomes (5.23 × 1010±1.99 × 109 per ml) secreted by PLSC. PLSCExo treatment strongly inhibited the viability of enzalutamide-sensitive and -resistant PCa cell lines (C4-2B, CWR-R1, and LNCaP cells). Interestingly, PLSCExo treatment had no effect on the viability of a non-neoplastic human prostate cell line (PREC-1). Mass spectrometry (MS) analyses showed that PLSCExo are loaded with 241 proteins and mainly with saturated fatty acids. Further, Ingenuity Pathway Analysis analyses of proteins loaded in PLSCExo suggested the role of retinoic acid receptor/liver x receptor pathways in their biological effects. Together, these results suggest the novel selective anti-cancer effects of PLSCExo against aggressive PCa cells.

MicroRNAs (miRNAs) are single-stranded non-coding RNA molecules that play a regulatory role in gene expression and cancer cell signaling. We previously identified miR-628-5p (miR-628) as a potential biomarker in serum samples from men with prostate cancer (PCa) (Srivastava et al. in Tumour Biol 35:4867-4873, 10.1007/s13277-014-1638-1, 2014). This study examined the detailed cellular phenotypes and pathways regulated by miR-628 in PCa cells. Since obesity is a significant risk factor for PCa, and there is a correlation between levels of the obesity-associated hormone leptin and PCa development, here we investigated the functional relationship between leptin and miR-628 regulation in PCa. We demonstrated that exposure to leptin downregulated the expression of miR-628 and increased cell proliferation/migration in PCa cells. We next studied the effects on cancer-related phenotypes in PCa cells after altering miR-628 expression levels. Enforced expression of miR-628 in PCa cells inhibited cell proliferation, reduced PCa cell survival/migration/invasion/spheroid formation, and decreased markers of cell stemness. Mechanistically, miR-628 binds with the JAG1-3'UTR and inhibits the expression of Jagged-1 (JAG1). JAG1 inhibition by miR-628 downregulated Notch signaling, decreased the expression of Snail/Slug, and modulated epithelial-mesenchymal transition and invasiveness in PC3 cells. Furthermore, expression of miR-628 in PCa cells increased sensitivity towards the drugs enzalutamide and docetaxel by induction of cell apoptosis. Collectively our data suggest that miR-628 is a key regulator of PCa carcinogenesis and is modulated by leptin, offering a novel therapeutic opportunity to inhibit the growth of advanced PCa.

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.

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.