Intermittent Fasting Attenuates Obesity-Induced Triple-Negative Breast Cancer Progression by Disrupting Cell Cycle, Epithelial-Mesenchymal Transition, Immune Contexture, and Proinflammatory Signature.

Obesity is associated with one-fifth of cancer deaths, and breast cancer is one of the obesity-related cancers. Triple-negative breast cancer (TNBC) lacks estrogen and progesterone receptors and human epidermal growth factor receptor 2, leading to the absence of these therapeutic targets, followed by poor overall survival. We investigated if obesity could hasten TNBC progression and intermittent fasting (IF) could attenuate the progression of obesity-related TNBC. Our meta-analysis of the TNBC outcomes literature showed that obesity led to poorer overall survival in TNBC patients. Fasting-mimicking media reduced cell proliferation disrupted the cell cycle, and decreased cell migration and invasion. IF decreased body weight in obese mice but no change in normal mice. Obese mice exhibited elevated plasma glucose and cholesterol levels, increased tumor volume and weight, and enhanced macrophage accumulation in tumors. The obesity-exacerbated TNBC progression was attenuated after IF, which decreased cyclin B1 and vimentin levels and reduced the proinflammatory signature in the obesity-associated tumor microenvironment. IF attenuated obesity-induced TNBC progression through reduced obesity and tumor burdens in cell and animal experiments, supporting the potential of a cost-effective adjuvant IF therapy for TNBC through lifestyle change. Further evidence is needed of these IF benefits in TNBC, including from human clinical trials.

Resumption of Spermatogenesis and Fertility Post Withdrawal of Hydroxyurea Treatment.

Hydroxyurea (HU), a drug for treating cancers of the blood and the management of sickle cell anemia, induces hypogonadism in males. However, the impact of HU on testicular architecture and function, as well as its effects on the resumption of male fertility following treatment withdrawal, remain poorly understood. We used adult male mice to determine whether HU-induced hypogonadism is reversible. Fertility indices of mice treated with HU daily for ~1 sperm cycle (2 months) were compared with those of their control counterparts. All indices of fertility were significantly reduced among mice treated with HU compared to controls. Interestingly, significant improvements in fertility indices were apparent after a 4-month withdrawal from HU treatment (testis weight: month 1 post-HU withdrawal (M1): HU, 0.09 ± 0.01 vs. control, 0.33 ± 0.03; M4: HU, 0.26 ± 0.03 vs. control, 0.37 ± 0.04 g); sperm motility (M1: HU,12 vs. 59; M4: HU, 45 vs. control, 61%; sperm density (M1: HU, 1.3 ± 0.3 vs. control, 15.7 ± 0.9; M4: HU, 8.1 ± 2.5 vs. control, 16.8 ± 1.9 million). Further, circulating testosterone increased in the 4th month following HU withdrawal and was comparable to that of controls. When a mating experiment was conducted, recovering males sired viable offspring with untreated females albeit at a lower rate than control males (p < 0.05); therefore, qualifying HU as a potential candidate for male contraception.

How Does Ginsenoside Rh2 Mitigate Adipogenesis in Cultured Cells and Obese Mice?

Ginsenoside Rh2, an intermediate metabolite of ginseng, but not naturally occurring, has recently drawn attention because of its anticancer effect. However, it is not clear if and how Rh2 inhibits preadipocytes differentiation. In the present study, we hypothesized that ginsenoside Rh2 attenuates adipogenesis through regulating the peroxisome proliferator-activated receptor gamma (PPAR-γ) pathway both in cells and obese mice. Different concentrations of Rh2 were applied both in 3T3-L1 cells and human primary preadipocytes to determine if Rh2 inhibits cell differentiation. Dietary Rh2 was administered to obese mice to determine if Rh2 prevents obesity in vivo. The mRNA and protein expression of PPAR-γ pathway molecules in cells and tissues were measured by real-time polymerase chain reaction (RT-PCR) and Western blot, respectively. Our results show that Rh2 dose-dependently (30-60 µM) inhibited cell differentiation in 3T3-L1 cells (44.5% ± 7.8% of control at 60 µM). This inhibitory effect is accompanied by the attenuation of the protein and/or mRNA expression of adipogenic markers including PPAR-γ and CCAAT/enhancer binding protein alpha, fatty acid synthase, fatty acid binding protein 4, and perilipin significantly (p < 0.05). Moreover, Rh2 significantly (p < 0.05) inhibited differentiation in human primary preadipocytes at much lower concentrations (5-15 µM). Furthermore, dietary intake of Rh2 (0.1 g Rh2/kg diet, w/w for eight weeks) significantly (p < 0.05) reduced protein PPAR-γ expression in liver and hepatic glutathione reductase and lowered fasting blood glucose. These results suggest that ginsenoside Rh2 dose-dependently inhibits adipogenesis through down-regulating the PPAR-γ pathway, and Rh2 may be a potential agent in preventing obesity in vivo.

