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.

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 Multi-level Model to Understand Cervical Cancer Disparities in Appalachia.

The Appalachian region experiences higher incidence and mortality due to cervical cancer compared with other regions of the United States. The goal of the Ohio State University Center for Population Health and Health Disparities (CPHHD), called the Community Awareness Resources and Education (CARE) project, was to understand reasons for this disparity. The first wave (2003-2008) of funding included three projects focusing on the known risk factors for cervical cancer, lack of screening, smoking, and infection with human papillomavirus (HPV). On the basis of the results of these projects, the second wave (2011-2017) included four projects, designed to address a multi-level model of factors contributing to cervical disparities in Appalachia. The results of these projects were then used to refine a multi-level model that explains cervical cancer disparities in Appalachia. Future funded projects will take these multi-level explanations for cervical disparities and focus on implementation science strategies to reduce the burden of cervical cancer morbidity and mortality in Appalachia.See all articles in this Special Collection Honoring Paul F. Engstrom, MD, Champion of Cancer Prevention.

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.

Metabolic Regulation of Glycolysis and AMP Activated Protein Kinase Pathways during Black Raspberry-Mediated Oral Cancer Chemoprevention.

Oral cancer is a public health problem with an incidence of almost 50,000 and a mortality of 10,000 each year in the USA alone. Black raspberries (BRBs) have been shown to inhibit oral carcinogenesis in several preclinical models, but our understanding of how BRB phytochemicals affect the metabolic pathways during oral carcinogenesis remains incomplete. We used a well-established rat oral cancer model to determine potential metabolic pathways impacted by BRBs during oral carcinogenesis. F344 rats were exposed to the oral carcinogen 4-nitroquinoline-1-oxide in drinking water for 14 weeks, then regular drinking water for six weeks. Carcinogen exposed rats were fed a 5% or 10% BRB supplemented diet or control diet for six weeks after carcinogen exposure. RNA-Seq transcriptome analysis on rat tongue, and mass spectrometry and NMR metabolomics analysis on rat urine were performed. We tentatively identified 57 differentially or uniquely expressed metabolites and over 662 modulated genes in rats being fed with BRB. Glycolysis and AMPK pathways were modulated during BRB-mediated oral cancer chemoprevention. Glycolytic enzymes Aldoa, Hk2, Tpi1, Pgam2, Pfkl, and Pkm2 as well as the PKA-AMPK pathway genes Prkaa2, Pde4a, Pde10a, Ywhag, and Crebbp were downregulated by BRBs during oral cancer chemoprevention. Furthermore, the glycolysis metabolite glucose-6-phosphate decreased in BRB-administered rats. Our data reveal the novel metabolic pathways modulated by BRB phytochemicals that can be targeted during the chemoprevention of oral cancer.

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.

Impact of Microcystin-LR on Liver Function Varies by Dose and Sex in Mice.

Microcystin (MC) exposure is an increasing concern because more geographical locations are covered with cyanobacterial blooms as eutrophication and bloom-favoring environmental factors become more prevalent worldwide. Acute MC exposure has been linked to gastrointestinal distress, liver toxicity, and death in extreme circumstances. The goal of this study was to provide an accurate and comprehensive description of MC-LRs impacts on liver pathology, clinical chemistry, and gap junction intercellular communication (GJIC) in CD-1 male and female mice. Mice were exposed to 0, 3000, and 5000/4000 µg/kg/day MC-LR, daily for 7 days, and were necropsied on Day 8. Blood samples for clinical chemistry analysis were processed to serum, while liver sections were fixed for histopathology or evaluated for GJIC using fluorescent cut-load dye. Results show a dose-dependent relationship with MC-LR exposure and hepatocellular hypertrophy, degradation, and necrosis. Clinical chemistry parameters alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total bilirubin, and cholesterol increased significantly in MC-LR exposed mice. Clinical chemistry parameter analysis showed significantly increased susceptibility to MC-LR in females compared to males. Changes in GJIC were not noted, but localization of hepatotoxicity near the central veins and midlobular areas was seen. Future toxicity studies involving MCs should consider response differences across sexes, differing MC congeners, and combinatorial exposures involving other cyanotoxins.

Characterization of the Functional Changes in Mouse Gut Microbiome Associated with Increased Akkermansia muciniphila Population Modulated by Dietary Black Raspberries.

