Adriamycin-mediated nitration of manganese superoxide dismutase in the central nervous system: insight into the mechanism of chemobrain

Adriamycin (ADR), a potent anti-tumor agent, produces reactive oxygen species (ROS) in cardiac tissue. Treatment with ADR is dose-limited by cardiotoxicity. However, the effect of ADR in the other tissues, including the brain, is unclear because ADR does not pass the blood-brain barrier. Some cancer patients receiving ADR treatment develop a transient memory loss, inability to handle complex tasks etc., often referred to by patients as chemobrain. We previously demonstrated that ADR causes CNS toxicity, in part, via systemic release of cytokines and subsequent generation of reactive oxygen and nitrogen species (RONS) in the brain. Here, we demonstrate that treatment with ADR led to an increased circulating level of tumor necrosis factor-alpha in wild-type mice and in mice deficient in the inducible form of nitric oxide (iNOSKO). However, the decline in mitochondrial respiration and mitochondrial protein nitration after ADR treatment was observed only in wild-type mice, not in the iNOSKO mice. Importantly, the activity of a major mitochondrial antioxidant enzyme, manganese superoxide dismutase (MnSOD), was reduced and the protein was nitrated. Together, these results suggest that NO is an important mediator, coupling the effect of ADR with cytokine production and subsequent activation of iNOS expression. We also identified the mitochondrion as an important target of ADR-induced NO-mediated CNS injury.     

Inhibition of oral cancer cell growth by adenovirusMnSOD plus BCNU treatment

We hypothesized that inhibitors of peroxide removal, such as BCNU, an indirect inhibitor of glutathione peroxidase (GPx), and 3-amino-1,2,4-triazole (AT), a direct inhibitor of catalase (CAT), should cause toxicity to cancer cells after manganese superoxide dismutase (MnSOD) overexpression due to elevated peroxide levels. In vitro, hamster cheek pouch carcinoma cells (HCPC-1) and human oral squamous carcinoma cells (SCC-25) were infected with various combinations of adenovirus containing MnSOD cDNA (AdMnSOD). Cells were then treated with or without BCNU and assayed for viability using Annexin/PI staining and flow cytometry. In AdMnSOD plus BCNU-treated SCC-25 and HCPC-1 cells, a 30-60% decrease in cell viability was observed compared to BCNU alone. In vivo, HCPC-1 and SCC-25 xenografts were allowed to grow to approximately 70 mm(3) and 10(9) plaque forming units (pfu) of AdMnSOD were injected directly into the tumors. Two days later, 15 or 30 mg/kg BCNU was injected intratumorally. Tumor growth was greatly inhibited (4- to 20-fold) by this combined treatment, as well as increasing animal survival. Tumor volume could be decreased further by giving multiple doses of AdMnSOD or inhibiting catalase activity with AT. These results suggest that, by using these combination therapies, a significant decrease in tumor mass can be achieved.     

Role of antioxidant enzymes in cell immortalization and transformation

Summary The role of antioxidant enzymes, particularly superoxide dismutase (SOD), in immortalization and malignant transformation is discussed. SOD (generally MnSOD) has been found to be lowered in a wide variety of tumor types when compared to an appropriate normal cell control. Levels of immunoreactive MnSOD protein and mRNA for MnSOD also appear to be lowered in tumor cells. Tumor cells have the capacity to produce superoxide radical, the substrate for SOD. This suggests that superoxide production coupled with diminished amounts of MnSOD may be a general characteristic of tumor cells. The levels of MnSOD in certain cells correlates with their degree of differentiation; non-differentiating cells, whether normal or malignant, appear to have lost the ability to undergo MnSOD induction. These observations are used to elucidate a two-step model of cancer. This model involves not only the antioxidant enzymes, but also organelle (particularly mitochondria and peroxisomes) function as a dominant theme in carcinogenesis.     

