Phosphodiesterase 4 Inhibition as a Therapeutic Target for Alcoholic Liver Disease: From Bedside to Bench.

Alcoholic liver disease (ALD) is a major cause of liver-related mortality. There is still no US Food and Drug Administration-approved therapy for ALD, and therefore, identifying therapeutic targets is needed. Our previous work demonstrated that ethanol exposure leads to up-regulation of cAMP-degrading phosphodiesterase 4 (PDE4) expression, which compromises normal cAMP signaling in monocytes/macrophages and hepatocytes. This effect of ethanol on cAMP signaling contributes to dysregulated inflammatory response and altered lipid metabolism. It is unknown whether chronic alcohol consumption in humans alters hepatic PDE4 expression and cAMP signaling and whether inadequate cAMP signaling plays a pathogenic role in alcohol-induced liver injury. Our present work shows that expression of the PDE4 subfamily of enzymes is significantly up-regulated and cAMP levels are markedly decreased in hepatic tissues of patients with severe ALD. We also demonstrate the anti-inflammatory efficacy of roflumilast, a clinically available PDE4 inhibitor, on endotoxin-inducible proinflammatory cytokine production ex vivo in whole blood of patients with alcoholic hepatitis. Moreover, we demonstrate that ethanol-mediated changes in hepatic PDE4 and cAMP levels play a causal role in liver injury in in vivo and in vitro models of ALD. This study employs a drug delivery system that specifically delivers the PDE4 inhibitor rolipram to the liver to avoid central nervous system side effects associated with this drug. Our results show that PDE4 inhibition significantly attenuates ethanol-induced hepatic steatosis and injury through multiple mechanisms, including reduced oxidative and endoplasmic reticulum stress both in vivo and in vitro. Conclusion: Increased PDE4 plays a pathogenic role in the development of ALD; hence, directed interventions aimed at inhibiting PDE4 might be an effective treatment for ALD.

A natural molecule, urolithin A, downregulates androgen receptor activation and suppresses growth of prostate cancer.

Androgen ablation therapy is the primary therapeutic option for locally advanced and metastatic castration-resistant prostate cancer (CRPC). We investigated therapeutic effect of a dietary metabolite Urolithin A (UroA) and dissected the molecular mechanism in CRPC cells. Treatment with UroA inhibited cell proliferation in both androgen receptor-positive (AR+ ) (C4-2B) and androgen receptor-negative (AR- ) (PC-3) cells however, AR+ CaP cells were more sensitive to UroA treatment as compared with AR- CaP cells. Inhibition of the AR signaling was responsible for the UroA effect on AR+ CaP cells. Ectopic expression of AR in PC-3 cells sensitized them to UroA treatment as compared to the vector-expresseing PC-3 cells, which suggests that AR could be a target of UroA. Similarly, in enzalutamide-resistant C4-2B cells, a downregulation of AR expression also suppressed cell proliferation which was observed with the UroA treatment. Oral administration of UroA significantly suppressed the growth of C4-2B xenografts (P = 0.05) compared with PC-3 xenografts (P = 0.069) without causing toxicity to animals. Immunohistochemistry analysis confirmed in vitro findings such as downregulation of AR/pAKT signaling in UroA-treated C4-2B tumors, which suggests that UroA may be a potent chemo-preventive and therapeutic agent for CRPC.

Development of a goat model for evaluation of withaferin A: Cervical implants for the treatment of cervical intraepithelial neoplasia.

Cervical cancer is caused by human papillomavirus (HPV). The disease develops over many years through a series of precancerous lesions. Cervical cancer can be prevented by HPV-vaccination, screening and treatment of precancer before development of cervical cancer. The treatment of high-grade cervical dysplasia (CIN 2+) has traditionally been by cervical conization. Surgical procedures are associated with increased risk of undesirable side effects including bleeding, infection, scarring (stenosis), infertility and complications in later pregnancies. An inexpensive, non-invasive method of delivering therapeutics locally will be favorable to treat precancerous cervical lesions without damaging healthy tissue. The feasibility and safety of a sustained, continuous drug-releasing cervical polymeric implant for use in clinical trials was studied using a large animal model. The goat (Capra hircus), non-pregnant adult female Boer goats, was chosen due to similarities in cervical dimensions to the human. Estrus was induced with progesterone CIDR® vaginal implants for 14days followed by the administration of chorionic gonadotropins 48h prior to removal of the progesterone implants to relax the cervix to allow for the placement of the cervical implant. Cervical implants, containing 2% and 4% withaferin A (WFA), with 8 coats of blank polymer, provided sustained release for a long duration and were used for the animal study. The 'mushroom'-shaped cervical polymeric implant, originally designed for women required redesigning to be accommodated within the goat cervix. The cervical implants were well tolerated by the animals with no obvious evidence of discomfort, systemic or local inflammation or toxicity. In addition, we developed a new method to analyze tissue WFA levels by solvent extractions and LS/MS-MS. WFA was found to be localized to the target and adjacent tissues with 12-16ng WFA/g tissue, with essentially no detectable WFA in distant tissues. This study suggests that the goat is a good large animal model for the future development and evaluation of therapeutic efficacy of continuous local drug delivery by cervical polymeric implants to treat precancerous cervical lesions.

