Chemical enhancers of posttranscriptional gene silencing in Arabidopsis.

RNAi mediated by small-interfering RNAs (siRNAs) operates via transcriptional (TGS) and posttranscriptional gene silencing (PTGS). In Arabidopsis thaliana, TGS relies on DICER-LIKE-3 (DCL3)-dependent 24-nt siRNAs loaded into AGO4-clade ARGONAUTE effector proteins. PTGS operates via DCL4-dependent 21-nt siRNAs loaded into AGO1-clade proteins. We set up and validated a medium-throughput, semi-automatized procedure enabling chemical screening, in a 96-well in vitro format, of Arabidopsis transgenic seedlings expressing an inverted-repeat construct from the phloem companion cells. The ensuing quantitative PTGS phenotype was exploited to identify molecules, which, upon topical application, either inhibit or enhance siRNA biogenesis/activities. The vast majority of identified modifiers were enhancers, among which Sortin1, Isoxazolone, and [5-(3,4-dichlorophenyl)furan-2-yl]-piperidine-1-ylmethanethione (DFPM) provided the most robust and consistent results, including upon their application onto soil-grown plants in which their effect was nonautonomous and long lasting. The three molecules increased the RNAi potency of the inverted-repeat construct, in large part by enhancing 21-nt siRNA accumulation and loading into AGO1, and concomitantly reducing AGO4 and DCL3 levels in planta. A similar, albeit not identical effect, was observed on 22-nt siRNAs produced from a naturally occurring inverted-repeat locus, demonstrating that the molecules also enhance endogenous PTGS. In standardized assays conducted in seedling extracts, the three enhancers selectively increased DCL4-mediated processing of in vitro-synthesized double-stranded RNAs, indicating the targeting of a hitherto unknown PTGS component probably independent of the DCL4-cofactor DOUBLE-STRANDED RNA-BINDING 4 (DRB4). This study establishes the proof-of-concept that RNAi efficacy can be modulated by chemicals in a whole organism. Their potential applications and the associated future research are discussed.

Acute cyanotoxin poisoning reveals a marginal effect on mouse gut microbiome composition but indicates metabolic shifts related to liver and gut inflammation.

Freshwater harmful algal blooms (HABs) are a major environmental health problem worldwide. HABs are caused by a predominance of cyanobacteria, some of which produce potent toxins. The most ubiquitous cyanotoxin is microcystin (MC) and the congener MC-LR is the most studied due to its toxicity. Short-term exposure to toxins can cause gut microbiome disturbances, but this has not been well described with MC-LR exposure. This study investigated the gut microbial communities of mice from a prior study, which identified significant liver toxicity from ingestion of MC-LR daily for 8 days. CD-1 mice were divided into three dosage groups: control, low exposure (sub-lethal MC-LR concentration), and high exposure (near-lethal MC-LR concentration). Fecal samples were analyzed using 16S rRNA sequencing. Results revealed that at population level, there were no significant shifts in bacterial diversity or the microbial community structure over the exposure period. However, there were significant differences between male and female mice. Predictive functional gene analysis indicated that several metabolic pathways were significantly different in the high dose group before exposure and following 7 doses of MC-LR, as well as between the control and high dose groups on Day 8. Significant differentially abundant taxa were also identified contributing to these pathways. Several pathways, including superpathway of N-acetylneuraminate degradation, were related to liver and gut inflammation. The outcome of this study suggests a need for in-depth investigation of metabolic activity and other functions in the gut in future studies, as well as potential consideration of the role of sex in MC-LR toxicity.

Impact of an Antifungal Insect Defensin on the Proteome of the Phytopathogenic Fungus Botrytis cinerea.