In vitro modulation of antioxidant enzyme levels in normal hamster kidney and estrogen-induced hamster kidney tumor

Antioxidant enzyme (AE) activities were studied in normal hamster kidney proximal tubules and in estrogen-induced hamster kidney cancer. In vivo, kidney tumor had lower activities of manganese superoxide dismutase (MnSOD), copper, zinc superoxide dismutase, catalase, and glutathione peroxidase than kidney proximal tubules. Differences in AE activities were, in general, maintained in tissue culture, with AE activities remaining low in tumor cells compared to normal cells. Normal proximal tubular cells showed significant induction of MnSOD activity as a function of time in culture of following exposure to diethylstilbestrol, a synthetic estrogen, while MnSOD activity remained low in tumor cells under these conditions. Our results suggest that antioxidant enzymes, particularly MnSOD, are regulated differently in estrogen-induced hamster kidney tumor cells than in normal kidney proximal tubular cells, demonstrating that cancers arising from hormonal influence have similar AE profiles to those previously described in cancers arising from viral or chemical etiologies.     

Robust anti-tumor immunity and memory in Rag-1-deficient mice following adoptive transfer of cytokine-primed splenocytes and tumor CD80 expression

Successful immunotherapy of solid tumors has proven difficult to achieve. The aim of the current study was to further investigate the effects of peripheral CD80-mediated co-stimulation on the efficacy of polyclonal anti-tumor effector CTL in an adoptive transfer model. Splenocytes obtained from wild-type mice immunized with CD80-transduced EL4 tumor cells were expanded in vitro in the presence of either IL-12 or IL-15 and irradiated CD80-transduced EL4 tumor cells. Polyclonal CD8 T cells were the major subset in the effector population. Primed effector cells were adoptively transferred into immuno-deficient Rag-1-deficient mice which were then challenged with syngeneic vector-control or CD80-transduced EL4 tumor cells. Expression of CD80 enhanced the elimination of EL4 tumors and mouse survival. Both IL-12 and IL-15 cultured cells had enhanced cytotoxicity. Importantly, anti-tumor memory was maintained without tumor evasion following re-challenge with either CD80-transduced and vector-control EL4 cells. We also show, using antibody-mediated depletion, that endogenous NK cells present in Rag-1-deficent mice exert anti-EL4 tumor activity that is enhanced by CD80 expression. Collectively these data show that peripheral co-stimulation by tumor expression of CD80 results in enhanced anti-tumor efficacy of NK and polyclonal effector T cells, and suggest that TCR repertoire diversity helps protect against tumor escape and provides memory with resultant robust immunity to subsequent tumor challenge irrespective of CD80 status.     

MnSOD drives neuroendocrine differentiation, androgen independence, and cell survival in prostate cancer cells

An increase in neuroendocrine (NE) cell number has been associated with progression of prostate tumor, one of the most frequent cancers among Western males. We previously reported that mitochondrial manganese superoxide dismutase (MnSOD) increases during the NE differentiation process. The goal of this study was to find whether MnSOD up-regulation is enough to induce NE differentiation. Several human prostate cancer LNCaP cell clones stably overexpressing MnSOD were characterized and two were selected (MnSOD-S4 and MnSOD-S12). MnSOD overexpression induces NE morphological features as well as coexpression of the NE marker synaptophysin. Both MnSOD clones exhibit lower superoxide levels and higher H(2)O(2) levels. MnSOD-overexpressing cells show higher proliferation rates in complete medium, but in steroid-free medium MnSOD-S12 cells are still capable of proliferation. MnSOD up-regulation decreases androgen receptor and prevents its nuclear translocation. MnSOD also induces up-regulation of Bcl-2 and prevents docetaxel-, etoposide-, or TNF-induced cell death. Finally, MnSOD-overexpressing cells enhance growth of androgen-independent PC-3 cells but reduce growth of androgen-dependent cells. These results indicate that redox modulation caused by MnSOD overexpression explains most NE-like features, including morphological changes, NE marker expression, androgen independence, inhibition of apoptosis, and enhancement of cell growth. Many of these events can be associated with the androgen dependent-independent transition during prostate cancer progression.     