Chemoprevention of Rat Mammary Carcinogenesis by Apiaceae Spices.

Scientific evidence suggests that many herbs and spices have medicinal properties that alleviate symptoms or prevent disease. In this study, we examined the chemopreventive effects of the Apiaceae spices, anise, caraway, and celery seeds against 17ß-estrogen (E2)-mediated mammary tumorigenesis in an ACI (August-Copenhagen Irish) rat model. Female ACI rats were given either control diet (AIN 93M) or diet supplemented with 7.5% (w/w) of anise, caraway, or celery seed powder. Two weeks later, one half of the animals in each group received subcutaneous silastic implants of E2. Diet intake and body weight were recorded weekly, and animals were euthanized after 3 and 12 weeks. E2-treatment showed significantly (2.1- and 3.4-fold) enhanced growth of pituitary gland at 3 and 12 weeks, respectively. All test spices significantly offset the pituitary growth by 12 weeks, except celery which was effective as early as three weeks. Immunohistochemical analysis for proliferative cell nuclear antigen (PCNA) in mammary tissues showed significant reduction in E2-mediated mammary cell proliferation. Test spices reduced the circulating levels of both E2 and prolactin at three weeks. This protection was more pronounced at 12 weeks, with celery eliciting the highest effect. RT-PCR and western blot analysis were performed to determine the potential molecular targets of the spices. Anise and caraway diets significantly offset estrogen-mediated overexpression of both cyclin D1 and estrogen receptor α (ERα). The effect of anise was modest. Likewise, expression of CYP1B1 and CYP1A1 was inhibited by all test spices. Based on short-term molecular markers, caraway was selected over other spices based on its enhanced effect on estrogen-associated pathway. Therefore, a tumor-end point study in ACI rats was conducted with dietary caraway. Tumor palpation from 12 weeks onwards revealed tumor latency of 29 days in caraway-treated animals compared with first tumor appearance at 92 days in control group. At the end of the study (25 weeks), the tumor incidence was 96% in the control group compared with only 70% in the caraway group. A significant reduction in tumor volume (661 ± 123 vs. 313 ± 81 mm³) and tumor multiplicity (4.2 ± 0.4 vs. 2.5 ± 0.5 tumors/animal) was also observed in the caraway group compared with the control group. Together, our data show dietary caraway can significantly delay and prevent the hormonal mammary tumorigenesis by modulating different cellular and molecular targets.

Tributyrin Mitigates Ethanol-Induced Lysine Acetylation of Histone-H3 and p65-NFκB Downregulating CCL2 Expression and Consequent Liver Inflammation and Injury.

PURPOSE: Chemokine-driven leukocyte infiltration and sustained inflammation contribute to alcohol-associated liver disease (ALD). Elevated hepatic CCL2 expression, seen in ALD, is associated with disease severity. However, mechanisms of CCL2 regulation are not completely elucidated. Post-translational modifications (PTMs) of proteins, particularly acetylation, modulate gene expression. This study examined the acetylation changes of promoter-associated histone-H3 and key transcription factor-NFκB in regulating hepatic CCL2 expression and subsequent inflammation and injury. Further, the effect of therapeutic modulation of the acetylation state by tributyrin (TB), a butyrate prodrug, was assessed. METHODS: Hepatic CCL2 expression was assessed in mice fed control (PF) or an ethanol-containing Lieber-DeCarli (5% v/v, EF) diet for 7 weeks with or without oral administration of tributyrin (TB, 2 g/kg, 5 days/week). A chromatin immunoprecipitation (ChIP) assay evaluated promoter-associated modifications. Nuclear association between SIRT1, p300, and NFκB-p65 and acetylation changes of p65 were determined using immunoprecipitation and Western blot analyses. A Student's t-test and one-way ANOVA determined the significance. RESULTS: Ethanol significantly increased promoter-associated histone-H3-lysine-9 acetylation (H3K9Ac), reflecting a transcriptionally permissive state with a resultant increase in hepatic CCL2 mRNA and protein expression. Moreover, increased lysine-310-acetylation of nuclear RelA/p65 decreased its association with SIRT1, a class III HDAC, but concomitantly increased with p300, a histone acetyltransferase. This further led to enhanced recruitment of NF-κB/p65 and RNA polymerase-II to the CCL2 promoter. Oral TB administration prevented ethanol-associated acetylation changes, thus downregulating CCL2 expression, hepatic neutrophil infiltration, and inflammation/ injury. CONCLUSION: The modulation of a protein acetylation state via ethanol or TB mechanistically regulates hepatic CCL2 upregulation in ALD.