Combination of curcumin and luteolin synergistically inhibits TNF-α-induced vascular inflammation in human vascular cells and mice.

Emerging evidence shows that phytochemicals, the secondary plant metabolites present in a large variety of foods, have the potential ability in reducing the risk of cardiovascular diseases. However, the dosages of phytochemicals in the cellular and animal studies are too high to reach in humans by relevant foods or dietary supplement intake. The aims of this study were to investigate whether and how combined curcumin and luteolin synergistically inhibit tumor necrosis factor-alpha (TNF-α)-induced monocytes adhesion endothelium, a crucial step of the development of endothelial dysfunction, both in human vascular cells and mouse aortic endothelium. Our results show that combined curcumin (1 µM) and luteolin (0.5 µM) synergistically (combination index is 0.60) inhibited TNF-α-induced monocytes adhesion to human EA.hy926 endothelial cells while the individual chemicals did not have such effect at the selected concentrations. We also found that TNF-α-enhanced protein expressions of vascular cell adhesion molecule 1 (VCAM-1), monocyte chemotactic protein-1 (MCP-1) and nuclear factor (NF)-κB translocation were synergistically reduced by the combined curcumin and luteolin in EA.hy 926 cells while the individual chemical did not have this inhibitory effect. Consistently, 2 weeks dietary intake of combined curcumin (500 mg/kg) and luteolin (500 mg/kg) in C57BL/6 mice synergistically prevented TNF-α-stimulated adhesion of mouse monocytes to aortic endothelium ex vivo as well as the TNF-α-increased aortic protein expression of MCP-1 and VCAM-1. Therefore, combined curcumin and luteolin at physiological concentrations synergistically inhibits TNF-α-induced monocytes adhesion to endothelial cells and expressions of MCP-1 and VCAM-1 via suppressing NF-κB translocation into the nucleus.

Synergistic anti-inflammatory effects and mechanisms of combined phytochemicals.

The anti-inflammatory effects of phytochemicals, bioactive components from plants having health benefits, have been heavily investigated in the last several decades. However, the gap between the high dosage demands (µM) of phytochemicals in vitro studies and the low bioavailability (nM) of most phytochemicals after consuming relevant foods/supplements in humans undermines the application of these phytochemicals in the prevention of chronic inflammation and its related chronic diseases in humans. One of the approaches to bridging this gap is to combine two or more phytochemicals/foods to synergistically prevent chronic inflammation. While increasing combinations of phytochemicals on anti-inflammation studies have been reported, there is no report dedicating why combining two or more phytochemicals synergistically attenuates chronic inflammation. In the present review, we summarized different types of combinations exerting synergistic anti-inflammatory effects such as the combination of phytochemicals from the same foods, and the combination of phytochemicals from different foods/plants. Particularly, we proposed five mechanisms including enhancing the bioavailability of phytochemicals, increasing antioxidant capacity, interacting with gut microbiome and targeting same and different signaling pathways, to understand how the combination of phytochemicals exerts synergistic anti-inflammatory effects in cells, animals, and humans. This review provides clues to boost more studies to combine several phytochemicals/foods to reduce chronic inflammation and prevent chronic diseases in humans.

Implication of calcium activated RasGRF2 in Annexin A6-mediated breast tumor cell growth and motility.

The role of AnxA6 in breast cancer and in particular, the mechanisms underlying its contribution to tumor cell growth and/or motility remain poorly understood. In this study, we established the tumor suppressor function of AnxA6 in triple negative breast cancer (TNBC) cells by showing that loss of AnxA6 is associated with early onset and rapid growth of xenograft TNBC tumors in mice. We also identified the Ca2+ activated RasGRF2 as an effector of AnxA6 mediated TNBC cell growth and motility. Activation of Ca2+ mobilizing oncogenic receptors such as epidermal growth factor receptor (EGFR) in TNBC cells or pharmacological stimulation of Ca2+ influx led to activation, subsequent degradation and altered effector functions of RasGRF2. Inhibition of Ca2+ influx or overexpression of AnxA6 blocked the activation/degradation of RasGRF2. We also show that AnxA6 acts as a scaffold for RasGRF2 and Ras proteins and that its interaction with RasGRF2 is modulated by GTP and/or activation of Ras proteins. Meanwhile, down-regulation of RasGRF2 and treatment of cells with the EGFR-targeted tyrosine kinase inhibitor (TKI) lapatinib strongly attenuated the growth of otherwise EGFR-TKI resistant AnxA6 high TNBC cells. These data not only suggest that AnxA6 modulated Ca2+ influx and effector functions of RasGRF2 underlie at least in part, the AnxA6 mediated TNBC cell growth and/or motility, but also provide a rationale to target Ras-driven TNBC with EGFR targeted therapies in combination with inhibition of RasGRF2.

