cyp21a2 Knockout Tadpoles Survive Metamorphosis Despite Low Corticosterone.

Corticosteroids are so vital for organ maturation that reduced corticosteroid signaling during postembryonic development causes death in terrestrial vertebrates. Indeed, death occurs at metamorphosis in frogs lacking proopiomelanocortin (pomc) or the glucocorticoid receptor (GR; nr3c1). Some residual corticosteroids exist in pomc mutants to activate the wild-type (WT) GR and mineralocorticoid receptor (MR), and the elevated corticosteroids in GR mutants may activate MR. Thus, we expected a more severe developmental phenotype in tadpoles with inactivation of 21-hydroxylase, which should eliminate all interrenal corticosteroid biosynthesis. Using CRISPR/Cas9 in Xenopus tropicalis, we produced an 11-base pair deletion in cyp21a2, the gene encoding 21-hydroxylase. Growth and development were delayed in cyp21a2 mutant tadpoles, but unlike the other frog models, they survived metamorphosis. Consistent with an absence of 21-hydroxylase, mutant tadpoles had a 95% reduction of aldosterone in tail tissue, but they retained some corticosterone (∼40% of WT siblings), an amount, however, too low for survival in pomc mutants. Decreased corticosteroid signaling was evidenced by reduced expression of corticosteroid-response gene, klf9, and by impaired negative feedback in the hypothalamus-pituitary-interrenal axis with higher messenger RNA expression levels of crh, pomc, star, and cyp11b2 and an approximately 30-fold increase in tail content of progesterone. In vitro tail-tip culture showed that progesterone can transactivate the frog GR. The inadequate activation of GR by corticosterone in cyp21a2 mutants was likely compensated for by sufficient corticosteroid signaling from other GR ligands to allow survival through the developmental transition from aquatic to terrestrial life.

Comparative Effects of E-Cigarette Aerosol on Periodontium of Periodontitis Patients.

Introduction: Tobacco use is one of the main causes of periodontitis. E-cigarette are gaining in popularity, and studies are needed to better understand the impact of e-cigarettes on oral health. Objective: To perform a longitudinal study to evaluate the adverse effects of e-cigarettes on periodontal health. Methods: Naïve E-cigarette users, cigarette smokers, and non-smokers were recruited using newspaper and social media. Age, gender, and ethnicity, were recorded. Participants were scheduled for two visits 6 months apart. At each visit, we collected data on the frequency and magnitude of e-cigarette and cigarette use, and alcohol consumption. Carbon monoxide (CO) levels, cotinine levels, salivary flow rate, periodontal probing depth (PD), bleeding on probing (BoP), and clinical attachment loss (CAL) were also determined at both baseline and follow-up visits and compared between groups with two-way repeated measures ANOVA. Periodontal diagnosis and other categorical variables were compared between groups with the chi-square statistic and logistic regression. Results: We screened 159 subjects and recruited 119 subjects. One-hundred-one subjects (31 cigarette smokers, 32 e-cigarette smokers, and 38 non-smokers) completed every assessment in both visits. The retention and compliance rate of subjects was 84.9%. The use of social media and craigslist was significant in recruiting e-cigarette subjects. Ethnicity and race differed between groups, as did average age in the male subjects. Carbon monoxide and salivary cotinine levels were highest among cigarette smokers. Bleeding on probing and average PDs similarly increased over time in all three groups, but CAL uniquely increased in e-cigarette smokers. Rates of severe periodontal disease were higher in cigarette smokers and e-cigarette users than non-smokers, but interpretation is confounded by the older age of the cigarette smokers. Conclusion: Among the recruited participants, CAL after 6 months was significantly worse only in the e-cigarette smokers. This study design and protocol will assist in future larger studies on e-cigarette and oral health.

Electronic Cigarette Aerosol Modulates the Oral Microbiome and Increases Risk of Infection.

