Suppression of tumorigenesis in mitochondrial NADP+-dependent isocitrate dehydrogenase knock-out mice

The tumor host microenvironment is increasingly viewed as an important contributor to tumor growth and suppression. Cellular oxidative stress resulting from high levels of reactive oxygen species (ROS) contributes to various processes involved in the development and progress of malignant tumors including carcinogenesis, aberrant growth, metastasis, and angiogenesis. In this regard, the stroma induces oxidative stress in adjacent tumor cells, and this in turn causes several changes in tumor cells including modulation of the redox status, inhibition of cell proliferation, and induction of apoptotic or necrotic cell death. Because the levels of ROS are determined by a balance between ROS generation and ROS detoxification, disruption of this system will result in increased or decreased ROS level. Recently, we demonstrated that the control of mitochondrial redox balance and cellular defense against oxidative damage is one of the primary functions of mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDH2) that supplies NADPH for antioxidant systems. To explore the interactions between tumor cells and the host, we evaluated tumorigenesis between IDH2-deficient (knock-out) and wild-type mice in which B16F10 melanoma cells had been implanted. Suppression of B16F10 cell tumorigenesis was reproducibly observed in the IDH2-deficient mice along with significant elevation of oxidative stress in both the tumor and the stroma. In addition, the expression of angiogenesis markers was significantly down-regulated in both the tumor and the stroma of the IDH2-deficient mice. These results support the hypothesis that redox status-associated changes in the host environment of tumor-bearing mice may contribute to cancer progression.     

Redox regulation of matrix metalloproteinase gene family in small cell lung cancer cells

It has been implicated that reactive oxygen species (ROS) play important roles in modulating tumor progression. However, the mechanisms by which redox-regulated tumor progression are largely unknown. We previously demonstrated that reduced intracellular redox conditions could be achieved in stably transfected small cell lung cancer cells with gamma-glutamylcysteine synthetase (gamma-GCSh) cDNA which encodes a rate-limiting enzyme in the biosynthesis of glutathione (GSH), a major physiological redox regulator. In the present study, using DNA microarray analyses, we compared the expression profiles between the gamma-GCSh-transfected cells and their nontransfected counterpart. We observed downregulation of several matrix metalloproteinases (MMPs), i.e., MMPI and MMP3, and MMP10 in the transfected cells. Dot blot and Northern blot hybridizations confirmed that, among the 18 MMP gene family members and four tissue inhibitors of matrix metalloprotein family (TIMP) analyzed, the expression levels of these three MMPs were consistently reduced. Transiently increased gamma-GCSh expression using tetracycline-inducible gamma-GCSh adenoviral expression system also showed down-regulation of MMP3 and MMP10, but not MMP1. Our results demonstrated that redox regulation of MMP1, MMP3 and MMP10 expression depend upon different modes of redox manipulation. These results bear implication that antioxidant modulation of antitumor progression may be contributed at least in part by the downregulation of a subset of metrix metalloproteins.     

IDH2 deficiency impairs cutaneous wound healing via ROS-dependent apoptosis

Dermal fibroblasts are mesenchymal cells found between the skin epidermis and subcutaneous tissue that play a pivotal role in cutaneous wound healing by synthesizing fibronectin (a component of the extracellular matrix), secreting angiogenesis factors, and generating strong contractile forces. In wound healing, low concentrations of reactive oxygen species (ROS) are essential in combating invading microorganisms and in cell-survival signaling. However, excessive ROS production impairs fibroblasts. Mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDH2) is a key enzyme that regulates the mitochondrial redox balance and reduces oxidative stress-induced cell injury through the generation of NADPH. In the present study, the downregulation of IDH2 expression resulted in an increase in cell apoptosis in mouse skin through ROS-dependent ATM-mediated p53 signaling. IDH2 deficiency also delayed cutaneous wound healing in mice and impaired dermal fibroblast function. Furthermore, pretreatment with the mitochondria-targeted antioxidant mito-TEMPO alleviated the apoptosis induced by IDH2 deficiency both in vitro and in vivo. Together, our findings highlight the role of IDH2 in cutaneous wound healing in association with mitochondrial ROS.     