Ginseng and obesity: observations and understanding in cultured cells, animals and humans.

Ginseng, a traditional medical herb, has been reported having beneficial effects in fatigue, heart diseases, diabetes, immune function and erectile dysfunction. In recent years, increasing investigations have been conducted on ginseng in preventing and treating of obesity, one of the major worldwide escalating public health concerns. However, the effect and the relevant mechanisms behind how ginseng works as an antiobesity treatment are still controversial. In this review, we briefly discussed the chemical structures, metabolism and pharmacokinetics of ginseng and its major bioactive components ginsenosides. The major focus is on the antiobesity effects and the physiological, cellular and molecular mechanisms of ginseng and its ginsenosides in cultured cells, animal models and humans. We particularly compared the ginsenosides profiles, the antiobesity effects and the mechanisms between Asian ginseng (Panax ginseng) and American ginseng (Panax quinquefolius), the two major ginseng species having opposite medical effects in traditional Chinese medicine. Our unpublished data on the ginseng antiobesity in cultured cells and mice were also included. We further addressed the current problems and future directions of the ginseng antiobesity research.

High corn oil dietary intake improves health and longevity of aging mice.

Corn oil has been recommended as a replacement for saturated fats because of its high levels of poly- and mono-unsaturated fatty acids. In the present study, we tested whether very high levels of corn oil (58.6% fat-derived calories, FDC) intake improve health and longevity of aging mice. Twelve month old male C57BL/6 mice were fed a normal diet (10% FDC of corn oil, N) or a high fat diet (58.6% FDC of corn oil, HF) for 13-15 months. Our results show that a HF diet significantly increased the longevity of the aged mice (at 25 months of age, 53.8% of mice died in the N group, whereas the mortality rate was only 23.2% in the HF group). High corn oil also reversed aging-increased blood lipids including triglyceride, total cholesterol and LDL. Similarly, high corn oil intake overturned aging-raised pro-inflammatory markers including IL-1ß, IL-6, and monocyte chemotactic protein-1 (MCP-1) in the blood. In addition, corn oil intake reversed aging-damaged rotarod performance and liver function. Interestingly, the HF group was significantly heavier than the N group (53.6g/mouse vs. 41.3g/mouse); however, both HF and N groups had the same calorie intake (12.48 kcal/d/mouse vs. 12.24 kcal/d/mouse). Although, the HF group's food consumption was lower than that of the N group (2.4 g/d/mouse vs. 3.4 g/d/mouse). These results suggest that if total calorie consumption stays in the normal range, very high levels of corn oil intake improve health and longevity of aging mice.

Activated charcoal and the absorption of ferrous sulfate in rats.

The treatment of iron poisoning has typically not included the administration of activated charcoal due to the lack of evidence supporting its efficacy. Several in vitro studies have demonstrated good adsorption of iron in a variety of pH ranges that were comparable to those found with other drugs for which activated charcoal is clinically used. This study was designed to determine whether activated charcoal altered the gastrointestinal absorption of toxic doses of iron as ferrous sulfate in an in vivo model. Seventy-five male Sprague-Dawley rats were randomly assigned into 5 groups: control given only distilled water; 100 mg elemental Iron and water; 1:1 charcoal to iron; 2:1 charcoal to iron; and 4:1 charcoal to iron. All treatments were administered consecutively by gavage within 5 min. Physiological measurements and blood samples were taken at 0, 1, 4 and 8 h after treatment. There were no consistent differences in physiological measurements among the 5 groups. Mean serum iron concentrations did not differ among Groups 2, 3, 4 and 5 at the 4 sampling times except at I h between Groups 4 and 5. The area under the curve for serum iron concentrations did not differ among the treatment groups. Activated charcoal did not alter the extent of iron absorption in the experimental model.

Chronic restraint via tail immobilization of mice: effects on corticosterone levels and other physiologic indices of stress.

Chronic venous cannulation in mice is an acceptable and useful technique for repeated blood sampling or continuous intravenous administration of substances, for which mild restraint of the animal may be necessary. Because chronic restraint has drawn considerable attention in the animal welfare community, the purpose of this study was to evaluate physiologic indices of stress in mice restrained by using an established tail restraint method. Serum corticosterone levels and body, thymus, adrenal, and spleen weights on days 2, 5, 8, and 12 were compared between tail-restrained and unrestrained mice. There were no significant differences between the two groups at the time points evaluated. Corticosterone levels were highest on day 8 for both groups and were significantly different from those on days 2 (P<0.009) and 5 (P<0.04) for restrained mice and on day 2 (P<0.02) for unrestrained mice. Levels in both groups declined by day 12, suggesting habitation. Weight loss was observed in all mice whether restrained or unrestrained. Significant differences in body, thymus, adrenal, and spleen weights were not evident between restrained and unrestrained mice. This study provides important information for balancing issues of prolonged restraint, animal well-being, and research goals.