SmartPill™ Administration to Assess Gastrointestinal Function after Spinal Cord Injury in a Porcine Model-A Preliminary Study.

Gastrointestinal (GI) complications, including motility disorders, metabolic deficiencies, and changes in gut microbiota following spinal cord injury (SCI), are associated with poor outcomes. After SCI, the autonomic nervous system becomes unbalanced below the level of injury and can lead to severe GI dysfunction. The SmartPill™ is a non-invasive capsule that, when ingested, transmits pH, temperature, and pressure readings that can be used to assess effects in GI function post-injury. Our minipig model allows us to assess these post-injury changes to optimize interventions and ultimately improve GI function. The aim of this study was to compare pre-injury to post-injury transit times, pH, and pressures in sections of GI tract by utilizing the SmartPill™ in three pigs after SCI at 2 and 6 weeks. Tributyrin was administered to two pigs to assess the influences on their gut microenvironment. We observed prolonged GET (Gastric Emptying Time) and CTT (Colon Transit Time), decreases in contraction frequencies (Con freq) in the antrum of the stomach, colon, and decreases in duodenal pressures post-injury. We noted increases in Sum amp generated at 2 weeks post-injury in the colon, with corresponding decreases in Con freq. We found transient changes in pH in the colon and small intestine at 2 weeks post-injury, with minimal effect on stomach pH post-injury. Prolonged GETs and CTTs can influence the absorptive profile in the gut and contribute to pathology development. This is the first pilot study to administer the SmartPill™ in minipigs in the context of SCI. Further investigations will elucidate these trends and characterize post-SCI GI function.

Controlled-release systemic delivery - a new concept in cancer chemoprevention.

Many chemopreventive agents have encountered bioavailability issues in pre-clinical/clinical studies despite high oral doses. We report here a new concept utilizing polycaprolactone implants embedded with test compounds to obtain controlled systemic delivery, circumventing oral bioavailability issues and reducing the total administered dose. Compounds were released from the implants in vitro dose dependently and for long durations (months), which correlated with in vivo release. Polymeric implants of curcumin significantly inhibited tissue DNA adducts following the treatment of rats with benzo[a]pyrene, with the total administered dose being substantially lower than typical oral doses. A comparison of bioavailability of curcumin given by implants showed significantly higher levels of curcumin in the plasma, liver and brain 30 days after treatment compared with the dietary route. Withaferin A implants resulted in a nearly 60% inhibition of lung cancer A549 cell xenografts, but no inhibition occurred when the same total dose was administered intraperitoneally. More than 15 phytochemicals have been tested successfully by this formulation. Together, our data indicate that this novel implant-delivery system circumvents oral bioavailability issues, provides continuous delivery for long durations and lowers the total administered dose, eliciting both chemopreventive/chemotherapeutic activities. This would also allow the assessment of activity of minor constituents and synthetic metabolites, which otherwise remain uninvestigated in vivo.

Oxidative DNA adducts detected in vitro from redox activity of cigarette smoke constituents.

