Jennifer L. Steiner

Exercise training increases mitochondrial biogenesis in the brain

Increased muscle mitochondria are largely responsible for the increased resistance to fatigue and health benefits ascribed to exercise training. However, very little attention has been given to the likely benefits of increased brain mitochondria in this regard. We examined the effects of exercise training on markers of both brain and muscle mitochondrial biogenesis in relation to endurance capacity assessed by a treadmill run to fatigue (RTF) in mice. Male ICR mice were assigned to exercise (EX) or sedentary (SED) conditions (n = 16-19/group). EX mice performed 8 wk of treadmill running for 1 h/day, 6 days/wk at 25 m/min and a 5% incline. Twenty-four hours after the last training bout a subgroup of mice (n = 9-11/group) were euthanized, and brain (brain stem, cerebellum, cortex, frontal lobe, hippocampus, hypothalamus, and midbrain) and muscle (soleus) tissues were isolated for analysis of mRNA expression of peroxisome proliferator-activated receptor-gamma coactivator-1-alpha (PGC-1α), Silent Information Regulator T1 (SIRT1), citrate synthase (CS), and mitochondrial DNA (mtDNA) using RT-PCR. A different subgroup of EX and SED mice (n = 7-8/group) performed a treadmill RTF test. Exercise training increased PGC-1α, SIRT1, and CS mRNA and mtDNA in most brain regions in addition to the soleus (P < 0.05). Mean treadmill RTF increased from 74.0 ± 9.6 min to 126.5 ± 16.1 min following training (P < 0.05). These findings suggest that exercise training increases brain mitochondrial biogenesis, which may have important implications, not only with regard to fatigue, but also with respect to various central nervous system diseases and age-related dementia that are often characterized by mitochondrial dysfunction.

Linking tumor-associated macrophages, inflammation, and intestinal tumorigenesis: role of MCP-1

Tumor-associated macrophages are associated with poor prognosis in certain cancers. Monocyte chemoattractant protein 1 (MCP-1) is thought to be the most important chemokine for recruitment of macrophages to the tumor microenvironment. However, its role on tumorigenesis in a genetic mouse model of colon cancer has not been explored. We examined the role of MCP-1 on tumor-associated macrophages, inflammation, and intestinal tumorigenesis. Male Apc(Min/+), Apc(Min/+)/MCP-1(-/-) or wild-type mice were euthanized at 18 wk of age and intestines were analyzed for polyp burden, apoptosis, proliferation, β-catenin, macrophage number and phenotype, markers for cytotoxic T lymphocytes and regulatory T cells, and inflammatory mediators. MCP-1 deficiency decreased overall polyp number by 20% and specifically large polyp number by 45% (P < 0.05). This was consistent with an increase in apoptotic cells (P < 0.05), but there was no change detected in proliferation or β-catenin. MCP-1 deficiency decreased F4/80-positive cells in both the polyp tissue and surrounding intestinal tissue (P < 0.05) as well as expression of markers associated with M1 (IL-12 and IL-23) and M2 macrophages (IL-13, CD206, TGF-β, and CCL17) (P < 0.05). MCP-1 knockout was also associated with increased cytotoxic T lymphocytes and decreased regulatory T cells (P < 0.05). In addition, MCP-1(-/-) offset the increased mRNA expression of IL-1β and IL-6 in intestinal tissue and IL-1β and TNF-α in polyp tissue (P < 0.05), and prevented the decrease in SOCS1 expression (P < 0.05). We demonstrate that MCP-1 is an important mediator of tumor growth and immune regulation that may serve as an important biomarker and/or therapeutic target in colon cancer.