Enhancement of anti-tumor CD8 immunity by IgG1-mediated targeting of Fc receptors

Dendritic cells (DCs) function as professional antigen presenting cells and are critical for linking innate immune responses to the induction of adaptive immunity. Many current cancer DC vaccine strategies rely on differentiating DCs, feeding them tumor antigens ex vivo, and infusing them into patients. Importantly, this strategy relies on prior knowledge of suitable “tumor-specific” antigens to prime an effective anti-tumor response. DCs express a variety of receptors specific for the Fc region of immunoglobulins, and antigen uptake via Fc receptors is highly efficient and facilitates antigen presentation to T cells. Therefore, we hypothesized that expression of the mouse IgG1 Fc region on the surface of tumors would enhance tumor cell uptake by DCs and other myeloid cells and promote the induction of anti-tumor T cell responses. To test this, we engineered a murine lymphoma cell line expressing surface IgG1 Fc and discovered that such tumor cells were taken up rapidly by DCs, leading to enhanced cross-presentation of tumor-derived antigen to CD8+ T cells. IgG1-Fc tumors failed to grow in vivo and prophylactic vaccination of mice with IgG1-Fc tumors resulted in rejection of unmanipulated tumor cells. Furthermore, IgG1-Fc tumor cells were able to slow the growth of an unmanipulated primary tumor when used as a therapeutic tumor vaccine. Our data demonstrate that engagement of Fc receptors by tumors expressing the Fc region of IgG1 is a viable strategy to induce efficient and protective anti-tumor CD8+ T cell responses without prior knowledge of tumor-specific antigens.     

D-factor and growth hormone enhance tumor necrosis factor-induced increase of Mn superoxide dismutase mRNA and oxygen tolerance.

D-Factor (differentiation-inducing factor or leukemia inhibitory factor) and growth hormone are proteins that regulate growth and differentiation of cells. In this study, we demonstrated that recombinant human D-factor and growth hormone caused a slight but significant protection of adult rats against oxygen toxicity without affecting levels of pulmonary manganous superoxide dismutase (MnSOD) mRNA. D-Factor and growth hormone also markedly enhanced tumor necrosis factor (TNF)-induced oxygen tolerance. This latter effect was associated with a marked enhancement of TNF-mediated induction of pulmonary MnSOD mRNA.     

Immune modulation by melanoma and ovarian tumor cells through expression of the immunosuppressive molecule CD200

Background and Objective Immune escape by tumors can occur by multiple mechanisms, each a significant barrier to immunotherapy. We previously demonstrated that upregulation of the immunosuppressive molecule CD200 on chronic lymphocytic leukemia cells inhibits Th1 cytokine production required for an effective cytotoxic T cell response. CD200 expression on human tumor cells in animal models prevents human lymphocytes from rejecting the tumor; treatment with an antagonistic anti-CD200 antibody restored lymphocyte-mediated tumor growth inhibition. The current study evaluated CD200 expression on solid cancers, and its effect on immune response in vitro. Methods and Results CD200 protein was expressed on the surface of 5/8 ovarian cancer, 2/4 melanoma, 2/2 neuroblastoma and 2/3 renal carcinoma cell lines tested, but CD200 was absent on prostate, lung, breast, astrocytoma, or glioblastoma cell lines. Evaluation of patient samples by immunohistochemistry showed strong, membrane-associated CD200 staining on malignant cells of melanoma (4/4), ovarian cancer (3/3) and clear cell renal cell carcinoma (ccRCC) (2/3), but also on normal ovary and kidney. CD200 expression on melanoma metastases was determined by RT-QPCR, and was found to be significantly higher in jejunum metastases (2/2) and lung metastases (2/6) than in normal samples. Addition of CD200-expressing, but not CD200-negative solid tumor cell lines to mixed lymphocyte reactions downregulated the production of Th1 cytokines. Inclusion of antagonistic anti-CD200 antibody restored Th1 cytokine responses. Conclusion These data suggest that melanoma, ccRCC and ovarian tumor cells can express CD200, thereby potentially suppressing anti-tumor immune responses. CD200 blockade with an antagonistic antibody may permit an effective anti-tumor immune response in these solid tumor types.     

