High Cytoplasmic Expression of Krüppel-like Factor 4 Is an Independent Prognostic Factor of Better Survival in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality in the world. Hepatocarcinogenesis is complex, with an extraordinary molecular heterogeneity. Krüppel-like factor 4 (KLF4) plays an important role in cell proliferation and differentiation, and it can function as a tumor suppressor or an oncoprotein, depending on tissue type. The role of KLF4 in HCC remains controversial. The aim of this study was to explore the clinical significance of KLF4 expression in HCC. The study included 205 patients with surgical resection. We performed immunostaining for KLF4 and Ki-67 to investigate the correlations of the clinicopathological parameters of HCC and to examine the proliferative index. KLF4 staining was observed in the cytoplasm of non-tumorous hepatocytes and tumor cells. We subdivided the immunohistological staining results for KLF4 into low expression (Staining 0 and 1+) and high expression (Staining 2+ and 3+) subgroups. The expression of KLF4 was significantly correlated with tumor differentiation (p = 0.001). The Ki-67 proliferative index was significantly lower in well-differentiated HCCs (0.781% ± 1.02% vs. 2.16% ± 3.14%, p = 0.012), but not significantly different between low-KLF4 expression and high-KLF4 expression (1.87% ± 2.93% vs. 2.51% ± 3.28%, p = 0.32). Kaplan–Meier analysis showed that a high expression of KLF4 was significantly correlated with a longer disease-specific survival (p = 0.019). Univariate and multivariate analyses showed that high KLF4 expression was an independent predictor of a better disease-specific survival (p = 0.017; hazard ratio = 0.398; 95% confidence interval: 0.19–0.85). High cytoplasmic expression of KLF4 was associated with better disease-specific survival and was an independently favorable prognostic factor in hepatocellular carcinoma. These promising results suggest that KLF4 may play an anti-oncogenic role in hepatocarcinogenesis.     

The Peroxisome Proliferator-Activated Receptor (PPAR) α Agonist Fenofibrate Suppresses Chemically Induced Lung Alveolar Proliferative Lesions in Male Obese Hyperlipidemic Mice

Activation of peroxisome proliferator-activated receptor (PPAR) α disrupts growth-related activities in a variety of human cancers. This study was designed to determine whether fenofibrate, a PPARα agonist, can suppress 4-nitroquinoline 1-oxide (4-NQO)-induced proliferative lesions in the lung of obese hyperlipidemic mice. Male Tsumura Suzuki Obese Diabetic mice were subcutaneously injected with 4-NQO to induce lung proliferative lesions, including adenocarcinomas. They were then fed a diet containing 0.01% or 0.05% fenofibrate for 29 weeks, starting 1 week after 4-NQO administration. At week 30, the incidence and multiplicity (number of lesions/mouse) of pulmonary proliferative lesions were lower in mice treated with 4-NQO and both doses of fenofibrate compared with those in mice treated with 4-NQO alone. The incidence and multiplicity of lesions were significantly lower in mice treated with 4-NQO and 0.05% fenofibrate compared with those in mice treated with 4-NQO alone (p < 0.05). Both doses of fenofibrate significantly reduced the proliferative activity of the lesions in 4-NQO-treated mice (p < 0.05). Fenofibrate also significantly reduced the serum insulin and insulin-like growth factor (IGF)-1 levels, and decreased the immunohistochemical expression of IGF-1 receptor (IGF-1R), phosphorylated Akt, and phosphorylated Erk1/2 in lung adenocarcinomas. Our results indicate that fenofibrate can prevent the development of 4-NQO-induced proliferative lesions in the lung by modulating the insulin-IGF axis.     

Is PTSD-Phenotype Associated with HPA-Axis Sensitivity?: The Endocannabinoid System in Modulating Stress Response in Rats

Endocannabinoids play a role in adaptation to stress and regulate the release of glucocorticoids in stressed and unstressed conditions. We recently found that basal corticosterone pulsatility may significantly impact the vulnerability for developing post-traumatic-stress-disorder (PTSD), suggesting that the endocannabinoid system may contribute to its development. To examine this, we exposed rats to predator scent stress (PSS). Behavioral reactions were recorded seven days post-PSS. Cerebrospinal fluid (CSF) was collected from anesthetized rats shortly after PSS exposure to determine the levels of 2-arachidonoyl glycerol (2-AG) and anandamide (AEA). To correlate between endocannabinoids and corticosterone levels, rats were placed in metabolic cages for urine collection. To assess the levels of endocannabinoids in specific brain regions, rats’ brains were harvested one day after behavioral analysis for staining and fluorescence quantification. Moreover, 2-AG was elevated in the CSF of PTSD-phenotype rats as compared with other groups and was inversely correlated with corticosterone urinary secretion. Eight days post-PSS exposure, hippocampal and hypothalamic 2-AG levels and hippocampal AEA levels were significantly more reduced in the PTSD-phenotype group compared to other groups. We posit that maladaptation to stress, which is propagated by an abnormal activation of endocannabinoids, mediates the subsequent stress-induced behavioral disruption, which, later, reduces neuronal the expression of endocannabinoids, contributing to PTSD symptomology.     