ETD151, an analogue of the antifungal insect defensin heliomicin, is an antifungal peptide active against yeasts and filamentous fungi. To decipher the mechanisms underlying its molecular action on the phytopathogenic fungus Botrytis cinerea, a necrotrophic pathogen responsible for gray mold disease, we investigated the changes in 3 day-old mycelia upon treatment with different concentrations of ETD151. Optical and fluorescence microscopies were used prior to establishing the peptide/protein profiles through two mass spectrometry approaches: MALDI profiling, to generate molecular mass fingerprints as peptide signatures, and a gel-free bottom-up proteomics approach. Our results show that a concentration of ETD151 above the half-maximal inhibitory concentration can alter the integrity of the mycelial structure of B. cinerea. Furthermore, reproducible modifications of the peptide/protein composition were demonstrated in the presence of ETD151 within a 1500-16,000 mass (m/z) range. After the robustness of LC-ESI-MS/MS analysis on B. cinerea mycelial extracts was confirmed, our analyses highlighted 340 significantly modulated proteins upon treatment with ETD151 within a 4.8-466 kDa mass range. Finally, data mapping on KEGG pathways revealed the molecular impact of ETD151 on at least six pathways, namely, spliceosome, ribosome, protein processing in endoplasmic reticulum, endocytosis, MAPK signaling pathway, and oxidative phosphorylation.

Microcystis toxin-mediated tumor promotion and toxicity lead to shifts in mouse gut microbiome.

Microcystins (MCs) are the most prevalent cyanotoxins reported in freshwater. While numerous studies have examined the toxicological impacts of MCs on mammalian systems, very few have examined the chronic impacts of MCs on the gut microbiome of exposed organisms. Our understanding of the relationship of MCs, especially lysed toxic cyanobacteria, and the gut microbiota is very limited. The objective of this study was to determine the impacts of MC-LR and Microcystis lysate ingestion on the gut microbiome in a hepatocellular carcinoma mouse model, simulating a high-risk population and exposure at an environmentally relevant MC level. Mice were assigned to 4 groups (MC-LR; Microcystis lysate; Negative control; Positive (liver carcinogen) control). Fecal samples were collected every 8 weeks. Bacterial community and colony counts were analyzed. The abundance of Firmicutes in the positive control and lysate group was higher than the negative control and MC group. Exposure to MC-LR or lysate was associated with significantly decreased bacterial diversity. A distinct separation of the three groups (MC-LR/lysate/carcinogen) from the negative was much more apparent in their gut microbiome as the exposure time increased. The MC-LR and lysate groups showed gut microbiome structure responding to lipid metabolism disturbance and high stress. Bacterial colony count was significantly lower in all the treated groups than the negative control. Our study highlights that chronic exposure to MC-LR and Microcystis lysate negatively impacts gut microbiome succession and altered the bacterial community structure into the one similar to the carcinogen group, which may indicate that the change favors progression of hepatocellular carcinoma. In a future study, more in-depth investigation is warranted to better understand the liver-gut nexus in promoting liver cancer among those exposed to MC and toxic cyanobacteria.

Inherited alterations of TGF beta signaling components in Appalachian cervical cancers.

PURPOSE: This study examined targeted genomic variants of transforming growth factor beta (TGFB) signaling in Appalachian women. Appalachian women with cervical cancer were compared to healthy Appalachian counterparts to determine whether these polymorphic alleles were over-represented within this high-risk cancer population, and whether lifestyle or environmental factors modified the aggregate genetic risk in these Appalachian women. METHODS: Appalachian women's survey data and blood samples from the Community Awareness, Resources, and Education (CARE) CARE I and CARE II studies (n = 163 invasive cervical cancer cases, 842 controls) were used to assess gene-environment interactions and cancer risk. Polymorphic allele frequencies and socio-behavioral demographic measurements were compared using t tests and χ2 tests. Multivariable logistic regression was used to evaluate interaction effects between genomic variance and demographic, behavioral, and environmental characteristics. RESULTS: Several alleles demonstrated significant interaction with smoking (TP53 rs1042522, TGFB1 rs1800469), alcohol consumption (NQO1 rs1800566), and sexual intercourse before the age of 18 (TGFBR1 rs11466445, TGFBR1 rs7034462, TGFBR1 rs11568785). Interestingly, we noted a significant interaction between "Appalachian self-identity" variables and NQO1 rs1800566. Multivariable logistic regression of cancer status in an over-dominant TGFB1 rs1800469/TGFBR1 rs11568785 model demonstrated a 3.03-fold reduction in cervical cancer odds. Similar decreased odds (2.78-fold) were observed in an over-dominant TGFB1 rs1800469/TGFBR1 rs7034462 model in subjects who had no sexual intercourse before age 18. CONCLUSIONS: This study reports novel associations between common low-penetrance alleles in the TGFB signaling cascade and modified risk of cervical cancer in Appalachian women. Furthermore, our unexpected findings associating Appalachian identity and NQO1 rs1800566 suggests that the complex environmental exposures that contribute to Appalachian self-identity in Appalachian cervical cancer patients represent an emerging avenue of scientific exploration.

