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Purpose Gallic acid, a natural agent present in a wide-range of fruits and vegetables, has been of potential interest as an anti-cancer agent; herein, we evaluated its efficacy in androgen-independent DU145 and androgen-dependent-22Rv1 human prostate cancer (PCa) cells. Materials and Methods Cell viability was determined by MTT and apoptosis by Annexin V-PI assays. In vivo anti-cancer efficacy was assessed by DU145 and 22Rv1 xenograft growth in nude mice given normal drinking water or one supplemented with 0.3% or 1% (w/v) gallic acid. PCNA, TUNEL and CD31 immunostaining was performed in tumor tissues for in vivo anti-proliferative, apoptotic and anti-angiogenic effects of gallic acid. Results Gallic acid decreased cell viability in a dose-dependent manner in both DU145 and 22Rv1 cells largely via apoptosis induction. In tumor studies, gallic acid feeding inhibited the growth of DU145 and 22Rv1 PCa xenografts in nude mice. Immunohistochemical analysis revealed significant inhibition of tumor cell proliferation, induction of apoptosis, and reduction of microvessel density in tumor xenografts from gallic acid-fed mice as compared to controls in both DU145 and 22Rv1 models. Conclusion Taken together, our findings show the anti-PCa efficacy of gallic acid and provide a rationale for additional studies with this naturally-occurring agent for its efficacy against PCa.

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.

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. In this work, we characterized the proteins loaded in exosomes isolated from the serum of African American prostate cancer patients and identified a novel protein signature.

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.

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.

Malignant gliomas are one of the most devastating and incurable tumors. Sustained excessive angiogenesis by glioma cells is the major reason for their uncontrolled growth and resistance toward conventional therapies resulting in high mortality. Therefore, targeting angiogenesis should be a logical strategy to prevent or control glioma cell growth. Earlier studies have shown that Asiatic Acid (AsA), a pentacyclic triterpenoid, is effective against glioma and other cancer cells; however, its efficacy against angiogenesis remains unknown. In the present study, we examined the anti-angiogenic efficacy of AsA using human umbilical vein endothelial cells (HUVEC) and human brain microvascular endothelial cells (HBMEC). Our results showed that AsA (5-20 µM) inhibits HUVEC growth and induces apoptotic cell death by activating caspases (3 and 9) and modulating the expression of apoptosis regulators Bad, survivin and pAkt-ser473. Further, AsA showed a dose-dependent inhibition of HUVEC migration, invasion and capillary tube formation, and disintegrated preformed capillary network. AsA also inhibited the VEGF-stimulated growth and capillary tube formation by HUVEC and HBMEC. Next, we analyzed the angiogenic potential of conditioned media collected from human glioma LN18 and U87-MG cells treated with either DMSO (control conditioned media, CCM) or AsA 20 µM (AsA20 conditioned media, AsA20CM). CCM from glioma cells significantly enhanced the capillary tube formation in both HUVEC and HBMEC, while capillary tube formation in both endothelial cell lines was greatly compromised in the presence of AsA20CM. Consistent with these results, VEGF expression was lesser in AsA20CM compared to CCM, and indeed AsA strongly inhibited VEGF level (both cellular and secreted) in glioma cells. AsA also showed dose-dependent anti-angiogenic efficacy in Matrigel plug assay, and inhibited the glioma cells potential to attract HUVEC/HBMEC. Overall, the present study clearly showed the strong anti-angiogenic potential of AsA and suggests its usefulness against malignant gliomas.

Silymarin, a defined mixture of natural flavonoid, has recently been shown to have potent cancer chemopreventive efficacy against colon carcinogenesis in rat model; however, the mechanism of such efficacy is not elucidated. Here, using pure active agent in silymarin, namely silibinin, we show its antiproliferative and apoptotic effects, and associated molecular alterations in human colon carcinoma HT-29 cells. Silibinin treatment of cells at 50–100  μ g/ml doses resulted in a moderate to very strong growth inhibition in a dose- and a time-dependent manner, which was largely due to a G0/G1 arrest in cell cycle progression; higher dose and longer treatment time also caused a G2/M arrest. In mechanistic studies related its effect on cell cycle progression, silibinin treatment resulted in an upregulation of Kip1/p27 and Cip1/p21 protein as well as mRNA levels, and decreased CDK2, CDK4, cyclin E and cyclin D1 protein levels together with an inhibition in CDK2 and CDK4 kinase activities. In other studies, we observed that G2/M arrest by silibinin was associated with a decrease in cdc25C, cdc2/p34 and cyclin B1 protein levels, as well as cdc2/p34 kinase activity. In the studies assessing biological fate of silibinin-treated cells, silibinin-induced cell cycle arrest and growth inhibition were not associated with cellular differentiation, but caused apoptotic death. The quantitative apoptosis analysis showed up to 15% apoptotic cell death after 48 h of silibinin treatment. Interestingly, silibinin-induced apoptosis in HT-29 cells was independent of caspases activation, as all caspases inhibitor did not reverse silibinin-induced apoptosis. This observation was further confirmed by the findings showing a lack in caspases activity increase and caspases and PARP cleavage as well as a lack in cytochrome c release in cytosol following silibinin treatment of HT-29 cells. Additional studies conducted in mice showed that silibinin doses found effective in HT-29 cells are achievable in plasma, which increases the significance of the present findings and their possible translation in in vivo anticancer efficacy of silibinin against colon cancer. Together, these results identify molecular mechanisms of silibinin efficacy as a cell cycle regulator and apoptosis inducer in human colon carcinoma HT-29 cells, and justify further studies to investigate potential usefulness of this nontoxic agent in colon cancer prevention and intervention.