Linking inflammation to tumorigenesis in a mouse model of high-fat-diet-enhanced colon cancer

Many observational epidemiologic studies suggest an association between high-fat-diet (HFD) and colon cancer risk. However, the lack of controlled experimental studies that examine this relationship and the mechanisms involved weaken the basis for inferring a causal relationship. Inflammation plays a role in colon cancer progression and HFDs have been reported to increase inflammation; however, the inflammatory effects of HFD in colon cancer have yet to be firmly established. We examined the effects of a novel HFD that closely mimics the standard American diet (12% and 40% of total caloric intake from saturated fat and total fat, respectively) on macrophage markers and inflammatory mediators in a mouse model of intestinal tumorigenesis and relate this to polyp characteristics as well as measures of adiposity. Male Apc(Min/+) mice (7-8/group) were fed a Control Diet (Con) or novel high-fat-diet (HFD) from 4 to 12weeks of age. Body weight and body composition were measured weekly and monthly, respectively. Intestinal tissue was analyzed for polyp burden (number and size). Gene expression of macrophage markers and inflammatory mediators were examined in the adipose tissue and polyps. The HFD increased the expression of macrophage markers and inflammatory mediators in the adipose tissue (F4/80, CD11c, TLR-4 and MCP-1) and tumor microenvironment (IL-12, MCP-1, IL-6 and TNF-α). As expected, the HFD increased body weight, body fat percent, fat mass and blood glucose (P<0.05), and was associated with an increase in the number of large polyps (P<0.05) but not total polyps. In summary, consumption of a HFD, similar in macronutrient composition to the standard American diet, altered the expression of macrophage phenotypic markers and inflammatory mediators in adipose tissue and intestinal polyps and this was associated with increased tumorigenesis.

Intestinal inflammatory cytokine response in relation to tumorigenesis in the ApcMin/+ mouse

The etiology of colon cancer is a complex phenomenon that involves both genetic and environmental factors. However, only about 20% have a familial basis with the largest fraction being attributed to environmental causes that can lead to chronic inflammation. While the link between inflammation and colon cancer is well established, the temporal sequence of the inflammatory response in relation to tumorigenesis has not been characterized. We examined the timing and magnitude of the intestinal inflammatory cytokine response in relation to tumorigenesis in the Apc(Min/+) mouse. Apc(Min/+) mice and wildtype mice were sacrificed at one of 4 time-points: 8, 12, 16, and 20 weeks of age. Intestinal tissue was analyzed for polyp burden (sections 1, 4 and 5) and mRNA expression and protein concentration of MCP-1, IL-1β, IL-6 and TNF-α (sections 2 and 3). The results show that polyp burden was increased at 12, 16 and 20 weeks compared to 8 weeks (P<0.05). Gene expression (mRNA) of MCP-1, IL-1β, IL-6 and TNF-α was increased in sections 2 and 3 starting at week 12 (P<0.05), with further increases in MCP-1, IL-1β and IL-6 at 16 weeks (P<0.05). Protein concentration for these cytokines followed a similar pattern in section 3. Similarly, circulating MCP-1 was increased at 12 weeks (P<0.05) and then again at 20 weeks (P<0.05). In general, overall polyp number and abundance of large polyps were significantly correlated with the inflammatory cytokine response providing further support for a relationship between polyp progression and these markers. These data confirm the association between intestinal cytokines and tumorigenesis in the Apc(Min/+) mouse and provide new information on the timing and magnitude of this response in relation to polyp development. These findings may lead to the development of inflammatory mediators as important biomarkers for colon cancer progression. Further, these data may be relevant in the design of future investigations of therapeutic interventions to effectively target inflammatory processes in rodent models.

Benefits of exercise training on breast cancer progression and inflammation in C3(1)SV40Tag mice

