Tasquinimod inhibits prostate cancer growth in bone through alterations in the bone microenvironment.

BACKGROUND: Tasquinimod (ABR-215050) is an orally active quinoline-3-carboxamide analog that inhibits occurrence of experimental metastasis and delays disease progression of castration resistant prostate cancer in humans. Its mechanism of action is not fully elucidated, but previous studies show immunomodulatory and anti-angiogenic effects. The aim of the present study was to investigate the tumor inhibiting effect of tasquinimod in bone of castrated mice as well as to elucidate its working mechanism related to bone microenvironment. METHODS: Effects of tasquinimod on prostate cancer metastasis to bone was studied in an intratibial xenograft model. Animals were treated with tasquinimod and tumor establishment and growth, immunological status, as well as markers for bone remodeling were analyzed. Direct effects of tasquinimod on osteoblasts were studied in vitro. RESULTS: Establishment and growth of tumors in the bone after intratibial implantation in castrated mice was suppressed by tasquinimod treatment. The treatment effect was linked to decreased potential for immunosuppression in the pre-metastatic niche in bone (lower levels of CD206 and Arg1 expression in combination with increased iNOS expression) as well as in the tumor microenvironment (less Gr1 and CD206 staining). The shift to a pro-inflammatory, anti-tumorigenic milieu was also reflected in serum by increased levels of IFN-γ, CCL4, IL-5, LIX, IP-10, and MCP-1 as well as decreased TGF-ß. Tasquinimod treatment also affected expression of factors involved in the pre-metastatic niche in the bone microenvironment (Lox, Cdh2, Cdh11, and Cxcl12). In addition, tasquinimod treatment caused a decreased osteogenic response indicated by decreased expression of Ocn, Runx2, and Col1a2 and increased expression of osteoclast stimulating CSF2. In vitro studies on mouse osteoblasts showed impaired osteoblast mineralization upon tasquinimod treatment. CONCLUSIONS: The present study shows that tasquinimod reduces establishment and progression of tumor growth in bone likely through a combination of effects on the pre-metastatic niche, homing, immunological status, and osteogenesis. It was concluded that tasquinimod interferes with the metastatic process, presumably by inhibition of tumor establishment. Hence, our data suggest that tasquinimod might be most effective in inhibiting the occurrence of new metastatic lesions.

Oregonin reduces lipid accumulation and proinflammatory responses in primary human macrophages.

Inflammation in the vascular wall is important for the development of atherosclerosis. We have previously shown that inflammatory macrophages are more abundant in human atherosclerotic lesions than in healthy arteries. Activated macrophages produce reactive oxygen species (ROS) that promote local inflammation in atherosclerotic lesions. Here, we investigated the role of oregonin, a diarylheptanoid, on proinflammatory responses in primary human macrophages and found that oregonin decreased cellular lipid accumulation and proinflammatory cytokine secretion. We also found that oregonin decreased ROS production in macrophages. Additionally, we observed that treatment of lipopolysaccharide-exposed macrophages with oregonin significantly induced the expression of antioxidant-related genes, including Heme oxygenase-1 and NADPH dehydrogenase quinone 1. In summary, we have shown that oregonin reduces lipid accumulation, inflammation and ROS production in primary human macrophages, indicating that oregonin has anti-inflammatory bioactivities.

Macrophage CD14 expression in human carotid plaques is associated with complicated lesions, correlates with thrombosis, and is reduced by angiotensin receptor blocker treatment.

CD14 is a predictor of inflammation and associated with atherosclerosis. We analyzed 118 carotid plaques from patients with symptomatic carotid artery stenosis for expression of the macrophage markers CD14, CD68 and the angiotensin II type 1 receptor (AT1-R). CD14 staining was significantly increased in thrombotic carotid plaques. AT1-R staining was found in macrophage-rich areas, and AT1-R mRNA was detected in plaque macrophages isolated with anti-CD14 immunobeads. In patients treated with an angiotensin receptor blocker, expression of CD14 and CD68 in carotid plaque and serum levels of inflammatory markers were lower than in untreated patients. In vitro, expression of CD14 in human monocyte-derived macrophages was increased by exposure to lipopolysaccharide and decreased by exposure to an angiotensin receptor blocker. Thus, inhibition of the innate immune responsive lipopolysaccharide receptor CD14 in macrophages, rather than AT1-R inhibition, may help explain the anti-inflammatory effects of angiotensin receptor blockade.

