Emodin reduces tumor burden by diminishing M2-like macrophages in colorectal cancer.

Emodin, a natural anthraquinone, has been shown to have antitumorigenic properties and may be an effective therapy for colorectal cancer (CRC). However, its clinical development has been hampered by a poor understanding of its mechanism of action. The purpose of this study was to 1) evaluate the efficacy of emodin in mouse models of intestinal/colorectal cancer and 2) to examine the impact of emodin on macrophage behavior in the context of CRC. We used a genetic model of intestinal cancer (ApcMin/+) and a chemically induced model of CRC [azoxymethane/dextran sodium sulfate (AOM/DSS)]. Emodin was administered orally (40 or 80 mg/kg in AOM/DSS and 80 mg/kg in ApcMin/+) three times a week to observe its preventative effects. Emodin reduced polyp count and size in both rodent models (P < 0.05). We further analyzed the colon microenvironment of AOM/DSS mice and found that mice treated with emodin exhibited lower protumorigenic M2-like macrophages and a reduced ratio of M2/M1 macrophages within the colon (P < 0.05). Despite this, we did not detect any significant changes in M2-associated cytokines (IL10, IL4, and Tgfb1) nor M1-associated cytokines (IL6, TNFα, IL1ß, and IFNγ) within excised polyps. However, there was a significant increase in NOS2 expression (M1 marker) in mice treated with 80 mg/kg emodin (P < 0.05). To confirm emodin's effects on macrophages, we exposed bone marrow-derived macrophages (BMDMs) to C26 colon cancer cell conditioned media. Supporting our in vivo data, emodin reduced M2-like macrophages. Overall, these data support the development of emodin as a natural compound for prevention of CRC given its ability to target protumor macrophages.NEW & NOTEWORTHY Our study confirms that emodin is an effective primary therapy against the onset of genetic and chemically induced sporadic colorectal cancer. We established that emodin reduces the M2-like protumorigenic macrophages in the tumor microenvironment. Furthermore, we provide evidence that emodin may be acting to antagonize the P2X7 receptor within the bone tissue and consequently decrease the activation of proinflammatory cells, which may have implications for recruitment of cells to the tumor microenvironment.

Understanding chemotherapy-induced intestinal mucositis and strategies to improve gut resilience.

Intestinal mucositis remains one of the most debilitating side effects related to chemotherapy. The onset and persistence of mucositis is an intricate physiological process involving cross-communication between the specific chemotherapeutic drug, the immune system, and gut microbes that results in a loss of mucosal integrity leading to gut-barrier dysfunction. Intestinal mucositis has a severe impact on a patient's quality of life and negatively influences the outcome of treatment. Most importantly, intestinal mucositis is a major contributor to the decreased survival rates and early onset of death associated with certain chemotherapy treatments. Understanding the pathophysiology and symptomology of intestinal mucositis is important in reducing the negative consequences of this condition. Prophylaxis, early diagnosis, and proper symptom management are essential to improved survival outcomes in patients with cancer. This review focuses on the pathobiology of intestinal mucositis that accompanies chemotherapy treatments. In addition, we will discuss the therapeutic potential of select strategies that have shown promise in mitigating chemotherapies' off-target effects without hampering their anticancer efficacy.NEW & NOTEWORTHY Intestinal mucositis, or damage to the intestinal mucosa, is a common side effect of chemotherapy. In this review, we describe the pathobiology of intestinal mucositis that is associated with chemotherapy treatments. In addition, we discuss the efficacy of several potential therapeutic strategies that have shown some potential in alleviating chemotherapies' off-target effects.

Safety of natural anthraquinone emodin: an assessment in mice.