Dietary administration of black raspberries modulates arsenic biotransformation and reduces urinary 8-oxo-2'-deoxyguanosine in mice.

Arsenic in drinking water is a worldwide public health problem due to its pathogenic induction of oxidative stress in various organ systems. Phytochemicals present in polyphenolic-rich fruits such as black raspberries (BRBs) have diverse health benefits, including antioxidation and modulation of enzymes in xenobiotic metabolism. We used a mouse model combined with a standardized BRB-rich diet to investigate the impact of BRB consumption on arsenic biotransformation. We observed a significant reduction of urinary 8-oxo-2'-deoxyguanosine (8-oxodG) together with elevated levels of methylation and urinary excretion of arsenic in mice concurrently fed BRBs upon arsenic exposure. Moreover, enzyme expression and liver metabolites involved in arsenic metabolism were found to be different between mice on BRB and control diets with arsenic exposure. These data indicate that BRB consumption affected arsenic biotransformation in vivo likely via alterations in related metabolic enzymes and cofactors, providing evidence on reduction of arsenic toxicity by consumption of BRBs.

Deletion of macrophage migration inhibitory factor inhibits murine oral carcinogenesis: Potential role for chronic pro-inflammatory immune mediators.

Oral cancer kills about 1 person every hour each day in the United States and is the sixth most prevalent cancer worldwide. The pro-inflammatory cytokine 'macrophage migration inhibitory factor' (MIF) has been shown to be expressed in oral cancer patients, yet its precise role in oral carcinogenesis is not clear. In this study, we examined the impact of global Mif deletion on the cellular and molecular process occurring during oral carcinogenesis using a well-established mouse model of oral cancer with the carcinogen 4-nitroquinoline-1-oxide (4NQO). C57BL/6 Wild-type (WT) and Mif knock-out mice were administered with 4NQO in drinking water for 16 weeks, then regular drinking water for 8 weeks. Mif knock-out mice displayed fewer oral tumor incidence and multiplicity, accompanied by a significant reduction in the expression of pro-inflammatory cytokines Il-1ß, Tnf-α, chemokines Cxcl1, Cxcl6 and Ccl3 and other molecular biomarkers of oral carcinogenesis Mmp1 and Ptgs2. Further, systemic accumulation of myeloid-derived tumor promoting immune cells was inhibited in Mif knock-out mice. Our results demonstrate that genetic Mif deletion reduces the incidence and severity of oral carcinogenesis, by inhibiting the expression of chronic pro-inflammatory immune mediators. Thus, targeting MIF is a promising strategy for the prevention or therapy of oral cancer.

Alterations in RD(INK4/ARF) -mediated en bloc regulation of the INK4-ARF locus in human squamous cell carcinoma of the head and neck.

