Plumbagin-induced apoptosis in lymphocytes is mediated through increased reactive oxygen species production, upregulation of Fas, and activation of the caspase cascade

Extracts from plants containing plumbagin (PLB) continue to be used as a treatment of a number of chronic immunologically-based diseases. However, most of these claims are supported only by anecdotal evidence with few scientific reports describing the mechanism of action or the efficacy of plumbagin in the suppression of the immune response. In the current study, we tested the hypothesis that plumbagin-induced suppression of the immune response was mediated through the induction of apoptosis. Splenocytes from C57BL/6 mice cultured in the presence of 0.5 mu M or greater concentrations of PLB significantly reduced proliferative responses to mitogens, including anti-CD3 mAbs, concanavalin A (Con A), lipopolysaccharide (LPS) and staphylococcal enterotoxin B (SEB) in vitro. Exposure of naive and activated splenocytes to PLO led to a significant increase in the levels of apoptosis. In addition, PLB treatment led to a significant increase in the levels of reactive oxygen species (ROS) in naive and activated splenocytes. Furthermore, treatment with the ROS scavenger, N-acetylcysteine (NAC), prevented PLB-induced apoptosis, suggesting a role of ROS in PLO-induced apoptosis. PLB-induced apoptosis led to ROS-mediated activation of both the extrinsic and intrinsic apoptotic pathways. In addition, plumbagin led to increased expression of Fas. Finally, treatment of mice with PLO (5 mg/kg) led to thymic and splenic atrophy as well as a significant suppression of the response to SEB and dinitroflourobenzene (DNFB) in vivo. Together, these results suggest that plumbagin has significant immunosuppressive properties which are mediated by generation of ROS, upregulation of Fas, and the induction of apoptosis. (C) 2010 Elsevier Inc. All rights reserved

Chemotherapy enhances tumor cell susceptibility to CTL-mediated killing during cancer immunotherapy in mice

Cancer immunotherapy faces a serious challenge because of low clinical efficacy. Recently, a number of clinical studies have reported the serendipitous finding of high rates of objective clinical response when cancer vaccines are combined with chemotherapy in patients with different types of cancers. However, the mechanism of this phenomenon remains unclear. Here, we tested in mice several cancer vaccines and an adoptive T cell transfer approach to cancer immunotherapy in combination with several widely used chemotherapeutic drugs. We found that chemotherapy made tumor cells more susceptible to the cytotoxic effect of CTLs through a dramatic perforin-independent increase in permeability to GrzB released by the CTLs. This effect was mediated via upregulation of mannose-6-phosphate receptors on the surface of tumor cells and was observed in mouse and human cells. When combined with chemotherapy, CTLs raised against specific antigens were able to induce apoptosis in neighboring tumor cells that did not express those antigens. These data suggest that small numbers of CTLs could mediate a potent antitumor effect when combined with chemotherapy. In addition, these results provide a strong rationale for combining these modalities for the treatment of patients with advanced cancers

Oxidized lipids block antigen cross-presentation by dendritic cells in cancer

Cross-presentation is one of the main features of dendritic cells (DCs), which is critically important for the development of spontaneous and therapy-inducible antitumor immune responses. Patients, at early stages of cancer, have normal presence of DCs. However, the difficulties in the development of antitumor responses in patients with low tumor burden raised the question of the mechanisms of DC dysfunction. In this study, we found that, in differentiated DCs, tumor-derived factors blocked the cross-presentation of exogenous Ags without inhibiting the Ag presentation of endogenous protein or peptides. This effect was caused by intracellular accumulation of different types of oxidized neutral lipids: triglycerides, cholesterol esters, and fatty acids. In contrast, the accumulation of nonoxidized lipids did not affect cross-presentation. Oxidized lipids blocked cross-presentation by reducing the expression of peptide–MHC class I complexes on the cell surface. Thus, this study suggests the novel role of oxidized lipids in the regulation of cross-presentation

Tumor-infiltrating myeloid cells induce tumor cell resistance to cytotoxic T cells in mice

Cancer immunotherapeutic approaches induce tumor-specific immune responses, in particular CTL responses, in many patients treated. However, such approaches are clinically beneficial to only a few patients. We set out to investigate one possible explanation for the failure of CTLs to eliminate tumors, specifically, the concept that this failure is not dependent on inhibition of T cell function. In a previous study, we found that in mice, myeloid-derived suppressor cells (MDSCs) are a source of the free radical peroxynitrite (PNT). Here, we show that pre-treatment of mouse and human tumor cells with PNT or with MDSCs inhibits binding of processed peptides to tumor cell–associated MHC, and as a result, tumor cells become resistant to antigen-specific CTLs. This effect was abrogated in MDSCs treated with a PNT inhibitor. In a mouse model of tumor-associated inflammation in which the antitumor effects of antigen-specific CTLs are eradicated by expression of IL-1β in the tumor cells, we determined that therapeutic failure was not caused by more profound suppression of CTLs by IL-1β–expressing tumors than tumors not expressing this proinflammatory cytokine. Rather, therapeutic failure was a result of the presence of PNT. Clinical relevance for these data was suggested by the observation that myeloid cells were the predominant source of PNT in human lung, pancreatic, and breast cancer samples. Our data therefore suggest what we believe to be a novel mechanism of MDSC-mediated tumor cell resistance to CTLs