Many observational epidemiologic studies suggest an association between exercise and breast cancer risk. However, the lack of controlled experimental studies that examine this relationship and the mechanisms involved weaken the basis for inferring a causal relationship. Inflammation plays a role in breast cancer progression and exercise has been reported to reduce inflammation; however, the anti-inflammatory effects of exercise in breast cancer have yet to be established. We examined the relationship between exercise training and systemic inflammation in relation to breast cancer progression in C3(1)SV40Tag mice. Female C3(1)SV40Tag mice were assigned to either exercise (Ex) or sedentary (Sed) treatment (n=12-14/group). Beginning at 4 wks of age mice (Ex) were run on a treadmill for 60 min/d (20 m/min and 5% grade), 6 d/wk for a period of 20 wks. Mice were examined weekly for palpable tumors, and tumor number and volume were recorded. At 24 wks of age mice were sacrificed and a more direct measure of tumor number and volume, and spleen weight was recorded. Plasma was analyzed for MCP-1 and IL-6 concentration using ELISA. Ex reduced palpable tumor number at sacrifice (24 wks) by approximately 70% (P<0.05). Tumor volume was also reduced in Ex at 21-23 wks (P<0.05). This reduction in tumor progression by Ex was associated with a reduction in plasma concentration of MCP-1 and IL-6, and spleen weight (P<0.05). These data provide strong support for a beneficial effect of exercise training on tumor progression in the C3(1)SV40Tag mouse model of breast cancer that may be partly mediated by its anti-inflammatory potential.

Importance of chemokine (CC-motif) ligand 2 in breast cancer

Breast cancer is the leading cause of cancer-related death in women in the United States. Chemokine (CC-motif) ligand 2 (CCL2), an inflammatory cytokine and chemokine, is highly expressed within the tumor and stromal cell populations and has been associated with enhanced tumorigenesis. In breast cancer patients, CCL2 has been correlated with high tumor grade and has been shown to have significant prognostic value for relapse-free survival. CCL2 likely exerts its pro-tumorigenic effects through recruitment of tumor-associated macrophages (TAMs); TAMs promote a tumorigenic microenvironment through the induction of growth enhancers, angiogenic factors and inflammatory mediators. CCL2 may also stimulate angiogenesis independently of TAM recruitment as it is closely associated with several endothelial cell growth factors. Additionally, CCL2 has been implicated in several processes leading to metastatic establishment including the development of bone metastasis. It has also been reported to directly upregulate pro-tumorigenic inflammatory mediators, including regulated upon activation, normal T cell expressed and secreted (RANTES) and tumor necrosis factor-alpha (TNF-α). While there is emerging support for a tumor promoting role of CCL2 in breast cancer, additional research is required before CCL2 can be decisively established as a prognostic factor and/or treatment target in breast cancer.

Effects of the MCP-1 synthesis inhibitor bindarit on tumorigenesis and inflammatory markers in the C3(1)/SV40Tag mouse model of breast cancer

Breast cancer, the most deadly cancer in women, is characterized by elevated levels of inflammation within and surrounding the tumor, which can lead to accelerated growth, invasion and metastasis. Macrophages are central to the inflammatory milieu and are recruited to the tumor microenvironment by several factors including monocyte chemoattractant protein-1 (MCP-1). Using the anti-inflammatory molecule bindarit to target MCP-1, we investigated the role of this chemokine on macrophage related inflammation and mammary tumorigenesis in a transgenic mouse model of breast cancer. C3(1)/SV40Tag mice and wild type FVB/N were randomized to either control or 0.5% bindarit diet from 4 to 21weeks of age. Tumor number and volume were recorded over time and at sacrifice. Macrophage markers as well as inflammatory meditators were examined in the tumor tissue and mammary glands. Circulating MCP-1 and IL-6 were measured by ELISA. Bindarit treatment reduced tumor number (P<0.05), but did not affect tumor size, tumor weight or tumor latency in C3(1)/SV40Tag mice. Within the tumor, mRNA expression of bindarits primary targets, MCP-1 and IL-12/p35, were significantly decreased by bindarit treatment (P<0.05), and this was consistent with trends for reduced expression of TNF-α, IL-6, F4/80, CD206, and IL-10. In mammary tissue, expression of MCP-1, TNF-α, IL-6, F4/80, IL-10 and IL-12/p35 was significantly elevated in C3(1)/SV40Tag mice compared to wild type FVB/N mice, but IL-6 was the only marker decreased by bindarit treatment (P<0.05). Plasma MCP-1 was highly correlated with tumor volume (P<0.05); however, it was not affected by bindarit at 21weeks of age. Similarly, circulating IL-6 was increased in C3(1)/SV40Tag mice but there was no effect of bindarit treatment. These results show that tumor multiplicity in the C3(1)/SV40Tag mouse model of breast cancer is reduced by bindarit, however these effects are independent of changes in plasma levels of MCP-1 and IL-6, but may be related to the attenuated expression of MCP-1 along with several inflammatory mediators and macrophage markers within the tumor.