The presence of RD(INK4/ARF) (RD) enhancer in the INK4-ARF locus provides a novel mechanism to simultaneously increase the transcription of p15(INK4b) (p15), p14ARF (p14), and p16(INK4a) (p16). While such upregulation can be repressed through interactions between RD and oncoproteins CDC6 and BMI1, little is known about the involvement of RD in cancer. In this study we investigated RD deletions in 30 squamous cell carcinoma of the head and neck (SCCHN) and the patient-matched High At-Risk Mucosa specimens (HARM, "phenotypically normal" tissues neighboring SCCHN foci but beyond the surgical resection margin). RD was deleted (homozygously/heterozygously) in SCCHN and HARM at the incidence of 36.7% (11/30) and 13.3% (4/30), respectively. In comparison, no RD deletion was detected in 26 oral buccal brush biopsy specimens from healthy donors. Both p16 and p14 were lowly expressed in SCCHN and HARM, and their mRNA expression levels were positively associated with each other (P < 0.01). Moreover, BMI1 was highly expressed in both SCCHN and HARM, and BMI1 overexpression was associated with p16 downregulation in SCCHN (P < 0.05). These results indicate that RD deletion and BMI1 overexpression frequently occur in the early stage of oral carcinogenesis and BMI1 overexpression may downregulate the transcription of p16 and p14 through interfering with RD.

Bioactive compounds or metabolites from black raspberries modulate T lymphocyte proliferation, myeloid cell differentiation and Jak/STAT signaling.

Bioactive phytochemicals from natural products, such as black raspberries (BRB; Rubus occidentalis), have direct anticancer properties on malignant cells in culture and in xenograft models. BRB components inhibit cancer progression in more complex rodent carcinogenesis models. Although mechanistic targets for BRB phytochemicals in cancer cells are beginning to emerge, the potential role in modulating host immune processes impacting cancer have not been systematically examined. We hypothesized that BRB contain compounds capable of eliciting potent immunomodulatory properties that impact cellular mediators relevant to chronic inflammation and tumor progression. We studied both an ethanol extract from black raspberries (BRB-E) containing a diverse mixture of phytochemicals and two abundant phytochemical metabolites of BRB produced upon ingestion (Cyanidin-3-Rutinoside, C3R; Quercitin-3-Rutinoside, Q3R). BRB-E inhibited proliferation, and viability of CD3/CD28 activated human CD4(+) and CD8(+) T lymphocytes. BRB-E also limited in vitro expansion of myeloid-derived suppressor cells (MDSC) and their suppressive capacity. Pre-treatment of immune cells with BRB-E attenuated IL-6-mediated phosphorylation of signal transducer and activator of transcription-3 (STAT3) and IL-2-induced STAT5 phosphorylation. In contrast, pre-treatment of immune cells with the C3R and Q3R metabolites inhibited MDSC expansion, IL-6-mediated STAT3 signaling, but not IL-2-induced STAT5 phosphorylation and were less potent inhibitors of T cell viability. Together these data indicate that BRB extracts and their physiologically relevant metabolites contain phytochemicals that affect immune processes relevant to carcinogenesis and immunotherapy. Furthermore, specific BRB components and their metabolites may be a source of lead compounds for drug development that exhibits targeted immunological outcomes or inhibition of specific STAT-regulated signaling pathways.

Genetic alterations of RD(INK4/ARF) enhancer in human cancer cells.

Recent identification of an enhancer element, RD(INK4/ARF) (RD), in the prominent INK4/ARF locus provides a novel mechanism to simultaneously regulate the transcription of p15(INK4B) (p15), p14(ARF) , and p16(INK4A) (p16) tumor suppressor genes. While genetic inactivation of p15, p14(ARF) , and p16 in human tumors has been extensively studied, little is known about genetic alterations of RD and its impact on p15, p14(ARF) , and p16 in human cancer. The purpose of this study was to investigate the potential existence of genetic alterations of RD in human cancer cells. DNAs extracted from 17 different cancer cell lines and 31 primary pheochromocytoma tumors were analyzed for deletion and mutation of RD using real-time PCR and direct DNA sequencing. We found that RD was deleted in human cancer cell lines and pheochromocytoma tumors at frequencies of 41.2% (7/17) and 13.0% (4/31), respectively. While some of these RD deletion events occurred along with deletions of the entire INK4/ARF locus, other RD deletion events were independent of genetic alterations in p15, p14(ARF) , and p16. Furthermore, the status of RD was poorly associated with the expression of p15, p14(ARF) , and p16 in tested cancer cell lines and tumors. This study demonstrates for the first time that deletion of the RD enhancer is a prevalent event in human cancer cells. Its implication in carcinogenesis remains to be further explored.