BACKGROUND: Emodin, a natural anthraquinone, has shown potential as an effective therapeutic agent in the treatment of many diseases including cancer. However, its clinical development is hindered by uncertainties surrounding its potential toxicity. The primary purpose of this study was to uncover any potential toxic properties of emodin in mice at doses that have been shown to have efficacy in our cancer studies. In addition, we sought to assess the time course of emodin clearance when administered both intraperitoneally (I.P.) and orally (P.O.) in order to begin to establish effective dosing intervals. METHODS: We performed a subchronic (12 week) toxicity study using 3 different doses of emodin (~ 20 mg/kg, 40 mg/kg, and 80 mg/kg) infused into the AIN-76A diet of male and female C57BL/6 mice (n = 5/group/sex). Body weight and composition were assessed following the 12-week feeding regime. Tissues were harvested and assessed for gross pathological changes and blood was collected for a complete blood count and evaluation of alanine transaminase (ALT), aspartate transaminase (AST) and creatinine. For the pharmacokinetic study, emodin was delivered intraperitoneally I.P. or P.O. at 20 mg/kg or 40 mg/kg doses to male and female mice (n = 4/group/sex/time-point) and circulating levels of emodin were determined at 1, 4 and 12 h following administration via liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. RESULTS: We found that 12 weeks of low (20 mg/kg), medium (40 mg/kg), or high (80 mg/kg) emodin feeding did not cause pathophysiological perturbations in major organs. We also found that glucuronidated emodin peaks at 1 h for both I.P. and P.O. administered emodin and is eliminated by 12 h. Interestingly, female mice appear to metabolize emodin at a faster rate than male mice as evidenced by greater levels of glucuronidated emodin at the 1 h time-point (40 mg/kg for both I.P. and P.O. and 20 mg/kg I.P.) and the 4-h time-point (20 mg/kg I.P.). CONCLUSIONS: In summary, our studies establish that 1) emodin is safe for use in both male and female mice when given at 20, 40, and 80 mg/kg doses for 12 weeks and 2) sex differences should be considered when establishing dosing intervals for emodin treatment.

The Acute Effects of 5 Fluorouracil on Skeletal Muscle Resident and Infiltrating Immune Cells in Mice.

5 fluorouracil (5FU) has been a first-choice chemotherapy drug for several cancer types (e.g., colon, breast, head, and neck); however, its efficacy is diminished by patient acquired resistance and pervasive side effects. Leukopenia is a hallmark of 5FU; however, the impact of 5FU-induced leukopenia on healthy tissue is only becoming unearthed. Recently, skeletal muscle has been shown to be impacted by 5FU in clinical and preclinical settings and weakness and fatigue remain among the most consistent complaints in cancer patients undergoing chemotherapy. Monocytes, or more specifically macrophages, are the predominate immune cell in skeletal muscle which regulate turnover and homeostasis through removal of damaged or old materials as well as coordinate skeletal muscle repair and remodeling. Whether 5FU-induced leukopenia extends beyond circulation to impact resident and infiltrating skeletal muscle immune cells has not been examined. The purpose of the study was to examine the acute effects of 5FU on resident and infiltrating skeletal muscle monocytes and inflammatory mediators. Male C57BL/6 mice were given a physiologically translatable dose (35 mg/kg) of 5FU, or PBS, i.p. once daily for 5 days to recapitulate 1 dosing cycle. Our results demonstrate that 5FU reduced circulating leukocytes, erythrocytes, and thrombocytes while inducing significant body weight loss (>5%). Flow cytometry analysis of the skeletal muscle indicated a reduction in total CD45+ immune cells with a corresponding decrease in total CD45+CD11b+ monocytes. There was a strong relationship between circulating leukocytes and skeletal muscle CD45+ immune cells. Skeletal muscle Ly6cHigh activated monocytes and M1-like macrophages were reduced with 5FU treatment while total M2-like CD206+CD11c- macrophages were unchanged. Interestingly, 5FU reduced bone marrow CD45+ immune cells and CD45+CD11b+ monocytes. Our results demonstrate that 5FU induced body weight loss and decreased skeletal muscle CD45+ immune cells in association with a reduction in infiltrating Ly6cHigh monocytes. Interestingly, the loss of skeletal muscle immune cells occurred with bone marrow cell cycle arrest. Together our results highlight that skeletal muscle is sensitive to 5FU's off-target effects which disrupts both circulating and skeletal muscle immune cells.

Impact of weight loss and partial weight regain on immune cell and inflammatory markers in adipose tissue in male mice.