Silybin is a natural compound extensively studied for its hepatoprotective, neuroprotective and anticancer properties. Envisioning the enhancement of silybin potential by suitable modifications in its chemical structure, here, a series of new 7-O-alkyl silybins derivatives were synthesized by the Mitsunobu reaction starting from the silybins and tyrosol-based phenols, such as tyrosol (TYR, 3), 3-methoxytyrosol (MTYR, 4), and 3-hydroxytyrosol (HTYR, 5). This research sought to explore the antioxidant and anticancer properties of eighteen new derivatives and their mechanisms. In particular, the antioxidant properties of new derivatives outlined by the DPPH assay showed a very pronounced activity depending on the tyrosyl moiety (HTYR > MTYR >> TYR). A significant contribution of the HTYR moiety was observed for silybins and 2,3-dehydro-silybin-based derivatives. According to the very potent antioxidant activity, 2,3-dehydro-silybin derivatives 15ab, 15a, and 15b exerted the most potent anticancer activity in human prostate cancer PC-3 cells. Furthermore, flow cytometric analysis for cell cycle and apoptosis revealed that 15ab, 15a, and 15b induce strong G1 phase arrest and increase late apoptotic population in PC-3 cells. Additionally, Western blotting for apoptotic marker cleaved caspase-3 confirmed apoptosis induction by these silybin derivatives in PC-3 cells. These findings hold significant importance in the investigation of anticancer properties of silybin derivatives and strongly encourage swift investigation in pre-clinical models and clinical trials.

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.

A loss of functional androgen receptor and an enhanced expression of growth factor receptors and associated ligands are causal genetic events in prostate cancer (PCA) progression. These genetic alterations lead to an epigenetic mechanism where a feedback autocrine loop between membrane receptor and ligand (e.g. EGFR–TGF α ) results in a constitutive activation of MAPK-Elk1–AP1-mediated mitogenic signaling in human PCA at an advanced and androgen-independent stage. We rationalized that inhibiting these epigenetic events could be useful in controlling advanced PCA growth. Recently, we found that grape seed extract (GSE), a dietary supplement rich in flavonoid procyanidins, inhibits advanced and androgen-independent human PCA DU145 cell growth in culture and nude mice. Here, we performed detailed mechanistic studies to define the effect of GSE on EGFR–Shc–MAPK–Elk1–AP1-mediated mitogenic signaling in DU145 cells. Pretreatment of serum-starved cells with GSE resulted in 70% to almost complete inhibition of EGF-induced EGFR activation and 50% to complete inhibition of Shc activation, which corroborated with a comparable decrease in EGF-induced Shc binding to EGFR. Conversely, EGF-induced ERK1/2 phosphorylation was inhibited only by lower doses of GSE; in fact, higher doses showed an increase. Additional studies showed that GSE alone causes a dose- and time-dependent increase in ERK1/2 phosphorylation in starved DU145 cells that is inhibited by an MEK1 inhibitor PD98059. Independent of this increase in ERK1/2 phosphorylation, GSE showed a strong inhibition of ERK1/2 kinase activity to Elk1 in both cellular and cell-free systems. GSE treatment of cells also inhibited both EGF-induced and constitutively active Elk1 phosphorylation and AP1 activation. GSE treatment also showed DNA synthesis inhibition in starved and EGF-stimulated cells as well as loss of cell viability and apoptotic death that was further increased by adding MEK1 inhibitor. Since GSE strongly induced apoptosis independent of its affect on an increase in phospho-ERK1/2, we hypothesized that apoptotic effect of GSE could be by other mechanism(s) including its effect on stress-associated MAPK, the JNK. Indeed, GSE-treated cells showed a strong and sustained increase in phospho-JNK1/JNK2 levels, JNK activity and phospho-cJun levels. An inhibition of GSE-induced JNK activation by a novel JNK inhibitor SP600125 resulted in a significant reversal of GSE-induced apoptotic death suggesting the involvement of JNK activation by GSE in its apoptosis response. Together, these results suggest that anticancer effects of GSE in PCA be mediated via impairment of EGFR–ERK1/2–Elk1–AP1-mediated mitogenic signaling and activation of JNK causing growth inhibition and apoptosis, respectively.