IDH2 deficiency promotes mitochondrial dysfunction and cardiac hypertrophy in mice

Cardiac hypertrophy, a risk factor for heart failure, is associated with enhanced oxidative stress in the mitochondria, resulting from high levels of reactive oxygen species (ROS). The balance between ROS generation and ROS detoxification dictates ROS levels. As such, disruption of these processes results in either increased or decreased levels of ROS. In previous publications, we have demonstrated that one of the primary functions of mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDH2) is to control the mitochondrial redox balance, and thereby mediate the cellular defense against oxidative damage, via the production of NADPH. To explore the association between IDH2 expression and cardiac function, we measured myocardial hypertrophy, apoptosis, and contractile dysfunction in IDH2 knockout (idh2^−/−) and wild-type (idh2^+/+) mice. As expected, mitochondria from the hearts of knockout mice lacked IDH2 activity and the hearts of IDH2-deficient mice developed accelerated heart failure, increased levels of apoptosis and hypertrophy, and exhibited mitochondrial dysfunction, which was associated with a loss of redox homeostasis. Our results suggest that IDH2 plays an important role in maintaining both baseline mitochondrial function and cardiac contractile function following pressure-overload hypertrophy, by preventing oxidative stress.     

Antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells

Reactive oxygen species (ROS) generation is linked to dynamic actin cytoskeleton reorganization, which is involved in tumor cell motility and metastasis. Thus, inhibition of ROS generation and actin polymerization in tumor cells may represent an effective anticancer strategy. However, the molecular basis of this signaling pathway is currently unknown. Here, we show that the Ecklonia cava-derived antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells. Steady-state intracellular ROS levels were higher in malignant B16F10 cells than in parental, nonmetastatic B16F0 cells. Elevation of ROS by H₂O₂ treatment increased migration and invasion ability of B16F0 cells to level similar to that of B16F10 cells, suggesting that intracellular ROS signaling mediates the prometastatic properties of B16 mouse melanoma cells. ROS levels and the cell migration and invasion ability of B16 melanoma cells correlated with Rac1 activation and WAVE2 expression. Overexpression of dominant negative Rac1 and depletion of WAVE2 by siRNA suppressed H₂O₂-induced cell invasion of B16F0 and B16F10 cells. Similarly, dieckol attenuates the ROS-mediated Rac1 activation and WAVE2 expression, resulting in decreased migration and invasion of B16 melanoma cells. In addition, we found that dieckol decreases association between WAVE2 and NADPH oxidase subunit p47^phox. Therefore, this finding suggests that WAVE2 acts to couple intracellular Rac1/ROS signaling to the invasive migration of B16 melanoma cells, which is inhibited by dieckol.     

Enhanced metastasis in RNF13 knockout mice is mediated by a reduction in GM-CSF levels

RING finger protein 13 (RNF13) is a novel E3 ubiquitin ligase whose expression is associated with cancer development. However, its specific role in cancer progression and metastasis remains unclear. Here, a B16F10/LLC experimental pulmonary metastatic model was developed to examine the formation of metastatic foci in the lung. A greater number of tumor colonies were observed in the lungs of RNF13-knockout (KO) mice than in their wild-type (WT) littermates, whereas no significant differences in tumor size were observed between the two groups. In short-term experiments, the number of fluorescently-labeled B16F10 cells increased remarkably in RNF13-KO lungs at early time points, whereas clearance of tumor cells from the blood was not affected. These results indicated that RNF13 may inhibit the colonization of B16F10 cells in the lung. Assessment of the concentration of various cytokines in tumor bearing lungs and blood did not detect significant differences between the blood of RNF13-KO and WT mice; however the levels of GM-CSF were significantly reduced in RNF13-KO tumor bearing lungs, which may have guided more B16F10 cells to migrate to the lungs. This was confirmed by lower GM-CSF concentrations in conditioned media from the culture of RNF13-KO lung slices. Collectively, our results suggest that host RNF13 affects the concentration of GM-CSF in tumor-bearing lungs, leading to a reduction in the colonization of metastatic tumor cells in the lung.     

The small leucine-rich proteoglycan lumican inhibits melanoma progression

Lumican is a member of the small leucine-rich proteoglycan (SLRP) family. It contributes to the organisation of the collagen network and plays an important role in cell migration and tissue repair. The present study aimed to determine the influence of lumican expression on adhesion, anchorage-dependent and -independent growth, migration, in vitro invasion and in vivo melanoma growth. For that purpose, B16F1 mouse melanoma cells were stably transfected with an expression plasmid containing the complete lumican cDNA. Lumican expression by tumor cells did not change the proliferative activity of mouse melanoma cells in monolayer culture and did not influence either cell adhesion to extracellular matrix gel or type I collagen or cell spreading on these substrates. In contrast, lumican-transfected cells were characterized by a strong reduction of their anchorage-independent proliferation in agarose gel and capacity to invade extracellular matrix gel. After subcutaneous injections of transfected B16F1 cells in syngenic mice, lumican expression significantly decreased subcutaneous tumor formation in vivo, with a concomitant decrease of cyclin D1 expression. Lumican induced and/or increased the apoptosis of B16F1 cells. The results suggest that lumican is involved in the control of melanoma growth and invasion and may be considered, like decorin, as an anti-tumor factor from the extracellular matrix.     