Weight fluctuations are common among individuals with obesity and are associated with increased morbidity. We examined adipose tissue immune and inflammatory markers in mice following weight loss and partial weight regain. Male C57BL/6 mice were randomized into four groups (n = 8-10/group): low-fat diet for 32 wk (LFD), high-fat diet for 32 wk (HFD), LFD for 28 wk and then changed to a HFD for 4 wk (LFD→H), and HFD for 21 wk and then changed to LFD for 7 wk and then changed to HFD for 4 wk (HFD→L→H). LFD→H and HFD→L→H mice did not differ in body weight, fat mass, or fat percentage; however, these parameters were greater than in LFD (P < 0.05) but lower than in HFD (P < 0.05). HFD→L→H mice had smaller adipocytes than HFD and LFD→H (P < 0.05) but not LFD mice. Expressions of CD11c and CD8a genes were elevated in epididymal fat of HFD→L→H compared with LFD→H and LFD (P < 0.05)mice. However, CD11c was lower in HFD→L→H than in HFD mice (P < 0.05), but there was no difference in CD8a between these groups. TNFα and IFNγ expressions were increased in HFD→L→H compared with LFD and LFD→H mice (P < 0.05), although HFD→L→H had lower expression of these cytokines than HFD (P < 0.05). IL-1ß was greater in HFD→L→H compared with LFD (P < 0.05) but was not different from LFD→H or HFD mice. Monocyte chemoattractant protein-1 was lower (P < 0.05) in HFD→L→H than in LFD→H. These data reinforce the importance of maintaining a body weight in the range that is recommended for optimal health to reduce immune and inflammatory perturbations associated with obesity.NEW & NOTEWORTHY We examined the immune and inflammatory status of adipose tissue in mice after they underwent weight loss followed by partial weight regain. We show an increase in selected immune cells and inflammatory mediators, in high-fat diet-fed mice that had prior exposure to a high-fat diet. Although weight fluctuations appear to exacerbate immune cell abundance and inflammation in adipose tissue, severity is less than in mice that were exposed to sustained high-fat diet feedings.

Prolonged high-fat-diet feeding promotes non-alcoholic fatty liver disease and alters gut microbiota in mice.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) has become an epidemic largely due to the worldwide increase in obesity. While lifestyle modifications and pharmacotherapies have been used to alleviate NAFLD, successful treatment options are limited. One of the main barriers to finding safe and effective drugs for long-term use in NAFLD is the fast initiation and progression of disease in the available preclinical models. Therefore, we are in need of preclinical models that (1) mimic the human manifestation of NAFLD and (2) have a longer progression time to allow for the design of superior treatments. AIM: To characterize a model of prolonged high-fat diet (HFD) feeding for investigation of the long-term progression of NAFLD. METHODS: In this study, we utilized prolonged HFD feeding to examine NAFLD features in C57BL/6 male mice. We fed mice with a HFD (60% fat, 20% protein, and 20% carbohydrate) for 80 wk to promote obesity (Old-HFD group, n = 18). A low-fat diet (LFD) (14% fat, 32% protein, and 54% carbohydrate) was administered for the same duration to age-matched mice (Old-LFD group, n = 15). An additional group of mice was maintained on the LFD (Young-LFD, n = 20) for a shorter duration (6 wk) to distinguish between age-dependent and age-independent effects. Liver, colon, adipose tissue, and feces were collected for histological and molecular assessments. RESULTS: Prolonged HFD feeding led to obesity and insulin resistance. Histological analysis in the liver of HFD mice demonstrated steatosis, cell injury, portal and lobular inflammation and fibrosis. In addition, molecular analysis for markers of endoplasmic reticulum stress established that the liver tissue of HFD mice have increased phosphorylated Jnk and CHOP. Lastly, we evaluated the gut microbial composition of Old-LFD and Old-HFD. We observed that prolonged HFD feeding in mice increased the relative abundance of the Firmicutes phylum. At the genus level, we observed a significant increase in the abundance of Adercreutzia, Coprococcus, Dorea, and Ruminococcus and decreased relative abundance of Turicibacter and Anaeroplasma in HFD mice. CONCLUSION: Overall, these data suggest that chronic HFD consumption in mice can mimic pathophysiological and some microbial events observed in NAFLD patients.

Effects of high fat diet-induced obesity on mammary tumorigenesis in the PyMT/MMTV murine model.