Possible cytotoxic mechanisms of TNF in vitro

TNF is a protein originally isolated by its ability to induce hemorrhagic necrosis of some tumors in vivo, and cytotoxic effects against certain tumor cells, but not against normal cells in vitro. Though mechanisms of the cytotoxicity and selectivity of TNF action in vitro are not clear, there is much evidence that free radicals such as superoxide and hydroxyl radicals, mediated by TNF, are correlated with TNF cytotoxicity. Recently, manganous superoxide dismutase (MnSOD) in mitochondria has been demonstrated to be a rescue protein against TNF cytotoxicity and to be induced when the cells are exposed to TNF in the resistant cells. Two steroid hormones, glucocorticoid and 1,25-dihydroxyvitamin D3, block the cytotoxicity of TNF. Glucocorticoid may reduce TNF cytotoxicity by inhibiting phospholipase A2 and 1,25-dihydroxyvitamin D3 by inducing MnSOD in combination with TNF.     

CD47分子与抗肿瘤免疫

肿瘤进展与人免疫系统间的联系已经被广泛研究,有许多免疫分子已被证实参与其中。CD47（整合素相关蛋白）为一种免疫球蛋白超家族成员,在人免疫系统中发挥着重要功能。研究表明CD47在肿瘤细胞表面也有高表达,其高表达与肿瘤的生长、转移及复发等密切相关。肿瘤细胞表面的CD47与巨噬细胞表面的SIRPα相互作用,并发出“别吃我”的免疫抑制性信号,从而保护肿瘤细胞免受巨噬细胞吞噬。因此,开发以CD47为靶点的拮抗剂可阻断此抑制性信号,从而增强巨噬细胞的吞噬效应,以达到增强抗肿瘤免疫反应的目的。最新研究证实,CD47拮抗剂在T细胞介导的抗肿瘤免疫反应中也发挥了重要作用。本文将对CD47分子的结构功能、在抗肿瘤免疫反应中的作用及以其为靶点的拮抗剂研究进展进行综述,以期为进一步的药物开发及临床研究等提供参考。     

Differential effects of tumor necrosis factor and asbestos fibers on manganese superoxide dismutase induction and oxidant-induced cytotoxicity in human mesothelial cells

We compared induction of manganese superoxide dismutase (MnSOD) by asbestos fibers and tumor necrosis factor (TNF) using cultures human mesothelial cells. Transformed pleural mesothelial cells (MET 5A) were exposed for 48 h to amosite asbestos fibers (2 g/cm²), to TNF (10 Ng/ml), and to the combination of these two. TNF and amosite+TNF caused significant MnSOD mRNA upregulation. Similarly MnSOD specific activity was increased by TNF (290% increase) and the amosite+TNF combination (313% increase) but not by amosite alone. In cell injury experiments, amosite and amosite+TNF exposures caused significant cell membrane injury when assessed by lactate dehydrogenase release, which was 31% and 57% higher than in the unexposed cells. However, only the amosite+TNF combination caused significant depletion of cellular high-energy nucleotide when expressed as percentage of [¹⁴C]denine labeling in cellular high-energy nucleotides. The nucleotide levels were 91.5 ± 2.0% in the unexposed cells, 89.9 ± 3.9% in amosite-exposed cells, 90.1 ± 2.2% in TNF-exposed cells, and 79.8 ± 9.4% in amosite+TNF-exposed Amosite+TNF-exposed cells were also most sensitive to menadione (20 mol/L, 2 h), a compound which generates superoxide radicals intracellularly. In conclusion, our data suggests that in human mesothelial cells inflammatory cytokines but not asbestos fibers alone can cause MnSOD induction. In this study, however amosite asbestos+TNF treatment rendered these cells more vulnerable to oxidant-induced cell damage despite elevated MnSOD activity.Abbreviations MnSOD manganese superoxide dismutase - TNF tumor necrosis factor - LDH lactate dehydrogenase     