A Novel Triterpenoid Isolated from the Root Bark of Ailanthus excelsa Roxb (Tree of Heaven), AECHL-1 as a Potential Anti-Cancer Agent

Background

We report here the isolation and characterization of a new compound Ailanthus excelsa chloroform extract-1 (AECHL-1) (C₂₉H₃₆O₁₀; molecular weight 543.8) from the root bark of Ailanthus excelsa Roxb. The compound possesses anti-cancer activity against a variety of cancer cell lines of different origin.

Principal Findings

AECHL-1 treatment for 12 to 48 hr inhibited cell proliferation and induced death in B16F10, MDA-MB-231, MCF-7, and PC3 cells with minimum growth inhibition in normal HEK 293. The antitumor effect of AECHL-1 was comparable with that of the conventional antitumor drugs paclitaxel and cisplatin. AECHL-1-induced growth inhibition was associated with S/G₂-M arrests in MDA-MB-231, MCF-7, and PC3 cells and a G₁ arrest in B16F10 cells. We observed microtubule disruption in MCF-7 cells treated with AECHL-1 in vitro. Compared with control, subcutaneous injection of AECHL-1 to the sites of tumor of mouse melanoma B16F10 implanted in C57BL/6 mice and human breast cancer MCF-7 cells in athymic nude mice resulted in significant decrease in tumor volume. In B16F10 tumors, AECHL-1 at 50 µg/mouse/day dose for 15 days resulted in increased expression of tumor suppressor proteins P53/p21, reduction in the expression of the oncogene c-Myc, and downregulation of cyclin D1 and cdk4. Additionally, AECHL-1 treatment resulted in the phosphorylation of p53 at serine 15 in B16F10 tumors, which seems to exhibit p53-dependent growth inhibitory responses.

Conclusions

The present data demonstrate the activity of a triterpenoid AECHL-1 which possess a broad spectrum of activity against cancer cells. We propose here that AECHL-1 is a futuristic anti-cancer drug whose therapeutic potential needs to be widely explored for chemotherapy against cancer.     

Immunity to melanoma in mice immunized with transfected allogeneic mouse fibroblasts expressing melanoma-associated antigens

Summary Transfection of genomic DNA from B16 mouse melanoma into LM(TK^–) fibroblasts led to the generation of several clones of transfected cells that strongly expressed B 16 melanoma-associated antigens (MAA). The transfected cells retained their H-2^k markers and served as allogeneic cells with expressive MAA in C57BL/6 mice, syngeneic with the melanoma. The cells were capable of eliciting primary anti-B16 immune responses in vitro in spleen cells from C57BL/6 mice. Immunization of C57BL/6 mice with the transfected cells led to the generation of anti-B16 cytotoxic activity in spleen cells, and C57BL/6 mice immunized with the MAA-positive transfected cells were partially resistant to a lethal challenge with B16 melanoma cells. Under similar conditions, B16 cells were nonimmunogenic. Therefore, transfected allogeneic LM(TK^–) fibroblast cells expressing MAA served as more potent anti-melanoma immunogens than the parental B16 tumor cells themselves.     

siRNA干扰MMP-9和FAK双基因抑制小鼠黑色素瘤生长和在体迁移

目的:观察干扰MMP-9和FAK双基因对恶性黑色素瘤高转移细胞B16F10体内转移的影响。方法:构建PGV102-MMP9-siRNA、PGV102-FAK-siRNA重组质粒载体,脂质体^TM2000介导转染小鼠黑色素瘤B16F10细胞,RT-PCR检测基因的干扰效果;建立C57BL/6小鼠皮下移植瘤模型观察细胞在体成瘤和肿瘤的生长情况,常规组织切片,H&E染色观察肿瘤组织病理学特征;经C57BL/6小鼠尾静脉注射细胞5×10⁵个/只,24天后计数小鼠肺转移结节数评价肿瘤细胞在体迁移能力。结果:RT-PCR结果表明,重组质粒转染细胞组的MMP-9和FAK的mRNA水平显著低于正常细胞组(P<0.01),转染细胞组C57BL/6小鼠皮下成瘤的肿瘤生长速率、黑色素瘤肺转移结节数明显低于正常细胞组(P<0.01)。结论:干扰B16F10细胞MMP-9和FAK双基因可明显抑制小鼠体内恶性肿瘤的生长和迁移。     

Recombinant angioarrestin secreted from mouse melanoma cells inhibits growth of primary tumours