The trend of e-cigarette use among teens is ever increasing. Here we show the dysbiotic oral microbial ecology in e-cigarette users influencing the local host immune environment compared with non-smoker controls and cigarette smokers. Using 16S rRNA high-throughput sequencing, we evaluated 119 human participants, 40 in each of the three cohorts, and found significantly altered beta-diversity in e-cigarette users (p = 0.006) when compared with never smokers or tobacco cigarette smokers. The abundance of Porphyromonas and Veillonella (p = 0.008) was higher among vapers. Interleukin (IL)-6 and IL-1ß were highly elevated in e-cigarette users when compared with non-users. Epithelial cell-exposed e-cigarette aerosols were more susceptible for infection. In vitro infection model of premalignant Leuk-1 and malignant cell lines exposed to e-cigarette aerosol and challenged by Porphyromonas gingivalis and Fusobacterium nucleatum resulted in elevated inflammatory response. Our findings for the first time demonstrate that e-cigarette users are more prone to infection.

Targeting cellular metabolism to reduce head and neck cancer growth.

Head and neck squamous cell carcinoma (HNSCC) presents a major public health concern because of delayed diagnosis and poor prognosis. Malignant cells often reprogram their metabolism in order to promote their survival and proliferation. Aberrant glutaminase 1 (GLS1) expression enables malignant cells to undergo increased glutaminolysis and utilization of glutamine as an alternative nutrient. In this study, we found a significantly elevated GLS1 expression in HNSCC, and patients with high expression levels of GLS1 experienced shorter disease-free periods after therapy. We hypothesized that the GLS1 selective inhibitor, bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), which curtails cells' glutamine consumption, may inhibit HNSCC cell growth. Our results support the idea that BPTES inhibits HNSCC growth by inducing apoptosis and cell cycle arrest. Considering that metformin can reduce glucose consumption, we speculated that metformin would enhance the anti-neoplasia effect of BPTES by suppressing malignant cells' glucose utilization. The combination of both compounds exhibited an additive inhibitory effect on cancer cell survival and proliferation. All of our data suggest that GLS1 is a promising therapeutic target for HNSCC treatment. Combining BPTES with metformin might achieve improved anti-cancer effects in HNSSC, which sheds light on using novel therapeutic strategies by dually targeting cellular metabolism.

The Effects of Combinatorial Genistein and Sulforaphane in Breast Tumor Inhibition: Role in Epigenetic Regulation.

Dietary compounds that possess the properties of altering epigenetic processes are gaining popularity as targets for cancer prevention studies. These compounds when administered at optimal concentrations and especially in combination can have enhanced effects in cancer prevention or therapy. It is important to study the interaction of two or more compounds in order to assess their role in enhancing prevention. Genistein (GEN), found in soy, has been extensively studied for its role as an epigenetic modifier especially as a DNA methyltransferase (DNMT) inhibitor and sulforaphane (SFN), found in cruciferous vegetables, is known as a histone deacetylase (HDAC) inhibitor. However, very little is known about the effects of these two compounds in conjunction in breast cancer prevention or therapy. In our current study, we determined that, at certain doses, the compounds have synergistic effects in decreasing cellular viability of breast cancer cell lines. Our results indicate that the combination of GEN and SFN is much more effective than their single doses in increasing the rate of apoptosis and lowering the colony forming potential of these cells. We determined that these compounds inhibit cell cycle progression to G2 phase in MDA-MB-231 and G1 phase in MCF-7 breast cancer cell lines. Additionally, we determined that the combination is effective as an HDAC and histone methyltransferase (HMT) inhibitor. Furthermore, we demonstrated that this combination downregulates the levels of HDAC2 and HDAC3 both at the mRNA and protein levels. We also found that these compounds have the potential to downregulate KLF4 levels, which plays an important role in stem cell formation. The combination of GEN and SFN is also effective in downregulating hTERT levels, which is known to be activated when KLF4 binds to its promoter region. Our hypothesis is further strengthened by in vivo studies, where the combination is administered to transgenic mice in the form of genistein and SFN-enriched broccoli sprouts. We have demonstrated that the combination is more effective in preventing or treating mammary cancer via extending tumor latency and reducing tumor volumes/sizes than either of these dietary components administered alone. These results are consistent with our in vitro study suggesting potential preventive and therapeutic effects of this novel dietary combinatorial approach against breast cancer.

Withaferin A and sulforaphane regulate breast cancer cell cycle progression through epigenetic mechanisms.