Comprehensive evaluation of caspase-14 in vulvar neoplasia: an opportunity for treatment with black raspberry extract.

OBJECTIVE: The aim of this study is to determine the expression of caspase-14, a key protein in maturation of squamous epithelia, in archival malignant and premalignant vulvar squamous lesions and examine in-vitro effects of a black raspberry extract (BRB-E) on a vulvar squamous cell carcinoma (VSCC) cell line. METHODS: VSCC cell cultures were exposed to different BRB-E concentrations and used to create cell blocks. Immunohistochemistry for caspase-14 was performed on cell block sections, whole tissue sections, and a tissue microarray consisting of normal vulvar skin, lichen sclerosus (LS), classic and differentiated vulvar intraepithelial neoplasia (cVIN and dVIN respectively), and VSCC. RESULTS: LS demonstrated abnormal full thickness (5/11) or absent (1/11) caspase-14 staining. dVIN often showed markedly reduced expression (4/7), and cVIN occasionally demonstrated either absent or reduced caspase-14 (6/22). VSCC predominantly had absent or markedly reduced caspase-14 (26/28). VSCC cell cultures demonstrated a significant increase in caspase-14 (p=0.013) after BRB-E treatment: 7.3% (±2.0%) of untreated cells showed caspase-14 positivity, while 21.3% (±8.9%), 21.7% (±4.8%), and 22.6% (±5.3%) of cells were positive for caspase-14 after treatment with 200, 400, and 800 µg/mL BRB-E, respectively. Pair-wise comparisons between the treatment groups and the control demonstrated significant differences between no treatment with BRB-E and each of these treatment concentrations (Dunnett's adjusted p-values: 0.024, 0.021, and 0.014, respectively). CONCLUSIONS: Caspase-14 is frequently decreased in premalignant and malignant vulvar squamous lesions, and is upregulated in VSCC cell culture by BRB-E. BRB-E should be further explored and may ultimately be incorporated in topical preparations.

Evidence that P12, a specific variant of P16(INK4A), plays a suppressive role in human pancreatic carcinogenesis.

The INK4a-ARF locus plays a central role in the development of pancreatic tumors as evidenced by the fact that up to 98% of pancreatic tumor specimens harbored genetic alterations at the INK4a-ARF locus. Interestingly, in addition to the well-known P16(INK4A) (P16) and P14ARF tumor suppressors, the INK4a-ARF locus in pancreas encodes another protein, P12, whose structure, function, and contributions to pancreatic carcinogenesis remain to be elucidated. In the current study, we demonstrated that over-expression of p12 in human pancreatic cancer cells led to cell arrest at the G1 phase and such cell cycle arrest was related to down-regulation of a number of oncogenes, such as c-Jun, Fos, and SEI1. Furthermore, unlike P16, P12 did not retain any cyclin-dependent kinase 4 (CDK4)-inhibitory activity. Instead, P12 exhibited a transactivating activity not found in P16. We also examined the genetic status of p12 in a cohort of 40 pancreatic tumor specimens and found that p12 alteration was prevalent in pancreatic tumors with an incidence of 70% (28/40). These results support that P12 is a tumor suppressive protein distinct from P16, and its genetic inactivation is associated with pancreatic carcinogenesis.

Chemoprevention of mouse lung and colon tumors by suberoylanilide hydroxamic acid and atorvastatin.