Angioarrestin is a recently described anti-angiogenic protein whose expression is down-regulated in solid tumours of various origins. It has a sequence identical to angiopoietin related protein-1. In this study we investigated anti-tumour properties of angioarrestin in B16 (F10) melanoma tumour model. We constructed an expression vector encoding human angioarrestin under the control of EF-1alpha promoter. This vector was transferred to B16 (F10) cells and recombinant angioarrestin secreted from the transfected cells was tested for anti-angiogenic activity using endothelial cell proliferation assay. Finally, mice were injected subcutaneously with cells that had been transfected with either angioarrestin-encoding vector or empty vector and tumor growth was compared. The obtained recombinant angioarrestin inhibited proliferation of bovine aortic endothelial cells. Tumours derived from an angioarrestin-secreting B16 (F10) cell clone grew in vivo more slowly than tumours derived from a cell clone transfected with empty vector. These data show, to our knowledge for the first time, that angioarrestin can inhibit primary melanoma tumour growth.     

The addition of interleukin-2 to cyclophosphamide therapy can facilitate tumor growth of B16 melanoma

The role of interleukin-2 (IL-2) on tumor growth of B16F10 melanoma cells was assessed in two sets of mice with different immune status: normal (immunocompetent) mice and immunodeficient mice. The two sets of animals were treated with cyclophosphamide (CY) or IL-2 alone or with a combined therapy of CY+IL-2. On days 6 and 10 after tumor cell injection, we evaluated the incidence of hepatic B16 melanoma metastases and the percentage of hepatic volume occupied by metastatic tissue. We observed that the CY alone (300 mg/kg, days 3 and 8 post-tumoral inoculation) significantly reduced tumor growth in all treated mice; however, CY proved more effective in normal recipients than in immunodeficient hosts. On the other hand, whereas administration of IL-2 alone (10⁵ IU daily, from day 3 to day 7) in immunocompetent mice significantly reduced tumor growth on days 6 and 10, in immunodeficient mice, no significant differences were observed in tumor growth either on the 6th or on the 10th day, in comparison to control groups. Finally, when the combined CY+IL-2 therapy was administered, an antisynergistic effect between these therapeutic agents was achieved both in normal and in immunodeficient mice. Thus, the addition of low-dose IL-2 (25×10³ IU daily, from day 4 to day 7) to high-dose CY (300 mg/kg, days 3 and 8) significantly increased tumor growth in both the early and later periods, compared to the effect of CY alone. It is concluded that exogenous IL-2 can facilitate tumor growth of B16 melanoma cells in vivo.     

CD4<Superscript>+</Superscript> T cell response against a non-tumor antigen is unaffected in melanoma-bearing mice

The tumor microenvironment is complex and creates an immunosuppressive network to tolerize tumor-specific immune responses; however, little information is available regarding the response against non-tumor antigens in tumor-bearing individuals. The goal of the present study was to evaluate if tumor burden could influence a CD4⁺ T cell response against a soluble protein, not expressed by the tumor, in the absence of in vitro stimulation. Using an experimental system in which we can compare CD4⁺ T cell responses to the Ea antigen when it is either expressed by B16F10 melanoma cells (B16EaRFP cells) or is an exogenous, non-tumor antigen (soluble EaRFP protein), in immunizations of B16F10 tumor-bearing mice, we observed that the tumor can modulate the CD4⁺ T cell-specific response to the antigen when it is expressed by the tumor cells. TEa cells proliferated poorly and produced less IFN-γ in mice bearing B16F10 melanoma expressing Ea peptide, and tumor growth was impervious to this response. However, in mice bearing 7 days B16F10 tumors, not expressing the Ea antigen, priming of TEa cells was similar to that observed in tumor-free mice, based on the total number of cells recovered and proliferation assessed by CFSE dilution after EaRFP immunization. We also investigated if tumor burden could influence recall responses of already differentiated effector cells. We immunized mice with EaRFP antigen and after a few days injected B16F10 cells. After 10 days of tumor growth, we challenged the mice with the non-tumor antigen. We found that the number of TEa cells producing IFN-γ in tumor-bearing mice was not different compared to tumor-free mice. No differences in antigen presentation, assessed by YAe antibody staining, were verified in the draining lymph node of these two groups. Collectively, our data indicate that tumor burden does not affect immune responses to non-tumor antigens. These results have important implications in the design of anti-cancer therapy.     