Cigarette smoke contains a variety of carcinogens, cocarcinogens, mutagens, and tumor promoters. In addition to polycyclic aromatic carcinogens and tobacco-specific nitrosamines, cigarette smoke also contains an abundance of catechols, aldehydes, and other constituents, which are DNA damaging directly or indirectly; therefore, they can also contribute to cigarette smoke-mediated carcinogenicity. In this study, we investigated the potential of cigarette smoke constituents to induce oxidative damage to DNA through their capacity to redox cycle. When DNA (300 µg/mL) was incubated with cigarette smoke condensate (0.2 mg of tobacco particulate matter/mL) and CuCl(2) as a catalyst (50-100 µM), a variety of oxidative DNA adducts were detected by (32)P-postlabeling/TLC. Of the total adduct burden (2114 ± 419 adducts/10(6) nucleotides), over 40% of all adducts were attributed to the benchmark oxidative DNA lesion, 8-oxodeoxyguanosine (8-oxodG). Adducts were formed dose dependently. Essentially, similar adduct profiles were obtained when cigarette smoke condensate was substituted with ortho- and para-dihydroxybenzenes. Vehicle treatment with Cu(2+) or CSC alone did not induce any significant amount of oxidative DNA damage. Furthermore, coincubation of cigarette smoke condensate and ortho-dihydroxybenzene with DNA resulted in a higher amount of oxidative DNA adducts than obtained with the individual entity, suggesting that adducts presumably originated from catechols or catechol-like compounds in cigarette smoke condensate. Adducts resulting from both cigarette smoke condensate and pure dihydroxybenzenes were chromatographically identical to adducts formed by reaction of DNA with H(2)O(2), which is known to produce 8-oxodG, and many other oxidative DNA adducts. When the cigarette smoke condensate-DNA reaction was performed in the presence of ellagic acid, a known antioxidant, the adduct formation was inhibited dose dependently, further suggesting that adducts originated from oxidative pathway. Our data thus provide evidence of the capacity of catechols or catechol-like constituents in cigarette smoke to produce oxidative DNA damage, which may contribute to the tumor-promoting activity of cigarette smoke.

Oxidative DNA damage following microsome/Cu(II)-mediated activation of the estrogens, 17ß-estradiol, equilenin, and equilin: role of reactive oxygen species.

Experimental and epidemiological data associate the exposure of estrogens to cancer development in several tissues, particularly, the breast, endometrium, liver, and kidney. One plausible mechanism of estrogen-mediated carcinogenicity is DNA damage by redox cycling of estrogen catechols. Reports have shown that metabolism of estrogens results in 2- and 4-hydroxylation to catechol metabolites which can then redox cycle. We examined the capacity of the endogenous estrogen, 17ß-estradiol, and two equine estrogens which formulate a significant proportion of hormone replacement drugs, equilenin and equilin, to induce oxidatively generated DNA damage. Microsome/Cu(II)-mediated activation of all three estrogens resulted in numerous oxidation DNA adducts, as detected by (32)P-postlabeling/TLC. Essentially the same DNA oxidation pattern was also found when catechol estrogens were incubated with DNA in the presence of Cu(II) suggesting that redox cycling of catechol estrogens mediates the formation of these DNA adducts. Since the oxidation patterns induced by estrogen catechols and other chemically diverse catechols were chromatographically identical to those generated by Fenton-type chemistry and these adducts were inhibited by known ROS modifiers (up to 96%), this oxidatively generated DNA damage is believed to be the product of the attack of free radicals on DNA, rather than direct addition of the estrogen quinones. These data support a mechanistic role by endogenous and synthetic estrogens to induce oxidative DNA damage in addition to specific DNA adducts.

Vaginal cells of smokers are more resistant to human papillomavirus infection than that of non-smokers.

To evaluate effect of HPV and smoking on DNA double-strand breaks in vaginal samples, vaginal specimens collected from participants (n=76) were classified based on HPV and smoking status, and DNA double-strand breaks measured using comet assay. Mean tail length (31.2±18.7µm) and tail moment (2.4±2.8 arbitrary units) for HPV-positive patients were lower (p<0.001) compared with HPV-negative patients (61.7±22.6µm; 8.7±4.9AU). Never-smokers were found to have a higher level (p<0.001) of double-strand breaks (57.7±24.5µm, 7.5±5.5AU) compared with ever smokers (35.3±21.9µm; 3.4±3.7AU). Among HPV infected patients, never-smokers have more double-strand breaks compared to smokers (p<0.001) which correlated with age (p<0.001). Highly differentiated vaginal epithelium may be resistant to DNA damage associated with HPV infection and smoking, which may be attributed to adoptive survival mechanisms of vaginal epithelium.

Enhanced activity of punicalagin delivered via polymeric implants against benzo[a]pyrene-induced DNA adducts.