Atorvastatin and suberoylanilide hydroxamic acid (SAHA) were evaluated for chemoprevention of mouse lung tumors. In Experiment 1, lung tumors were induced by vinyl carbamate in strain A/J mice followed by 500 mg/kg SAHA, 60 or 180 mg/kg atorvastatin, and combinations containing SAHA and atorvastatin administered in their diet. SAHA and both combinations, but not atorvastatin, decreased the multiplicity of lung tumors, including large adenomas and adenocarcinomas with the combinations demonstrating the greatest efficacy. In Experiment 2, lung tumors were induced by 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanol in strain A/J mice followed by 180 mg/kg atorvastatin, 500 mg/kg SAHA, or both drugs administered in the diet. SAHA and the combination of both drugs, but not atorvastatin alone, decreased the multiplicity of lung tumors and large tumors, with the combination demonstrating greater efficacy. In Experiment 3, lung tumors were induced by 1,2-dimethylhydrazine in Swiss-Webster mice followed by 160 mg/kg atorvastatin, 400 mg/kg SAHA, or a combination of both drugs administered in the diet. SAHA and the combination, but not atorvastatin, decreased the multiplicity of lung tumors with the combination demonstrating greater efficacy. The multiplicity of colon tumors was decreased by SAHA, atorvastatin, and the combination, without any significant difference in their efficacy. mRNA expression analysis of lung tumor bearing mice suggested that the enhanced chemopreventive activity of the combination is related to atorvastatin modulation of DNA repair, SAHA modulation of angiogenesis, and both drugs modulating invasion and metastasis pathways. Atorvastatin demonstrated chemoprevention activity as indicated by the enhancement of the efficacy of SAHA to prevent mouse lung tumors.

Contrast enhancement of the brain by folate-conjugated gadolinium-diethylenetriaminepentaacetic acid-human serum albumin nanoparticles by magnetic resonance imaging.

Different from regular small molecule contrast agents, nanoparticle-based contrast agents have a longer circulation time and can be modified with ligands to confer tissue-specific contrasting properties. We evaluated the tissue distribution of polymeric nanoparticles (NPs) prepared from human serum albumin (HSA), loaded with gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA) (Gd-HSA-NP), and coated with folic acid (FA) (Gd-HSA-NP-FA) in mice by magnetic resonance imaging (MRI). FA increases the affinity of the Gd-HSA-NP to FA receptor-expressing cells. Clinical 3 T MRI was used to evaluate the signal intensities in the different organs of mice injected with Gd-DTPA, Gd-HSA-NP, or Gd-HSA-NP-FA. Signal intensities were measured and standardized by calculating the signal to noise ratios. In general, the NP-based contrast agents provided stronger contrasting than Gd-DTPA. Gd-HSA-NP-FA provided a significant contrast enhancement (CE) in the brain (p  =  .0032), whereas Gd-DTPA or Gd-HSA-NP did not. All studied MRI contrast agents showed significant CE in the blood, kidney, and liver (p < .05). Gd-HSA-NP-FA elicited significantly higher CE in the blood than Gd-HSA-NP (p  =  .0069); Gd-HSA-NP and Gd-HSA-NP-FA did not show CE in skeletal muscle and gallbladder; Gd-HSA-NP, but not Gd-HSA-NP-FA, showed CE in the cardiac muscle. Gd-HSA-NP-FA has potential as an MRI contrast agent in the brain.

Impact of Cyanotoxin Ingestion on Liver Cancer Development Using an At-Risk Two-Staged Model of Mouse Hepatocarcinogenesis.