Clinical studies provide strong evidence that obesity and associated adipose tissue (AT) inflammation are risk factors for breast cancer (BrCA); however, mechanistic knowledge of the interaction of obesity, BrCA, and menopausal status has proven to be not only lacking, but contradictory. Obesity-induced inflammation and elevated biosynthesis of estrogens, through aromatase-mediated metabolism of precursors, have been linked with hormone receptor positive (HP) postmenopausal BrCA but not previously associated with premenopausal BrCA risk. Thus, further delineation of the interaction of obesity, inflammation, and aromatase is required for the development of therapeutic treatment options. The purpose of this study was to examine the effect of high fat diet (HFD)-induced inflammation on tumorigenesis in a model of pre and postmenopausal HP BrCA. Female PyMT/MMTV ovary intact and ovariectomized mice were fed low and HFD diets to examine the role of obesity-induced inflammation and hormone production in the development of HP BrCA. Tumor statistics for number, volume, weight, histopathology scoring and gene expression of macrophage and inflammatory mediators were measured in the AT and mammary gland at sacrifice. HFD feedings of ovary intact mice resulted in increased adiposity and tumorigenesis, indicated by increased primary tumor volume, multiplicity, tumor burden, and increased tumor progression represented by histopathological scoring. HFD-induced obesity significantly upregulated aromatase and macrophage marker expression in the AT (F4/80 and CD11c) and mammary gland (Mertk) in a premenopausal model of BrCA. Conversely, HFD feedings had no significant effect on tumorigenesis in a postmenopausal model of BrCA despite large increases in adiposity in ovariectomized mice; however, limitations within the model may have precluded any significant findings. This data suggests that obesity-induced increases in inflammation and hormone production, via aromatase expression, is associated with increases in tumorigenesis in a model of premenopausal HP BrCA in the PyMT/MMTV strain.

miR155 deficiency aggravates high-fat diet-induced adipose tissue fibrosis in male mice.

Noncoding RNAs are emerging as regulators of inflammatory and metabolic processes. There is evidence to suggest that miRNA155 (miR155) may be linked to inflammation and processes associated with adipogenesis. We examined the impact of global miRNA-155 deletion (miR155-/-) on the development of high-fat diet (HFD)-induced obesity. We hypothesized that loss of miR155 would decrease adipose tissue inflammation and improve the metabolic profile following HFD feedings. Beginning at 4-5 weeks of age, male miR155-/- and wild-type (WT) mice (n = 13-14) on a C57BL/6 background were fed either a HFD or low-fat diet for 20 weeks. Body weight was monitored throughout the study. Baseline and terminal body composition was assessed by DEXA analysis. Adipose tissue mRNA expression (RT-qPCR) of macrophage markers (F4/80, CD11c, and CD206) and inflammatory mediators (MCP-1 and TNF-α) as well as adiponectin were measured along with activation of NFκB-p65 and JNK and PPAR-γ Adipose tissue fibrosis was assessed by picrosirius red staining and western blot analysis of Collagen I, III, and VI. Glucose metabolism and insulin resistance were assessed by Homeostatic Model Assessment - Insulin Resistance (HOMA-IR), and a glucose tolerance test. Compared to WT HFD mice, miR155-/- HFD mice displayed similar body weights, yet reduced visceral adipose tissue accumulation. However, miR155-/- HFD displayed exacerbated adipose tissue fibrosis and decreased PPAR-γ protein content. The loss of miR155 did not affect adipose tissue inflammation or glucose metabolism. In conclusion, miR155 deletion did not attenuate the development of the obese phenotype, but adipose tissue fibrosis was exacerbated, possibly through changes to adipogenic processes.

Phosphodiesterase 11A (PDE11A), Enriched in Ventral Hippocampus Neurons, is Required for Consolidation of Social but not Nonsocial Memories in Mice.

The capacity to form long-lasting social memories is critical to our health and survival. cAMP signaling in the ventral hippocampal formation (VHIPP) appears to be required for social memory formation, but the phosphodiesterase (PDE) involved remains unknown. Previously, we showed that PDE11A, which degrades cAMP and cGMP, is preferentially expressed in CA1 and subiculum of the VHIPP. Here, we determine whether PDE11A is expressed in neurons where it could directly influence synaptic plasticity and whether expression is required for the consolidation and/or retrieval of social memories. In CA1, and possibly CA2, PDE11A4 is expressed throughout neuronal cell bodies, dendrites (stratum radiatum), and axons (fimbria), but not astrocytes. Unlike PDE2A, PDE9A, or PDE10A, PDE11A4 expression begins very low at postnatal day 7 (P7) and dramatically increases until P28, at which time it stabilizes to young adult levels. This expression pattern is consistent with the fact that PDE11A is required for social long-term memory (LTM) formation during adolescence and adulthood. Male and female PDE11 knockout (KO) mice show normal short-term memory (STM) for social odor recognition (SOR) and social transmission of food preference (STFP), but no LTM 24 h post training. Importantly, PDE11A KO mice show normal LTM for nonsocial odor recognition. Deletion of PDE11A may impair memory consolidation by impairing requisite protein translation in the VHIPP. Relative to WT littermates, PDE11A KO mice show reduced expression of RSK2 and lowered phosphorylation of S6 (pS6-235/236). Together, these data suggest PDE11A is selectively required for the proper consolidation of recognition and associative social memories.