A bispecific antibody targeting CD47 and CD20 selectively binds and eliminates dual antigen expressing lymphoma cells

Agents that block the anti-phagocytic signal CD47 can synergize with pro-phagocytic anti-tumor antigen antibodies to potently eliminate tumors. While CD47 is overexpressed on cancer cells, its expression in many normal tissues may create an ‘antigen sink’ that could minimize the therapeutic efficacy of CD47 blocking agents. Here, we report development of bispecific antibodies (BsAbs) that co-target CD47 and CD20, a therapeutic target for non-Hodgkin lymphoma (NHL), that have reduced affinity for CD47 relative to the parental antibody, but retain strong binding to CD20. These characteristics facilitate selective binding of BsAbs to tumor cells, leading to phagocytosis. Treatment of human NHL-engrafted mice with BsAbs reduced lymphoma burden and extended survival while recapitulating the synergistic efficacy of anti-CD47 and anti-CD20 combination therapy. These findings serve as proof of principle for BsAb targeting of CD47 with tumor-associated antigens as a viable strategy to induce selective phagocytosis of tumor cells and recapitulate the synergy of combination antibody therapy. This approach may be broadly applied to cancer to add a CD47 blocking component to existing antibody therapies.     

Lorvotuzumab mertansine,a CD56-targeting antibody-drug conjugate with potent antitumor activity against small cell lung cancer in human xenograft models

Lorvotuzumab mertansine (LM) is an antibody-drug conjugate composed of a humanized anti-CD56 antibody, lorvotuzumab, linked via a cleavable disulfide linker to the tubulin-binding maytansinoid DM1. CD56 is expressed on most small cell lung cancers (SCLC), providing a promising therapeutic target for treatment of this aggressive cancer, which has a poor five-year survival rate of only 5–10%. We performed immunohistochemical staining on SCLC tumor microarrays, which confirmed that CD56 is expressed at high levels on most (~74%) SCLC tumors. Conjugation of lorvotuzumab with DM1 did not alter its specific binding to cells and LM demonstrated potent target-dependent cytotoxicity against CD56-positive SCLC cells in vitro. The anti-tumor activity of LM was evaluated against SCLC xenograft models in mice, both as monotherapy and in combination with platinum/etoposide and paclitaxel/carboplatin. Dose-dependent and antigen-specific anti-tumor activity of LM monotherapy was demonstrated at doses as low as 3 mg/kg. LM was highly active in combination with standard-of-care platinum/etoposide therapies, even in relatively resistant xenograft models. LM demonstrated outstanding anti-tumor activity in combination with carboplatin/etoposide, with superior activity over chemotherapy alone when LM was used in combinations at significantly reduced doses (6-fold below the minimally efficacious dose for LM monotherapy). The combination of LM with carboplatin/paclitaxel was also highly active. This study provides the rationale for clinical evaluation of LM as a promising novel targeted therapy for SCLC, both as monotherapy and in combination with chemotherapy.     