Exposure to cyanobacterial hepatotoxins has been linked to the promotion and increased incidence of liver cancer in pre-clinical and epidemiologic studies. The family of hepatotoxins, microcystins (MCs), are produced by over 40 cyanobacterial species found in harmful algal blooms (HABs) worldwide, with MC-LR being the most common and potent MC congener. In the current study, we hypothesized that the low-dose chronic ingestion of Microcystis cyanotoxins via drinking water would promote liver carcinogenesis in pre-initiated mice. Four groups of C3H/HeJ mice received one intraperitoneal (i.p.) injection of diethylnitrosamine (DEN) at 4 weeks of age. Three weeks later, the mice were administered ad libitum drinking water containing one of the following: (1) reverse osmosis, deionized water; (2) water containing 500 mg/L phenobarbital (PB500); (3) water with purified MC-LR (10 µg/L) added; or (4) water containing lysed Microcystis aeruginosa (lysate; 10 µg/L total MCs). The exposure concentrations were based on environmentally relevant concentrations and previously established Ohio EPA recreational water MC guidelines. Throughout the 30-week exposure, mouse weights, food consumption, and water consumption were not significantly impacted by toxin ingestion. We found no significant differences in the number of gross and histopathologic liver lesion counts across the treatment groups, but we did note that the PB500 group developed lesion densities too numerous to count. Additionally, the proportion of lesions classified as hepatocellular carcinomas in the MC-LR group (44.5%; p < 0.05) and lysate group (55%; p < 0.01) was significantly higher compared to the control group (14.9%). Over the course of the study, the mice ingesting the lysate also had a significantly lower survival probability (64.4%; p < 0.001) compared to water (96.8%), PB500 (95.0%), and MC-LR (95.7%) exposures. Using cyanotoxin levels at common recreational water concentration levels, we demonstrate the cancer-promoting effects of a single cyanotoxin MC congener (MC-LR). Furthermore, we show enhanced hepatocarcinogenesis and significant mortality associated with combinatorial exposure to the multiple MCs and bioactive compounds present in lysed cyanobacterial cells­a scenario representative of the ingestion exposure route, such as HAB-contaminated water and food.

A black raspberry extract inhibits proliferation and regulates apoptosis in cervical cancer cells.

OBJECTIVE: Cervical cancer is the second most common female cancer worldwide, and it remains a challenge to manage preinvasive and invasive lesions. Food-based cancer prevention entities, such as black raspberries and their derivatives, have demonstrated a marked ability to inhibit preclinical models of epithelial cancer cell growth and tumor formation. Here, we extend the role of black raspberry-mediated chemoprevention to that of cervical carcinogenesis. METHODS: Three human cervical cancer cell lines, HeLa (HPV16-/HPV18+, adenocarcinoma), SiHa (HPV16+/HPV18-, squamous cell carcinoma) and C-33A (HPV16-/HPV18-, squamous cell carcinoma), were treated with a lyophilized black raspberry ethanol extract (RO-ET) at 25, 50, 100 or 200µg/ml for 1, 3 and 5days, respectively. Cell proliferation was measured by WST1 (tetrazolium salt cleavage) assays. Flow cytometry (propidium iodide and Annexin V staining) and fluorescence microscopy analysis were used to measure apoptotic cell changes. RESULTS: We found that non-toxic levels of RO-ET significantly inhibited the growth of human cervical cancer cells, in a dose-dependent and time-dependent manner to a maximum of 54%, 52% and 67%, respectively (p<0.05). Furthermore, cell growth inhibition was persistent following short-term withdrawal of RO-ET from the culture medium. Flow cytometry and fluorescence microscopy demonstrated RO-ET-induced apoptosis in all cell lines. CONCLUSION: Black raspberries and their bioactive components represent promising candidates for future phytochemical-based mechanistic pathway-targeted cancer prevention strategies.

Comparison of intracellular accumulation and cytotoxicity of free mTHPC and mTHPC-loaded PLGA nanoparticles in human colon carcinoma cells.

The second generation photosensitizer mTHPC was approved by the European Medicines Agency (EMA) for the palliative treatment of advanced head and neck cancer in October 2001. It is known that mTHPC possesses a significant phototoxicity against a variety of human cancer cells in vitro but also exhibits dark toxicity and can cause adverse effects (especially skin photosensitization). Due to its poor water solubility, the administration of hydrophobic photosensitizer still presents several difficulties. To overcome the administration problems, the use of nanoparticles as drug carrier systems is much investigated. Nanoparticles based on poly(lactic-co-glycolic acid) (PLGA) have been extensively studied as delivery systems into tumours due to their biocompatibility and biodegradability. The goal of this study was the comparison of free mTHPC and mTHPC-loaded PLGA nanoparticles concerning cytotoxicity and intracellular accumulation in human colon carcinoma cells (HT29). The nanoparticles delivered the photosensitizer to the colon carcinoma cells and enabled drug release without losing its activity. The cytotoxicity assays showed a time- and concentration-dependent decrease in cell proliferation and viability after illumination. However, first and foremost mTHPC lost its dark toxic effects using the PLGA nanoparticles as a drug carrier system. Therefore, PLGA nanoparticles are a promising drug carrier system for the hydrophobic photosensitizer mTHPC.