A Polymorphism at the 3′-UTR Region of the Aromatase Gene Is Associated with the Efficacy of the Aromatase Inhibitor,Anastrozole, in Metastatic Breast Carcinoma

Estrogen-related genes and the fat mass and obesity-associated (FTO) gene play a critical role in estrogen metabolism, and those polymorphisms are associated with a poor prognosis in breast cancer. However, little is known about the association between these polymorphisms and the efficacy of anastrozole. The aim was to investigate the impact of the genetic polymorphisms, CYP19A1, 17-β-HSD-1 and FTO, on the response to anastrozole in metastatic breast carcinoma (MBC) and to evaluate the impact of those polymorphisms on various clinicopathologic features. Two-hundred seventy-two women with hormone receptor-positive MBC treated with anastrozole were identified retrospectively. DNA was extracted from peripheral blood and genotyped for five variants in three candidate genes. Time to progression was improved in patients carrying the variant alleles of rs4646 when compared to patients with the wild-type allele (16.40 months versus 13.52 months; p = 0.049). The rs4646 variant alleles were significantly associated with longer overall survival (37.3 months versus 31.6 months; p = 0.007). This relationship was not observed with the rs10046, rs2830, rs9926298 and rs9939609 polymorphisms. The findings of this study indicate that rs4646 polymorphism in the CYP19A1 gene may serve as a prognostic maker of the response to anastrozole in patients with MBC who are treated with anastrozole.     

Calcium-Sensing Receptor in Human Peripheral Blood T Lymphocytes Is Involved in the AMI Onset and Progression through the NF-κB Signaling Pathway

Acute myocardial infarction (AMI) is a condition triggered by an inflammatory process that seriously affects human health. Calcium-sensing receptor (CaSR) in T lymphocytes is involved during the inflammation reaction. However, the relationship between them is not very clear. In this study, we collected human peripheral blood T lymphocytes from patients with AMI and in different stages of percutaneous coronary intervention (PCI) (at the onset of AMI, the first day after PCI (PCI-1), PCI-3, and PCI-5) to study the CaSR and NF-κB pathway protein expression, cytokine release and T cell apoptosis. The results showed that the expressions of CaSR, P-p65, Caspase-12, and the secretions of Th-1 and Th-2 type cytokines were increased at the onset of AMI, especially on the PCI-1. Meanwhile, the apoptosis rate of CD³⁺, CD⁴⁺ and CD⁸⁺ T lymphocytes also increased. However, from PCI-3, all the indicators began to decline. In addition, we also found that positive CaSR small interfering RNA (siRNA) transfection in T lymphocytes and NF-κB pathway blocker Bay-11-7082 reversed the increased expressions of CaSR, P-p65, Caspase-12, reduced the secretions of Th-1 and Th-2 type cytokines, and decreased T lymphocytes apoptosis rate not only in the AMI patients but also in the normal controls. All of these results indicated that CaSR in the human peripheral blood T lymphocytes were involved in the AMI onset and progression, which probably was related to the NF-κB pathway. Our study demonstrated the relationship between AMI and CaSR, and will provide new effective prevention theory and new targets for drug treatment.     

Temporary Increase of PPAR-γ and Transient Expression of UCP-1 in Stromal Vascular Fraction Isolated Human Adipocyte Derived Stem Cells During Adipogenesis

In this study, cells from the stromal vascular fraction of human subcutaneous tissues were induced to differentiate toward adipose cells in vitro for 2 weeks. During adipogenic differentiation, we followed the chronological changes in their morphology with Coherent anti-Stokes Raman scattering (CARS) microscopy and checked the PPAR-γ and UCP-1 expression with RT-PCR. On day 4 after inducing adipogenic differentiation, CARS imaging showed multiple small lipid droplets (LD) distributed peripherally along the cellular membrane. PPAR-γ began to express at this time and increased until day 14 at a steady rate. On day 7, the cells appeared as brown adipocytes with numerous small LD throughout the cytoplasm, and the mRNA level of UCP-1 rose abruptly by 6- to 7-fold. After an additional 7 days, CARS imaging showed the development of a large LD, which is characteristic of white adipocytes, and the mRNA level of UCP-1 slumped significantly. These results demonstrate the possibility that ADSC pass through a brown adipocyte-like stage while differentiating into white adipocytes.     