二氢卟吩e6对小鼠艾氏移植瘤的声动力杀伤作用

目的通过研究聚焦超声结合二氢卟吩e6（chlorine e6,Ce6）对小鼠艾氏移植瘤的抑制效果,初步探讨其杀伤艾氏移植瘤的生物学机制。方法 测定不同处理对艾氏移植瘤小鼠肿瘤组织的生长抑制作用;利用酶化学方法检测处理后肿瘤组织内超氧化物歧化酶（superoxide dismutase,SOD）、谷胱甘肽过氧化物酶（glutathioneperoxidase,GSH-PX）和过氧化氢酶（catalase,CAT）活性的变化,利用硫代巴比妥酸法检测丙二醛（malonaldehyde,MDA）活力。结果单纯Ce6处理没有抑瘤效应;单纯超声处理对肿瘤有一定程度的抑制作用,且具有强度依赖性;强度为4、6 W/cm2的超声结合浓度为10 mg/kg的Ce6具有显著抑瘤效应。肿瘤组织内SOD、GSH-PX、CAT 3种酶活性均有不同程度下降,肿瘤组织内MDA水平显著性升高。结论超声结合Ce6对小鼠艾氏移植瘤的杀伤作用可能与移植瘤组织中抗氧化物酶活性降低、丙二醛水平升高有关。     

Lorvotuzumab mertansine,a CD56-targeting antibody-drug conjugate with potent antitumor activity against small cell lung cancer in human xenograft models

Lorvotuzumab mertansine (LM) is an antibody-drug conjugate composed of a humanized anti-CD56 antibody, lorvotuzumab, linked via a cleavable disulfide linker to the tubulin-binding maytansinoid DM1. CD56 is expressed on most small cell lung cancers (SCLC), providing a promising therapeutic target for treatment of this aggressive cancer, which has a poor five-year survival rate of only 5–10%. We performed immunohistochemical staining on SCLC tumor microarrays, which confirmed that CD56 is expressed at high levels on most (~74%) SCLC tumors. Conjugation of lorvotuzumab with DM1 did not alter its specific binding to cells and LM demonstrated potent target-dependent cytotoxicity against CD56-positive SCLC cells in vitro. The anti-tumor activity of LM was evaluated against SCLC xenograft models in mice, both as monotherapy and in combination with platinum/etoposide and paclitaxel/carboplatin. Dose-dependent and antigen-specific anti-tumor activity of LM monotherapy was demonstrated at doses as low as 3 mg/kg. LM was highly active in combination with standard-of-care platinum/etoposide therapies, even in relatively resistant xenograft models. LM demonstrated outstanding anti-tumor activity in combination with carboplatin/etoposide, with superior activity over chemotherapy alone when LM was used in combinations at significantly reduced doses (6-fold below the minimally efficacious dose for LM monotherapy). The combination of LM with carboplatin/paclitaxel was also highly active. This study provides the rationale for clinical evaluation of LM as a promising novel targeted therapy for SCLC, both as monotherapy and in combination with chemotherapy.     

Heme synthesis increases artemisinin-induced radical formation and cytotoxicity that can be suppressed by superoxide scavengers

Artemisinin (ART) is a sesquiterpene lactone natural product that is widely used to treat multi-drug resistant strains of malaria. Artemisinin and its derivatives are also selectively cytotoxic to cancer cells, which can be modulated by altering heme synthesis. Cytotoxicity to cancer cells is thought to involve generation of oxidative stress, although conflicting data exist. We have analyzed reactive oxygen species (ROS) generation using the fluorescent probes 2′,7′-dichlorodihydrofluorescein diacetate (DCF) and dihydroethidine (HET) upon exposure to dihydroartemisinin (DHA) in Molt-4 leukemia cells. HET fluorescence correlated with dose-dependent DHA-induced cytotoxicity, increased within 30 min of DHA exposure, and was significantly enhanced by increasing heme synthesis. Protein levels of copper,zinc-superoxide dismutase (CuZnSOD), manganese-superoxide dismutase (MnSOD), catalase, and glutathione peroxidases 1/2 were also found to increase with DHA exposure. 4-hydroxy-tempol (TEMPOL) and DF-Mn, MnSOD mimetics, could significantly inhibit ROS generation and reduce cell death. Production of superoxide appears to be a central mediator of cytotoxicity from DHA.