Exercise effects on polyp burden and immune markers in the ApcMin/+ mouse model of intestinal tumorigenesis

Many observational epidemiologic studies suggest an association between exercise and colon cancer risk. The mechanisms contributing to a preventative effect of exercise on colon cancer are complex and multifaceted. Altered immune system function is one possible mechanism that has been largely unexplored. Therefore, the purpose of this study was to examine the effects of exercise on markers associated with macrophages and select T cell populations in a mouse model of intestinal tumorigenesis and to relate this to polyp characteristics. Male ApcMin/+ mice were randomly assigned to either sedentary (Sed) or exercise (Ex) treatment (n=6–9/group). The exercise treatment consisted of treadmill running for 1 h/day and 6 days a week at 15 m/min from 4 until 16 weeks of age. Intestinal polyps were counted and categorized by size. Mucosal tissue was analyzed for mRNA expression of overall macrophages (F4/80), for genes associated with M1 (IL-12, IL-23 and Nos2) and M2 (CD206, IL-10, IL-4, CCL17, CCL22 and Arg-1) macrophages and the macrophage chemoattractants MCP-1, fetuin A and CXCL14. Markers for cytotoxic T cells (CTLs) and regulatory T cells were also examined by measuring mRNA expression of CD8 and Foxp3, respectively. While there was no significant difference in overall polyp number between the groups (Sed, 23.3±4.3; and Ex, 16.5±4.3), Ex did have a reduction in the number of large polyps (Sed, 6.1±1.1; and Ex, 3.0±0.6) (P<0.05). This was consistent with a decrease in spleen weight (P<0.05). Similarly, Ex reduced mRNA expression of overall macrophages (F4/80) as well as markers associated with both M1 (IL-12) and M2 (CD206, CCL22 and Arg-1) subtypes (P<0.05) but there was no significant decrease in macrophage chemoattractants. CD8 expression was increased while Foxp3 expression was decreased with Ex (P<0.05). Overall the data provide important new information on immune regulation as a possible mechanism for the documented benefits of exercise training on reducing colon cancer progression.

Effects of voluntary exercise on tumorigenesis in the C3(1)/SV40Tag transgenic mouse model of breast cancer

Epidemiologic studies suggest an association between physical activity (PA) and breast cancer risk. We examined the relationship between voluntary wheel running and breast cancer in C3(1)/SV40Tag mice. Female FVB/N and C3(1)/SV40Tag mice were assigned to either PA [C3(1)-PA] (n=12) or sedentary (Sed) [C3(1)-Sed] (n=15) treatment and were placed in a cage with access to a running wheel (PA) or without (Sed) from 4 to 24 weeks of age (sacrifice). Physical activity data were analyzed for running distance, time and speed. Body composition was examined at 12 weeks of age. Tumors were counted twice weekly and at sacrifice to assess multiplicity. Tumor volume was calculated using external calipers [0.52 x (largest diameter) x (smallest diameter)2]. Heart and body weight were also recorded at sacrifice. Results showed that voluntary wheel running reduced tumor volume per tumor [C3(1)-Sed, 422.3±89.9 mm(3); C3(1)-PA, 260.2±61.7 mm(3)] (P<0.05), but was associated with increased tumor number (P<0.05). Body composition analysis showed no differences in body fat between the groups. Heart weight/body weight ratio was increased following physical activity (P<0.05) providing evidence of a training effect. In conclusion, voluntary wheel running activity was effective at slowing tumor growth in the C3(1)/SV40Tag mouse model of breast cancer, but did not inhibit tumor initiation. These data provide support for further development of the C3(1)/SV40Tag mouse model for use in understanding the role of physical activity on breast cancer progression and the mechanisms for its effects.