Comparative Encapsulation Efficiency of Lutein in Micelles Synthesized via Batch and High Throughput Methods.

PURPOSE: Black raspberries (BRBs) and their anthocyanin-rich hydrophilic fractions (BRB-H) have exhibited significant chemopreventative activity across aerodigestive cancers. Lutein, the primary component of the BRB lipophilic fraction (BRB-L), also demonstrates bioactivity potential, but is less well characterized, in part because of its poor, innate bioavailability. For these lipophilic compounds to be accurately evaluated for anticancer efficacy, it is necessary to increase their functional bioavailability using delivery vehicles. Lutein has been delivered in commercial settings in emulsion form. However, emulsions are unstable, particularly in the gastrointestinal tract, which limit their use as an oral nutraceutical. Here, we evaluated lutein encapsulation and cellular uptake for nanoparticle (NP) delivery vehicles composed of three different materials synthesized via two different approaches. METHODS: Specifically, NPs were synthesized via smaller scale batch interfacial instability (II) sonication and semi-continuous high throughput electrohydrodynamic-mediated mixing nanoprecipitation (EM-NP) methods using polystyrene-polyethylene oxide (PSPEO) or polycaprolactone-polyethylene glycol (PCLPEG) block copolymers and PHOSPHOLIPON 90G® (P90G, Lipoid GmbH) lipids. Size distribution, lutein encapsulation efficiency (EE), and cellular uptake and delivery were evaluated for each NP formulation. RESULTS: NPs produced via high throughput EM-NP had higher EEs than NPs produced via batch II sonication, and P90G had the greatest EE (55%) and elicited faster cellular uptake in premalignant oral epithelial cells (SCC83) compared to other delivery systems. CONCLUSION: These qualities suggest P90G could be a beneficial candidate for future lutein in vitro delivery research and clinical translation for oral cancer prevention.

A machine learning-based chemoproteomic approach to identify drug targets and binding sites in complex proteomes.

Chemoproteomics is a key technology to characterize the mode of action of drugs, as it directly identifies the protein targets of bioactive compounds and aids in the development of optimized small-molecule compounds. Current approaches cannot identify the protein targets of a compound and also detect the interaction surfaces between ligands and protein targets without prior labeling or modification. To address this limitation, we here develop LiP-Quant, a drug target deconvolution pipeline based on limited proteolysis coupled with mass spectrometry that works across species, including in human cells. We use machine learning to discern features indicative of drug binding and integrate them into a single score to identify protein targets of small molecules and approximate their binding sites. We demonstrate drug target identification across compound classes, including drugs targeting kinases, phosphatases and membrane proteins. LiP-Quant estimates the half maximal effective concentration of compound binding sites in whole cell lysates, correctly discriminating drug binding to homologous proteins and identifying the so far unknown targets of a fungicide research compound.

Oral cancer in Appalachia.

Oral cancer accounts for 2.3% of malignancies in the U.S. and has one of the lowest five-year survival rates. An examination of oral cancer in Appalachia was motivated by the high incidence of lung and bronchial cancers in Appalachian states, the risk factors for which overlap with those for oral cancer. The incidence and mortality rates for oral cancer in 13 Appalachian states and the relative frequency of presumptive risk factors were examined and compared with national rates, using data from the National Program of Cancer Registries, Surveillance Epidemiology and End Results, Behavioral Risk Factor Surveillance System, the Appalachian Regional Commission, and the National Health Interview Survey. Combined incidence rates for oral cancer were higher in six of 12 Appalachian states, and mortality rates higher in 10 of 13, compared with the national average. Smoking was more prevalent than the national average in nine of 13 states, whereas alcohol consumption was the same or less in 11 Appalachian states. Only five of 13 states averaged fewer than the recommended five or more servings per day of fruits and vegetables.