We investigated the effect of punicalagin (PC) on benzo[a]pyrene (BP)-induced DNA adducts in vitro and in vivo. Incubation of BP (1 µM) with rat liver microsomes, appropriate co-factors and DNA in the presence of vehicle or punicalagin (1-40 µM) showed dose-dependent inhibition of the resultant DNA adducts, with essentially complete (97%) inhibition at 40 µM. However, PC failed to inhibit anti-BPDE-induced DNA adducts when tested in an in vitro non-microsomal system, suggesting that the inhibition of the microsomal BP-DNA adducts occurred due to inhibition of P450 1A1 by PC. To determine its efficacy in vivo, female S/D rats were administered punicalagin via the diet (1500 ppm; approximately 19 mg/day/animal) or subcutaneous polymeric implants (two 2-cm, 200mg with 20% drug load; 40 mg PC/implant) and then treated with continuous low-dose of BP by a subcutaneous polymeric implant (2 cm, 200mg with 10% load; 20mg BP/implant) and euthanized after 10 days. Analysis of the lung DNA by (32)P-postlabeling showed significant (60%; p=0.029) inhibition of DNA adducts by PC administered via the implants; the dietary route showed modest (34%) but statistically insignificant inhibition. Furthermore, total PC administered by implants was approximately 38-fold lower compared with the dietary route. Analysis of the lung microsomes showed significant inhibition of cytochrome P450 1A1 activity and induction of glutathione. Release of PC from the implants was found to be biphasic starting with a burst release, followed by a gradual decline. Ultra performance liquid chromatography analysis showed no detectable PC in the plasma but its hydrolyzed product, ellagic acid was readily detected. The plasma concentration of ellagic acid was over two orders of magnitude higher (589 ± 78 ng/mL) in the implant group compared with diet (4.36 ± 0.83 ng/mL). Together, our data show that delivery of PC by implants can reduce its effective dose substantially, and that the inhibition of DNA adducts in vivo occurred presumably due to the conversion of PC to ellagic acid.

Anti-proliferative activity and protection against oxidative DNA damage by punicalagin isolated from pomegranate husk.

Ellagitannins are the most abundant polyphenols in pomegranate (Punica granatum) husk and contribute greatly towards its biological properties. A pre-enriched pomegranate husk powder was extracted with water and then further purified by an Amberlite XAD-16 column. Punicalagin (PC) anomers were eluted using a gradient of methanol and water. Fractions eluted with 20% and 25% methanol yielded 1.08 g of light brown powder (purity > 97%) from a total of 40 g of extract. This fraction was identified as PC by HPLC-UV using reference compounds and confirmed by FTICR-MS analysis. PC (10-40 µM) was found to significantly inhibit oxidative DNA products, about 70% inhibition at 40 µM (p=0.0017), resulting from Cu2+-catalyzed redox cycling of 4-hydroxy-17ß-estradiol as analyzed by 32P-postlabeling. Evidence of high antioxidant activity of PC was also obtained based on ORAC assay (1556±79 µmol of TE/g), as well as by 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS)-, 2,2-diphenyl-1-picrylhydrazyl (DPPH)-, hydrogen peroxide (H2O2) scavenging and ferrous ion-chelating activities (IC50=1.1, 17.1, 24 and 45.4 µg/ml, respectively). Further, PC exhibited strong anti-proliferative activity against the human lung, breast and cervical cancer cell lines. Together, these data suggest that PC can be isolated in its purified form by simple column chromatography, inhibits oxidative DNA damage and possesses high anti-proliferative activity.

Sustained overexpression of CYP1A1 and 1B1 and steady accumulation of DNA adducts by low-dose, continuous exposure to benzo[a]pyrene by polymeric implants.