Identification and Expression Analysis of Candidate Genes Associated with Defense Responses to Phytophthora capsici in Pepper Line “PI 201234”

Phytophthora capsici (Leonian), classified as an oomycete, seriously threatens the production of pepper (Capsicum annuum). Current understanding of the defense responses in pepper to P. capsici is limited. In this study, RNA-sequencing analysis was utilized to identify differentially expressed genes in the resistant line “PI 201234”, with 1220 differentially expressed genes detected. Of those genes, 480 were up-regulated and 740 were down-regulated, with 211 candidate genes found to be involved in defense responses based on the gene annotations. Furthermore, the expression patterns of 12 candidate genes were further validated via quantitative real-time PCR (qPCR). These genes were found to be significantly up-regulated at different time points post-inoculation (6 hpi, 24 hpi, and 5 dpi) in the resistant line “PI 201234” and susceptible line “Qiemen”. Seven genes were found to be involved in cell wall modification, phytoalexin biosynthesis, symptom development, and phytohormone signaling pathways, thus possibly playing important roles in combating exogenous pathogens. The genes identified herein will provide a basis for further gene cloning and functional verification studies and will aid in an understanding of the regulatory mechanism of pepper resistance to P. capsici.     

<Emphasis Type="Italic">Trans</Emphasis> Fatty Acids Suppress TNF-α-Induced Inflammatory Gene Expression in Endothelial (HUVEC) and Hepatocellular Carcinoma (HepG2) Cells

Trans fatty acids (TFA) intake has been linked to cardiovascular diseases and liver diseases; yet the effect of TFA on inflammation remains controversial. Accordingly, the objective of this paper was to determine the in vitro effects of TFA on inflammatory gene expression. Human umbilical vein endothelial cells (HUVEC) and human hepatocellular carcinoma (HepG2) cells were treated for 24 h with either trans-vaccenic acid (tVA), trans-palmitoleic acid (tPA) or elaidic acid (EA) at concentrations of 5–150 µM, or with a mixture of tVA and tPA (150/50 µM). All TFA were highly incorporated into cell membranes, as determined by gas chromatography, representing 15–20% of total fatty acids in HUVEC and 3–8% in HepG2 cells. Incorporation of EA, a common industrial TFA, increased the ratio of the stearoyl-CoA desaturase (SCD-1), a key enzyme involved in fatty acid metabolism. Ruminant TFA, including tVA, tPA and the mixture of tVA and tPA, significantly reduced the TNF-α-induced gene expression of TNF, VCAM-1 and SOD2 in HUVEC, as well as TNF and IL-8 in HepG2 cells. EA also decreased inflammatory gene expression in HUVEC, but not in HepG2 cells. The inhibition of peroxisome proliferator-activated receptor (PPAR)-γ did not influence the effects of TFA on gene expression. Overall, physiological and supraphysiological concentrations of TFA, especially tVA and tPA, prevented inflammatory gene expression in vitro. This effect is independent of PPAR-γ activation and may be due to an alteration of fatty acid metabolism in cell membranes caused by the high incorporation of TFA.     

Antioxidant Enzymatic Activities and Gene Expression Associated with Heat Tolerance in the Stems and Roots of Two Cucurbit Species (“Cucurbita maxima” and “Cucurbita moschata”) and Their Interspecific Inbred Line “Maxchata”

The elucidation of heat tolerance mechanisms is required to combat the challenges of global warming. This study aimed to determine the antioxidant enzyme responses to heat stress, at the enzymatic activity and gene expression levels, and to investigate the antioxidative alterations associated with heat tolerance in the stems and roots of squashes using three genotypes differing in heat tolerance. Plants of heat-tolerant “C. moschata”, thermolabile “C. maxima” and moderately heat-tolerant interspecific inbred line “Maxchata” genotypes were exposed to moderate (37 °C) and severe (42 °C) heat shocks. “C. moschata” exhibited comparatively little oxidative damage, with the lowest hydrogen peroxide (H₂O₂), superoxide (O₂⁻) and malondialdehyde (MDA) contents in the roots compared to stems, followed by “Maxchata”. The enzyme activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and peroxidase (POD) were found to be increased with heat stress in tolerant genotypes. The significant inductions of FeSOD, MnSOD, APX2, CAT1 and CAT3 isoforms in tolerant genotypes suggested their participation in heat tolerance. The differential isoform patterns of SOD, APX and CAT between stems and roots also indicated their tissue specificity. Furthermore, despite the sequence similarity of the studied antioxidant genes among “C. maxima” and “Maxchata”, most of these genes were highly induced under heat stress in “Maxchata”, which contributed to its heat tolerance. This phenomenon also indicated the involvement of other unknown genetic and/or epigenetic factors in controlling the expression of these antioxidant genes in squashes, which demands further exploration.