Sex and diet-dependent gene alterations in human and rat brains with a history of nicotine exposure.

Introduction: Chronic nicotine exposure induces changes in the expression of key regulatory genes associated with metabolic function and neuronal alterations in the brain. Many bioregulatory genes have been associated with exposure to nicotine, but the modulating effects of sex and diet on gene expression in nicotine-exposed brains have been largely unexplored. Both humans and rodents display motivation for nicotine use and the emergence of withdrawal symptoms during abstinence. Research comparing pre-clinical models with human subjects provides an important opportunity to understand common biomarkers of the harmful effects of nicotine as well as information that may help guide the development of more effective interventions for nicotine cessation. Methods: Human postmortem dorsolateral prefrontal cortex (dLPFC) tissue BA9 was collected from female and male subjects, smokers and non-smokers (N = 12 per group). Rat frontal lobes were collected from female and male rats that received a regular diet (RD) or a high-fat diet (HFD) (N = 12 per group) for 14 days following implantation of a osmotic mini-pump (Alzet) that delivered nicotine continuously. Controls (control-s) received a sham surgical procedure. RNA was extracted from tissue from human and rat samples and reversed-transcribed to cDNA. Gene expression of CHRNA10 (Cholinergic receptor nicotinic alpha 10), CERKL (Ceramide Kinase-Like), SMYD1 (SET and MYD Domin Containing 1), and FA2H (Fatty Acid 2-Hydrolase) in humans was compared to rats in each subset of groups and quantified by qPCR methods. Additionally, protein expression of FA2H was analyzed by immunohistochemistry (IHC) in human dLPFC. Results: Humans with a history of smoking displayed decreased CHRNA10 (p = 0.0005), CERKL (p ≤ 0.0001), and SMYD1 (p = 0.0005) expression and increased FA2H (p = 0.0097) expression compared to non-smokers (p < 0.05). Similar patterns of results were observed in nicotine exposed vs. control rats. Interestingly, sex-related differences in gene expression for CERKL and FA2H were observed. In addition, ANCOVA analysis showed a significant effect of nicotine in a sex-different manner, including an increase in CERKL in male and female rats with RD or HFD. In rats exposed to an HFD, FA2H gene expression was lower in nicotine-treated rats compared to RD rats treated with nicotine. Protein expression of FA2H (p = 0.001) by IHC was significantly higher in smokers compared to non-smokers. Conclusion: These results suggest that a history of long-term nicotine exposure in humans alters the expression of sphingolipid metabolism-related (CERKL, SMYD1, and FA2H) and neuronal (CHRNA10) marker genes similarly as compared to rats. Sex- and diet-dependent differences appear in nicotine-exposed rats, critical in regulating sphingolipid metabolism and nicotinic acetylcholine receptors. This research enhances the construct validity of rat models of nicotine usage by showing a similar pattern of changes in gene expression in human subjects with a smoking history.

3D Tissue-Engineered Tumor Model for Ewing's Sarcoma That Incorporates Bone-like ECM and Mineralization.

The tumor microenvironment harbors essential components required for cancer progression including biochemical signals and mechanical cues. To study the effects of microenvironmental elements on Ewing's sarcoma (ES) pathogenesis, we tissue-engineered an acellular three-dimensional (3D) bone tumor niche from electrospun poly(ε-caprolactone) (PCL) scaffolds that incorporate bone-like architecture, extracellular matrix (ECM), and mineralization. PCL-ECM constructs were generated by decellularizing PCL scaffolds harboring cultures of osteogenic human mesenchymal stem cells. The PCL-ECM constructs simulated in vivo-like tumor architecture and increased the proliferation of ES cells compared to PCL scaffolds alone. Compared to monolayer controls, 3D environments facilitated the downregulation of the canonical insulin-like growth factor 1 receptor (IGF-1R) signal cascade through mechanistic target of rapamycin (mTOR), both of which are targets of recent clinical trials. In addition to the downregulation of canonical IGF-1R signaling, 3D environments promoted a reduction in the clathrin-dependent nuclear localization and transcriptional activity of IGF-1R. In vitro drug testing revealed that 3D environments generated cell phenotypes that were resistant to mTOR inhibition and chemotherapy. Our versatile PCL-ECM constructs allow for the investigation of the roles of various microenvironmental elements in ES tumor growth, cancer cell morphology, and induction of resistant cell phenotypes.