Many carcinogenesis and tumorigenesis studies reported in the past several decades have relied upon bolus dose(s) of test compounds to determine their DNA damage and carcinogenic potential. The high doses are far from the human scenario where exposure is almost always to low doses and for long duration. In this study, we report a novel polymeric implant system that provides continuous ("24/7") exposure to low doses using benzo[a]pyrene (BP) as a model carcinogen. Cylindrical implants (1 cm length, 3.2 mm diameter; 10 mg BP/100 mg implant) prepared from polycaprolactone:F68 (9:1) showed controlled release in vitro for long duration. To determine the rate of release and biochemical effects in vivo, groups of female Sprague-Dawley rats received either no treatment or subcutaneous sham or BP implants (1 cm, 10% load) and were euthanized after 6, 15, 30, and 180 days; the average dose of BP by the implant route was 16.7 ± 3 µg/rat. For comparison, rats were also treated with a single bolus dose of BP intraperitoneally (10 mg/rat) and euthanized at 6, 15, and 30 days. DNA adducts analyzed by (32)P-postlabeling in the lung and liver increased steadily with time with levels reaching 31 ± 3 and 17 ± 6 adducts/10(9) nucleotides, respectively, after 25 weeks; the adduct burden in the mammary tissue initially increased but then declined with time presumably due to high cell turn over. In contrast, the bolus dose treatment showed the highest DNA adduct levels after 6 days, followed by a steady decline. The steady accumulation of tissue DNA adducts in the implant groups corroborates the sustained overexpression of CYP1A1 and 1B1, the cytochrome P450s involved in the conversion of BP to its electrophilic metabolites. In contrast, the overexpression of CYP1A1 and 1B1 resulting from the bolus dose of BP lasted only for a few days. This is the first demonstration revealing that low-dose, continuous exposure to environmental polycyclic aromatic hydrocarbons such as BP can render sustained expression of CYPs and steady accumulation of tissue DNA adducts. On the basis of our recent study in which we showed the presence of 17ß-estradiol in the lung, the sustained overexpression of CYP1A1 and 1B1 due to continuous exposure to BP may increase the susceptibility to estrogen-mediated carcinogenicity.

Sustained systemic delivery of green tea polyphenols by polymeric implants significantly diminishes benzo[a]pyrene-induced DNA adducts.

The polyphenolics in green tea are believed to be the bioactive components. However, poor bioavailability following ingestion limits their efficacy in vivo. In this study, polyphenon E (poly E), a standardized green tea extract, was administered by sustained-release polycaprolactone implants (two, 2-cm implants; 20% drug load) grafted subcutaneously or via drinking water (0.8% w/v) to female S/D rats. Animals were treated with continuous low dose of benzo[a]pyrene (BP) via subcutaneous polymeric implants (2 cm; 10% load) and euthanized after 1 and 4 weeks. Analysis of lung DNA by (32)P-postlabeling resulted in a statistically significant reduction (50%; p = 0.023) of BP-induced DNA adducts in the implant group; however, only a modest (34%) but statistically insignificant reduction occurred in the drinking water group at 1 week. The implant delivery system also showed significant reduction (35%; p = 0.044) of the known BP diolepoxide-derived DNA adduct after 4 weeks. Notably, the total dose of poly E administered was >100-fold lower in the implant group than the drinking water group (15.7 versus 1,632 mg, respectively). Analysis of selected phase I, phase II, and nucleotide excision repair enzymes at both mRNA and protein levels showed no significant modulation by poly E, suggesting that the reduction in the BP-induced DNA adducts occurred presumably due to known scavenging of the antidiolepoxide of BP by the poly E catechins. In conclusion, our study demonstrated that sustained systemic delivery of poly E significantly reduced BP-induced DNA adducts in spite of its poor bioavailability following oral administration.

Development and in vitro-in vivo evaluation of polymeric implants for continuous systemic delivery of curcumin.

PURPOSE: The introduction of curcumin into clinics is hindered by its low water solubility and poor bioavailability. To overcome these limitations, we developed curcumin implants using poly (ε-caprolactone) as the polymeric matrix. METHODS: Implants were prepared by melt-extrusion method; in vitro drug release was optimized for effects of polymer composition, drug load, surface area and water-soluble additives. Implants were also tested under in vivo conditions for cumulative curcumin release, and liver concentration was correlated with its efficacy to modulate selected xenobiotic-metabolizing enzymes (CYP1A1 and GSTM). RESULTS: Drug release from implants followed biphasic release pattern with Higuchi kinetics and was proportional to the surface area of implants. Drug release increased proportionately from 2 to 10% (w/w) drug load, and incorporation of 10% (w/w) of water-soluble additives (F-68, PEG 8000 and cyclodextrin) did not significantly alter the drug release. In vivo drug release was found to be ~1.8 times higher than in vitro release. Curcumin was detected at 60 ± 20 ng/g in the liver after four days of implantation and was almost constant (8-15 ng/g) for up to 35 days. This time-dependent drop in curcumin level was found to be due to induction of CYP1A1 and GSTM (µ) enzymes which led to increased metabolism of curcumin. CONCLUSION: Our data showed that these implants were able to release curcumin for long duration and to modulate liver phase I and phase II enzymes, demonstrating curcumin's biological efficacy delivered via this delivery system.