Gut microbiome plays an essential role in host health through host-gut microbiota metabolic interactions. Desirable modulation of beneficial gut bacteria, such as Akkermansia muciniphila, can confer health benefits by altering microbiome-related metabolic profiles. The purpose of this study is to examine the effects of a black raspberry-rich diet to reshape the gut microbiome by selectively boosting A. muciniphila population in C57BL/6J mice. Remarkable changes of the mouse gut microbiome were revealed at both compositional and functional levels with an expected increase of A. muciniphila in concert with a profound impact on multiple gut microbiome-related functions, including vitamin biosynthesis, aromatic amino acid metabolism, carbohydrate metabolism, and oxidative stress. These functional alterations in the gut microbiome by an easily accessed freeze-dried black raspberry-supplemented diet may provide novel insights on the improvement of human health via gut microbiome modulation.

Storage conditions modulate the metabolomic profile of a black raspberry nectar with minimal impact on bioactivity.

Pre-clinical and clinical studies suggest black raspberries (BRBs) may inhibit the development of oral cancer. Lyophilized BRB powder is commonly used in these studies, but processed BRB products are more often consumed. The objective of this work was to understand how storage conditions influence the phytochemical profile and anti-proliferative activity of a BRB nectar beverage. Untargeted UHPLC-Q-TOF-MS based metabolomics analyses demonstrated that large chemical variation was introduced by storage above -20 °C over 60 days. However, minimal change in anti-proliferative activity was observed when stored nectar extracts were applied to SCC-83-01-82 premalignant oral epithelial cells. As proof of concept, cyanidin-3-O-rutinoside and its degradation product, protocatechuic acid, were administered in different ratios maintaining an equimolar dose, and anti-proliferative activity was maintained. This study shows the utility of metabolomics to profile global chemical changes in foods, while demonstrating that isolated phytochemicals do not explain the complete bioactivity of a complex food product.

Inhibition of Pro-inflammatory and Anti-apoptotic Biomarkers during Experimental Oral Cancer Chemoprevention by Dietary Black Raspberries.

Oral cancer continues to be a significant public health problem worldwide. Recently conducted clinical trials demonstrate the ability of black raspberries (BRBs) to modulate biomarkers of molecular efficacy that supports a chemopreventive strategy against oral cancer. However, it is essential that a preclinical animal model of black raspberry (BRB) chemoprevention which recapitulates human oral carcinogenesis be developed, so that we can validate biomarkers and evaluate potential mechanisms of action. We therefore established the ability of BRBs to inhibit oral lesion formation in a carcinogen-induced rat oral cancer model and examined potential mechanisms. F344 rats were administered 4-nitroquinoline 1-oxide (4NQO) (20 µg/ml) in drinking water for 14 weeks followed by regular drinking water for 6 weeks. At week 14, rats were fed a diet containing either 5 or 10% BRB, or 0.4% ellagic acid (EA), a BRB phytochemical. Dietary administration of 5 and 10% BRB reduced oral lesion incidence and multiplicity by 39.3 and 28.6%, respectively. Histopathological analyses demonstrate the ability of BRBs and, to a lesser extent EA, to inhibit the progression of oral cancer. Oral lesion inhibition by BRBs was associated with a reduction in the mRNA expression of pro-inflammatory biomarkers Cxcl1, Mif, and Nfe2l2 as well as the anti-apoptotic and cell cycle associated markers Birc5, Aurka, Ccna1, and Ccna2. Cellular proliferation (Ki-67 staining) in tongue lesions was inhibited by BRBs and EA. Our study demonstrates that, in the rat 4NQO oral cancer model, dietary administration of BRBs inhibits oral carcinogenesis via inhibition of pro-inflammatory and anti-apoptotic pathways.

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.

Suppression of Proinflammatory and Prosurvival Biomarkers in Oral Cancer Patients Consuming a Black Raspberry Phytochemical-Rich Troche.

