Primary Tumor Suppression and Systemic Immune Activation of Macrophages through the Sting Pathway in Metastatic Skin Tumor

Purpose@#Agonists of the stimulator of interferon genes (STING) play a key role in activating the STING pathway by promoting the production of cytokines. In this study, we investigated the antitumor effects and activation of the systemic immune response of treatment with DMXAA (5,6-dimethylxanthenone-4-acetic acid), a STING agonist, in EML4-ALK lung cancer and CT26 colon cancer. @*Materials and Methods@#The abscopal effects of DMXAA in the treatment of metastatic skin nodules were assessed. EML4-ALK lung cancer and CT26 colon cancer models were used to evaluate these effects after DMXAA treatment. To evaluate the expression of macrophages and T cells, we sacrificed the tumor-bearing mice after DMXAA treatment and obtained the formalin-fixed paraffin-embedded (FFPE) tissue and tumor cells. Immunohistochemistry and flow cytometry were performed to analyze the expression of each FFPE and tumor cell. @*Results@#We observed that highly infiltrating immune cells downstream of the STING pathway had increased levels of chemokines after DMXAA treatment. In addition, the levels of CD80 and CD86 in antigen-presenting cells were significantly increased after STING activation. Furthermore, innate immune activation altered the systemic T cell-mediated immune responses, induced proliferation of macrophages, inhibited tumor growth, and increased numbers of cytotoxic memory T cells. Tumor-specific lymphocytes also increased in number after treatment with DMXAA. @*Conclusion@#The abscopal effect of DMXAA treatment on the skin strongly reduced the spread of EML4-ALK lung cancer and CT26 colon cancer through the STING pathway and induced the presentation of antigens.

Cilostazol Attenuates 4-hydroxynonenal-enhanced CD36 Expression on Murine Macrophages via Inhibition of NADPH Oxidase-derived Reactive Oxygen Species Production

Although anti-atherogenic effects of cilostazol have been suggested, its effects on the expression of SR in macrophages are unclear. This study investigated the role of cilostazol on CD36 expression of murine macrophages enhanced by HNE, a byproduct of lipid peroxidation. The stimulation of macrophages with HNE led to an increased expression of CD36, which was significantly attenuated by NAC, an antioxidant. Moreover, the increased production of ROS by HNE was completely abolished by NADPH oxidase inhibitors, DPI and apocynin, as well as by the 5-LO inhibitor, MK886, but not by inhibitors for other oxidases. This suggested that NADPH-oxidase and 5-LO were major sources of ROS induced by HNE. In addition, HNE-enhanced expression of CD36 was reduced by these inhibitors, which indicated a role for NADPH oxidase and 5-LO on CD36 expression. In our present study, cilostazol was a significant inhibitor of ROS production, as well as CD36 expression induced by HNE. An increase in NADPH oxidase activity by HNE was significantly attenuated by cilostazol, however cilostazol had no effect on HNE-enhanced 5-LO activity. Together, these results suggest that cilostazol attenuates HNE-enhanced CD36 expression on murine macrophages thorough inhibition of NADPH oxidase-derived ROS generation.

Enhanced Expression of Cell Adhesion Molecules in the Aorta of Diabetic Mice is Mediated by gp91phox-derived Superoxide

Endothelial activation and subsequent recruitment of inflammatory cells are important steps in atherogenesis. The increased levels of cell adhesion molecules (CAM) have been identified in diabetic vasculatures, but the underlying mechanisms remain unclear. To determine the relationship among vascular production of superoxide, expression of CAM and diabetes, superoxide generation and expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E- and P-selectin in the aorta from control (C57BL/6J) and diabetic mice (ob/ob) were measured. In situ staining for superoxide using dihydroethidium showed an increased superoxide production in diabetic aorta, accompanied with an enhanced NAD (P) H oxidase activity. Immunohistochemical analysis revealed that the endothelial expression of ICAM-1 (3.5+/-0.4) and VCAM-1 (3.8+/-0.3) in diabetic aorta was significantly higher than those in control aorta (0.9+/-0.5 and 1.6+/-0.3, respectively), accompanied with the enhanced expression of gp91phox, a membrane subunit of NAD (P) H oixdase. Furthermore, there was a strong positive correlation (r=0.89, P< 0.01 in ICAM-1 and r=0.88, P< 0.01 in VCAM-1) between ICAM-1/VCAM-1 expression and vascular production of superoxide. The present data indicate that the increased production of superoxide via NAD (P) H oxidase may explain the enhanced expression of CAM in diabetic vasculatures.

Gene Transfer of Cu/ZnSOD to Cerebral Vessels Prevents Subarachnoid Hemorrhage-induced Cerebral Vasospasm

The preventive effects of gene transfer of human copper/zinc superoxide dismutase (Cu/ZnSOD) on the development of cerebral vasospasm after subarachnoid hemorrhage (SAH) were examined using a rat model of SAH. An experimental SAH was produced by injecting autologous arterial blood twice into the cisterna magna, and the changes in the diameter of the middle cerebral artery (MCA) were measured. Rats subjected to SAH exhibited a decreased diameter with an increased wall thickness of MCA that were significantly ameliorated by pretreatment with diphenyleneiodonium (DPI, 10microM), an inhibitor of NAD (P) H oxidase. Furthermore, application of recombinant adenovirus (100microliter of 1 x 1010 pfu/ml, intracisternally), which encodes human Cu/ZnSOD, 3 days before SAH prevented the development of SAH-induced vasospasm. Our findings demonstrate that SAH-induced cerebral vasospasm is closely related with NAD (P) H oxidase-derived reactive oxygen species, and these alterations can be prevented by the recombinant adenovirus-mediated transfer of human Cu/ZnSOD gene to the cerebral vasculature.

Involvement of NAD (P) H Oxidase in a Potential Link between Diabetes and Vascular Smooth Muscle Cell Proliferation

The cellular mechanisms that contribute to the acceleration of atherosclerosis in diabetes are poorly understood. Therefore, the potential mechanisms involved in the diabetes-dependent increase in vascular smooth muscle cell (VSMC) proliferation was investigated. Using primary culture of VSMC from streptozotocin-induced diabetic rat aorta, cell proliferation assay showed two-fold increase in cell number accompanied with enhanced superoxide generation compared to normal VSMC, 2 days after plating. Both the increased superoxide production and cell proliferation in diabetic VSMC were significantly attenuated by not only tiron (1 mM), a superoxide scavenger, but also by diphenyleneiodonium (DPI; 10micrometer), an NAD (P) H oxidase inhibitor. NAD (P) H oxidase activity in diabetic VSMC was significantly higher than that in control cell, accompanied with increased mRNA expression of p22phox, a membrane subunit of oxidase. Furthermore, inhibition of p22phox expression by transfection of antisense p22phox oligonucleotides into diabetic VSMC resulted in a decrease in superoxide production, which was accompanied by a significant inhibition of cell proliferation. Based on these results, it is suggested that diabetes-associated increase in NAD (P) H oxidase activity via enhanced expression of p22phox contributes to augmented VSMC proliferation in diabetic rats.