Tributyrin Inhibits Ethanol-Induced Epigenetic Repression of CPT-1A and Attenuates Hepatic Steatosis and Injury.

Ethanol-mediated down-regulation of carnitine palmitoyltransferase-1 (CPT-1A) gene expression plays a major role in the development of hepatic steatosis; however, the underlying mechanisms are not completely elucidated. Tributyrin, a butyrate prodrug that can inhibit histone deacetylase (HDAC) activity, attenuates hepatic steatosis and injury. The present study examined the beneficial effect of tributyrin/butyrate in attenuating ethanol-induced pathogenic epigenetic mechanisms affecting CPT-1A promoter-histone modifications and gene expression and hepatic steatosis/injury. METHODS: Mice were fed a liquid Lieber-DeCarli diet (Research Diet Inc, New Brunswick, NJ) with or without ethanol for 4 weeks. In a subset of mice, tributyrin (2 g/kg) was administered orally by gavage. Primary rat hepatocytes were treated with 50 mmol/L ethanol and/or 2 mmol/L butyrate. Gene expression and epigenetic modifications at the CPT-1A promoter were analyzed by chromatin immunoprecipitation analysis. RESULTS: In vivo, ethanol induced hepatic CPT-1A promoter histone H3K9 deacetylation, which is indicative of a repressive chromatin state, and decreased CPT-1A gene expression. Our data identified HDAC1 as the predominant HDAC causing CPT-1A promoter histone H3K9 deacetylation and epigenetic down-regulation of gene expression. Significantly, Specificity Protein 1 (SP1) and Hepatocyte Nuclear Factor 4 Alpha (HNF4α) participated in the recruitment of HDAC1 to the proximal and distal regions of CPT-1A promoter, respectively, and mediated transcriptional repression. Importantly, butyrate, a dietary HDAC inhibitor, attenuated ethanol-induced recruitment of HDAC1 and facilitated p300-HAT binding by enabling SP1/p300 interaction at the proximal region and HNF4α/peroxisomal proliferator-activated receptor-γ coactivator-1α/p300 interactions at the distal region, leading to promoter histone acetylation and enhanced CPT-1A transcription. CONCLUSIONS: This study identifies HDAC1-mediated repressive epigenetic mechanisms that underlie an ethanol-mediated decrease in CPT-1A expression. Importantly, tributyrin/butyrate inhibits HDAC1, rescues CPT-1A expression, and attenuates ethanol-mediated hepatic steatosis and injury, suggesting its potential use in therapeutic strategies for alcoholic liver disease.

Dietary berries and ellagic acid prevent oxidative DNA damage and modulate expression of DNA repair genes.

DNA damage is a pre-requisite for the initiation of cancer and agents that reduce this damage are useful in cancer prevention. In this study, we evaluated the ability of whole berries and berry phytochemical, ellagic acid to reduce endogenous oxidative DNA damage. Ellagic acid was selected based on >95% inhibition of 8-oxodeoxyguosine (8-oxodG) and other unidentified oxidative DNA adducts induced by 4-hydroxy-17ss-estradiol and CuCl(2) in vitro. Inhibition of the latter occurred at lower concentrations (10 microM) than that for 8-oxodG (100 microM). In the in vivo study, female CD-1 mice (n=6) were fed either a control diet or diet supplemented with ellagic acid (400 ppm) and dehydrated berries (5% w/w) with varying ellagic acid contents - blueberry (low), strawberry (medium) and red raspberry (high), for 3 weeks. Blueberry and strawberry diets showed moderate reductions in endogenous DNA adducts (25%). However, both red raspberry and ellagic acid diets showed a significant reduction of 59% (p < 0.001) and 48% (p < 0.01), respectively. Both diets also resulted in a 3-8 fold over-expression of genes involved in DNA repair such as xeroderma pigmentosum group A complementing protein (XPA), DNA excision repair protein (ERCC5) and DNA ligase III (DNL3). These results suggest that red raspberry and ellagic acid reduce endogenous oxidative DNA damage by mechanisms which may involve increase in DNA repair.

Mammary tumor induction in ACI rats exposed to low levels of 17beta-estradiol.