Little is known about the effects of combinatorial dietary compounds on the regulation of epigenetic mechanisms involved in breast cancer prevention. The human diet consists of a multitude of components, and there is a need to elucidate how certain compounds interact in collaboration. Withaferin A (WA), found in the Indian winter cherry and documented as a DNA methyltransferase (DNMT) inhibitor, and sulforaphane (SFN), a well-known histone deacetylase (HDAC) inhibitor found in cruciferous vegetables, are two epigenetic modifying compounds that have only recently been studied in conjunction. The use of DNMT and HDAC inhibitors to reverse the malignant expression of certain genes in breast cancer has shown considerable promise. Previously, we found that SFN + WA synergistically promote breast cancer cell death. Herein, we determined that these compounds inhibit cell cycle progression from S to G2 phase in MDA-MB-231 and MCF-7 breast cancer. Furthermore, we demonstrate that this unique combination of epigenetic modifying compounds down-regulates the levels of Cyclin D1 and CDK4, and pRB; conversely, the levels of E2F mRNA and tumor suppressor p21 are increased independently of p53. We find these events coincide with an increase in unrestricted histone methylation. We propose SFN + WA-induced breast cancer cell death is attributed, in part, to epigenetic modifications that result in the modulated expression of key genes responsible for the regulation of cancer cell senescence.

Impact of genistein on the gut microbiome of humanized mice and its role in breast tumor inhibition.

Since dietary polyphenols can have beneficial effects in prevention and treatment of cancer, we tested the hypothesis that breast cancer patients' intestinal microbiota is modulated by genistein (GE), an isoflavone found in soy, and that microbial alterations may offset the side effects brought about by chemotherapy. We demonstrated successful humanization of germ-free mice by transplanting fecal samples from breast cancer patients before and after chemotherapy. Mice were then grouped based on chemotherapy status and GE or control diet. We did not find any significant differences between pre-chemotherapy and post-chemotherapy bacterial composition and abundances. Germ-free mice on a GE diet showed differences in microbial composition as compared to mice on control diet. Four weeks after introduction of the customized GE diet, there was distinct clustering of GE-fed mice as compared to the control-fed group. In the gut microbiome of GE-treated humanized mice, there was an increase in abundance of genera Lactococcus and Eubacterium. Phylum Verrucomicrobia showed statistically significant (p = 0.02) differences in abundances between the GE-fed and control-fed groups. There was an increase in bacteria belonging to family Lachnospiraceae and Ruminococcaceae in GE-fed mice. Marked changes were observed in GE catabolism in mice humanized with fecal material from two of three patients' post-chemotherapy with complete disappearance of 4-ethylphenol and 2-(4-hydroxyphenol) propionic acid conjugates. The post-tumor samples did not show any distinct clustering of the gut microbiota between the two diet groups. There was an increase in latency of about 25% for tumor growth of the humanized mice that were on a GE diet as compared to humanized mice on a control diet. The average tumor size for the GE group was significantly decreased compared to the non-GE group. Collectively, our results suggest that the intestinal microbiota becomes altered with a GE diet before induction of tumor. Our findings indicate that GE modulates the microbiome in humanized mice that may contribute to its effects on increasing the latency of breast tumor and reducing tumor growth.

Influences of diet and the gut microbiome on epigenetic modulation in cancer and other diseases.

Epigenetic modulation of gene activity occurs in response to non-genetic factors such as body weight status, physical activity, dietary factors, and environmental toxins. In addition, each of these factors is thought to affect and be affected by the gut microbiome. A primary mechanism that links these various factors together in mediating control of gene expression is the production of metabolites that serve as critical cofactors and allosteric regulators of epigenetic processes. Here, we review the involvement of the gut microbiota and its interactions with dietary factors, many of which have known cellular bioactivity, focusing on particular epigenetic processes affected and the influence they have on human health and disease, particularly cancer and response to treatment. Advances in DNA sequencing have expanded the capacity for studying the microbiome. Combining this with rapidly improving techniques to measure the metabolome provides opportunities to understand complex relationships that may underlie the development and progression of cancer as well as treatment-related sequelae. Given broad reaching and fundamental biology, both at the cellular and organismal levels, we propose that interactive research programs, which utilize a wide range of mutually informative experimental model systems-each one optimally suited for answering particular questions-provide the best path forward for breaking ground on new knowledge and ultimately understanding the epigenetic significance of the gut microbiome and its response to dietary factors in cancer prevention and therapy.