Black raspberries (BRB) demonstrate potent inhibition of aerodigestive tract carcinogenesis in animal models. However, translational clinical trials evaluating the ability of BRB phytochemicals to impact molecular biomarkers in the oral mucosa remain limited. The present phase 0 study addresses a fundamental question for oral cancer food-based prevention: Do BRB phytochemicals successfully reach the targeted oral tissues and reduce proinflammatory and antiapoptotic gene expression profiles? Patients with biopsy-confirmed oral squamous cell carcinomas (OSCC) administered oral troches containing freeze-dried BRB powder from the time of enrollment to the date of curative intent surgery (13.9 ± 1.27 days). Transcriptional biomarkers were evaluated in patient-matched OSCCs and noninvolved high at-risk mucosa (HARM) for BRB-associated changes. Significant expression differences between baseline OSCC and HARM tissues were confirmed using a panel of genes commonly deregulated during oral carcinogenesis. Following BRB troche administration, the expression of prosurvival genes (AURKA, BIRC5, EGFR) and proinflammatory genes (NFKB1, PTGS2) were significantly reduced. There were no BRB-associated grade 3-4 toxicities or adverse events, and 79.2% (N = 30) of patients successfully completed the study with high levels of compliance (97.2%). The BRB phytochemicals cyanidin-3-rutinoside and cyanidin-3-xylosylrutinoside were detected in all OSCC tissues analyzed, demonstrating that bioactive components were successfully reaching targeted OSCC tissues. We confirmed that hallmark antiapoptotic and proinflammatory molecular biomarkers were overexpressed in OSCCs and that their gene expression was significantly reduced following BRB troche administration. As these molecular biomarkers are fundamental to oral carcinogenesis and are modifiable, they may represent emerging biomarkers of molecular efficacy for BRB-mediated oral cancer chemoprevention.

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.

Chemoprevention of oral cancer by topical application of black raspberries on high at-risk mucosa.

OBJECTIVE: To evaluate the preclinical efficacy of topical administration of freeze-dried black raspberries (BRBs) to inhibit the progression of premalignant oral lesions and modulate biomarkers of cancer development in high at-risk mucosa (HARM). STUDY DESIGN: Hamster cheek pouches (HCPs) were treated with carcinogen for 6 weeks to initiate a HARM microenvironment. Subsequently, right HCPs were topically administered a BRB suspension in short-term or long-term studies. After 12 weeks, squamous cell carcinoma (SCC) multiplicity, SCC incidence, and cell proliferation rates were evaluated. mRNA expression was measured in short-term treated pouches for selected oral cancer biomarkers. RESULTS: SCC multiplicity (-41.3%), tumor incidence (-37.1%), and proliferation rate (-6.9%) were reduced in HCPs receiving BRBs. Topical BRBs correlated with an increase in RB1 expression in developing oral lesions. CONCLUSIONS: Topical BRBs inhibit SCC development when targeted to HARM tissues. These results support the translational role of BRBs to prevent oral cancer development in humans.

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.

Coordinated expression of cyclin-dependent kinase-4 and its regulators in human oral tumors.

BACKGROUND/AIM: While aberrant expression of cyclin-dependent kinase-4 (CDK4) has been found in squamous cell carcinoma of the head and neck (SCCHN), the associations between CDK4 and its regulators, namely, cyclin D1, cyclin E, gankyrin, SEI1, and BMI1 in gene expression remain to be explored. Herein we investigated the mRNA profiles of these oncogenes and their interrelations in different oral lesion tissues. MATERIALS AND METHODS: Thirty SCCHN specimens and patient-matched high at-risk mucosa (HARM) and 16 healthy control specimens were subjected to quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses. RESULTS: The mRNA levels of CDK4, cyclin D1, gankyrin, SEI1, BMI1 were significantly elevated in both HARM and SCCHN (in comparison with control specimens), and statistically significant correlations were found among these markers in gene expression. CONCLUSION: Up-regulation of CDK4 and its regulators takes place in oral cancer progression in a coordinate manner, and HARM and SCCHN share a similar molecular signature within the CDK4-pRB pathway.

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.

The choice of reference gene affects statistical efficiency in quantitative PCR data analysis.

Quantitative polymerase chain reaction (qPCR), a highly sensitive method of measuring gene expression, is widely used in biomedical research. To produce reliable results, it is essential to use stably expressed reference genes (RGs) for data normalization so that sample-to-sample variation can be controlled. In this study, we examine the effect of different RGs on statistical efficiency by analyzing a qPCR data set that contains 12 target genes and 3 RGs. Our results show that choosing the most stably expressed RG for data normalization does not guarantee reduced variance or improved statistical efficiency. We also provide a formula for determining when data normalization will improve statistical efficiency and hence increase the power of statistical tests in data analysis.

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.