Animal models play a major role in understanding the etiology, molecular mechanisms, strategizing intervention and treatment of human diseases. ACI, an inbred line derived from August and Copenhagen strains, is unique for its susceptibility to estrogen-induced mammary tumors. Histologically and in many molecular aspects, the tumors formed in these rats are similar to human breast cancers. Previous studies have shown high mortality and significant weight loss in this model associated with pituitary gland abnormality. We hypothesized that this could be due to overwhelming the biological system with estrogen. Three groups of female ACI rats (7-8 weeks) received either 3-cm sham silastic implants, or the conventional 3-cm silastic implants containing 27 mg of 17beta-estradiol, or 1.2-cm silastic implants containing 9 mg 17beta-estradiol. The sham and 3-cm implant rats were euthanized at 180 days while the 1.2-cm implant rats were euthanized at 240 days. The 1.2-cm implants resulted in significantly reduced serum estrogen levels and pituitary gland size. Animals with 1.2-cm implants had 100% tumor incidence, while not all rats developed tumors with 3-cm implants. Both the tumor burden (from 1,011+/-402 to 2,324+/-454 mm(3); p=0.01) and tumor multiplicity (from 5.78+/-1.4 to 7.6+/-1.04) increased by lowering the estrogen dose, and the inter-animal variability in the tumor indices decreased. Finally, the weight of the pituitary gland was also significantly (p=0.0004) reduced (from 178+/-23.5 mg to 80+/-8.9 mg) and the mortality rate decreased from 42% to 0% (p=0.01). Our data indicate that the improvised model will provide valuable insights into the molecular alterations in the estrogen-induced mammary tumorigenesis and will be ideal for inhibition studies.

Exosomal formulation of anthocyanidins against multiple cancer types.

Over the last two decades, berries and berry bioactives, particularly anthocyanins and their aglycones anthocyanidins (Anthos) have demonstrated excellent anti-oxidant, anti-proliferative, apoptotic and anti-inflammatory properties. However, their physicochemical and pharmacokinetic limitations such as, low permeability, and poor oral bioavailability are considered as unfavorable properties for development as drugs. Therefore there is a need to develop systems for efficient systemic delivery and robust bioavailability. In this study we prepared nano-formulation of bilberry-derived Anthos using exosomes harvested from raw bovine milk. Exosomal formulation of Anthos enhanced antiproliferative and anti-inflammatory effects compared with the free Anthos against various cancer cells in vitro. Our data also showed significantly enhanced therapeutic response of exosomal-Anthos formulation compared with the free Anthos against lung cancer tumor xenograft in nude mice. The Anthos showed no signs of gross or systemic toxicity in wild-type mice. Thus, exosomes provide an effective alternative for oral delivery of Anthos that is efficacious, cost-effective, and safe, and this regimen can be developed as a non-toxic, widely applicable therapeutic agent.

Modulation of novel DNA adducts during human uterine cervix cancer progression.

Progressive accumulation of DNA lesions leads to genetic mutations that are central to the process of tumorigenesis. Human cervix provides an ideal system to determine progressive accumulation of DNA adducts in the target tissue because of its accessibility during routine diagnostic checkups. Uterine cervix samples from various pathologies, i.e. normal (n=13), inflammation (n=9), dysplasia (n=5) and different stages of invasive cancer (n=47), were analyzed for DNA adduct burden by modified 32P-postlabeling/TLC systems. Six subgroups of adducts were detected in the following descending order of polarities: P-1, P-2, PL-1, PL-2, L-1 and L-2 (P, polar; L, lipophilic; PL, between polar and lipophilic). No qualitative differences were observed in adduct profiles in the various cervix pathologies analyzed. However, significant quantitative differences were found. Previously known lipophilic adducts increased significantly from normal to cancer (144+/-61 to 503+/-51 adducts/10(9) nucleotides). Interestingly, the newly discovered polar adducts were present at 61- to 527-fold higher levels than lipophilic adducts. Of all the polar adducts, the known mutagenic lesion, 8-oxodeoxyguanosine, predominated in all cervix conditions. Notably, this lesion was elevated 27-fold in inflammation compared with normal cervix (51,058+/-9,863 versus 1,886+/-507 adducts/10(9) nucleotides). The P-1, PL-1, PL-2 and L-1 adducts were elevated 3- to 13-fold in inflammation compared with normal cervix, and were also higher in dysplasia and cancer. Our data suggest that inflammation may be involved in directing the course of disease progression by accumulating higher levels of DNA lesions. The data further suggest the biomarker potential of the newly detected array of DNA adducts.