The importance of GLUT3 for de novo lipogenesis in hypoxia-induced lipid loading of human macrophages.

Atherosclerotic lesions are characterized by lipid-loaded macrophages (foam cells) and hypoxic regions. Although it is well established that foam cells are produced by uptake of cholesterol from oxidized LDL, we previously showed that hypoxia also promotes foam cell formation even in the absence of exogenous lipids. The hypoxia-induced lipid accumulation results from increased triglyceride biosynthesis but the exact mechanism is unknown. Our aim was to investigate the importance of glucose in promoting hypoxia-induced de novo lipid synthesis in human macrophages. In the absence of exogenous lipids, extracellular glucose promoted the accumulation of Oil Red O-stained lipid droplets in human monocyte-derived macrophages in a concentration-dependent manner. Lipid droplet accumulation was higher in macrophages exposed to hypoxia at all assessed concentrations of glucose. Importantly, triglyceride synthesis from glucose was increased in hypoxic macrophages. GLUT3 was highly expressed in macrophage-rich and hypoxic regions of human carotid atherosclerotic plaques and in macrophages isolated from these plaques. In human monocyte-derived macrophages, hypoxia increased expression of both GLUT3 mRNA and protein, and knockdown of GLUT3 with siRNA significantly reduced both glucose uptake and lipid droplet accumulation. In conclusion, we have shown that hypoxia-induced increases in glucose uptake through GLUT3 are important for lipid synthesis in macrophages, and may contribute to foam cell formation in hypoxic regions of atherosclerotic lesions.

Arachidonate 15-lipoxygenase type B knockdown leads to reduced lipid accumulation and inflammation in atherosclerosis.

Inflammation in the vascular wall is important for development of atherosclerosis. We have shown previously that arachidonate 15-lipoxygenase type B (ALOX15B) is more highly expressed in human atherosclerotic lesions than in healthy arteries. This enzyme oxidizes fatty acids to substances that promote local inflammation and is expressed in lipid-loaded macrophages (foam cells) present in the atherosclerotic lesions. Here, we investigated the role of ALOX15B in foam cell formation in human primary macrophages and found that silencing of human ALOX15B decreased cellular lipid accumulation as well as proinflammatory cytokine secretion from macrophages. To investigate the role of ALOX15B in promoting the development of atherosclerosis in vivo, we used lentiviral shRNA silencing and bone marrow transplantation to knockdown mouse Alox15b gene expression in LDL-receptor-deficient (Ldlr(-/-)) mice. Knockdown of mouse Alox15b in vivo decreased plaque lipid content and markers of inflammation. In summary, we have shown that ALOX15B influences progression of atherosclerosis, indicating that this enzyme has an active proatherogenic role.

High expression of arachidonate 15-lipoxygenase and proinflammatory markers in human ischemic heart tissue.

A common feature of the ischemic heart and atherosclerotic plaques is the presence of hypoxia (insufficient levels of oxygen in the tissue). Hypoxia has pronounced effects on almost every aspect of cell physiology, and the nuclear transcription factor hypoxia inducible factor-1α (HIF-1α) regulates adaptive responses to low concentrations of oxygen in mammalian cells. In our recent work, we observed that hypoxia increases the proinflammatory enzyme arachidonate 15-lipoxygenase (ALOX15B) in human carotid plaques. ALOX15 has recently been shown to be present in the human myocardium, but the effect of ischemia on its expression has not been investigated. Here we test the hypothesis that ischemia of the heart leads to increased expression of ALOX15, and found an almost 2-fold increase in HIF-1α mRNA expression and a 17-fold upregulation of ALOX15 mRNA expression in the ischemic heart biopsies from patients undergoing coronary bypass surgery compared with non ischemic heart tissue. To investigate the effect of low oxygen concentration on ALOX15 we incubated human vascular muscle cells in hypoxia and showed that expression of ALOX15 increased 22-fold compared with cells incubated in normoxic conditions. We also observed increased mRNA levels of proinflammatory markers in ischemic heart tissue compared with non-ischemic controls. In summary, we demonstrate increased ALOX15 in human ischemic heart biopsies. Furthermore we demonstrate that hypoxia increases ALOX15 in human muscle cells. Our results yield important insights into the underlying association between hypoxia and inflammation in the human ischemic heart disease.