Galectin-9 suppresses tumor metastasis by blocking adhesion to endothelium and extracellular matrices

We previously described an inverse correlation between galectin-9 (Gal-9) expression and metastasis in patients with malignant melanoma and breast cancer. This study verified the ability of Gal-9 to inhibit lung metastasis in experimental mouse models using highly metastatic B16F10 melanoma and Colon26 colon cancer cells. B16F10 cells transfected with a secreted form of Gal-9 lost their metastatic potential. Intravenous Gal-9 administration reduced the number of metastases of both B16F10 and Colon26 cells in the lung, indicating that secreted Gal-9 suppresses metastasis. Analysis of adhesive molecule expression revealed that B16F10 cells highly express CD44, integrin alpha1, alpha 4, alpha V, and beta1, and that Colon26 cells express CD44, integrin alpha2, alpha 5, alpha V, and beta1, suggesting that Gal-9 may inhibit the adhesion of tumor cells to vascular endothelium and the extracellular matrix (ECM) by binding to such adhesive molecules. Indeed, Gal-9 suppressed the binding of hyaluronic acid to CD44 on both B16F10 and Colon26 cells, and also suppressed the binding of vascular cell adhesion molecule-1 to very late antigen-4 on B16F10 cells. Furthermore, Gal-9 inhibited the binding of tumor cells to ECM components, resulting in the suppression of tumor cell migration. The present results suggest that Gal-9 suppresses both attachment and invasion of tumor cells by inhibiting the binding of adhesive molecules on tumor cells to ligands on vascular endothelium and ECM.     

APC/C CDH1 ubiquitinates IDH2 contributing to ROS increase in mitosis

NADPH is a cofactor used by reactive oxygen species (ROS) scavenging enzymes to block ROS produced in cells. Recently, it was shown that in cancer cells, ROS progressively increases in tune to cell cycle leading to a peak in mitosis. Loss of IDH2 is known to cause severe oxidative stress in cell and mouse models as ROS increases in mitochondria. Therefore, we hypothesized that IDH2, a major NADPH-producing enzyme in mitochondria is ubiquitinated for ROS to increase in mitosis. To test this hypothesis, in cancer cells we examined IDH2 ubiquitination in mitosis and measured the ROS produced. We found that IDH2 is ubiquitinated in mitosis and on inhibiting anaphase-promoting complex/Cyclosome (APC/C) IDH2 was stabilized. Further, we observed that overexpressing APC/C coactivator CDH1 decreased IDH2, whereas depleting CDH1 decreased IDH2 ubiquitination. To understand the link between IDH2 ubiquitination and ROS produced in mitosis, we show that overexpressing mitochondria-targeted-IDH1 decreased ROS by increasing NADPH in IDH2 ubiquitinated cells. We conclude that APC/C CDH1 ubiquitinates IDH2, a major NADPH-producing enzyme in mitochondria contributing to ROS increase in mitosis. Based on our results, we suggest that mitosis can be a therapeutic window in mutant IDH2-linked pathologies.     

Reinstalling antitumor immunity by inhibiting tumor-derived immunosuppressive molecule IDO through RNA interference

Tumor-derived immune suppression is a major impediment to successful immune/gene cancer therapy. In the present study, we describe a novel strategy to disrupt tumor-derived immune suppression by silencing a tolerogenic molecule of tumor origin, IDO, using small interfering RNA (siRNA). Silencing of IDO in B16F10 cells in vitro using IDO-siRNA prevented catabolism of tryptophan and inhibited apoptosis of T cells. IDO-siRNA treatment of B16F10 cells in vitro inhibited subsequent growth, tumor formation, and the size of tumor formed, by those cells when transplanted into host mice. In vivo treatment of B16F10 tumor-bearing mice successfully postponed tumor formation time and significantly decreased tumor size. Furthermore, in vivo IDO-siRNA treatment resulted in recovery of T cells responses and enhancement of tumor-specific killing. Thus, silencing IDO may break tumor-derived immune suppression. These data indicate that RNA interference has potential to enhance cancer therapy by reinstalling anticancer immunity.     

GM-CSF-secreting cancer immunotherapies: preclinical analysis of the mechanism of action

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell immunotherapies have demonstrated long-lasting, and specific anti-tumor immune responses in animal models. The studies reported here specifically evaluate two aspects of the immune response generated by such immunotherapies: the persistence of irradiated tumor cells at the immunization site, and the breadth of the immune response elicited to tumor associated antigens (TAA) derived from the immunotherapy. To further define the mechanism of GM-CSF-secreting cancer immunotherapies, immunohistochemistry studies were performed using the B16F10 melanoma tumor model. In contrast to previous reports, our data revealed that the irradiated tumor cells persisted and secreted high levels of GM-CSF at the injection site for more than 21 days. Furthermore, dense infiltrates of dendritic cells were observed only in mice treated with GM-CSF-secreting B16F10 cells, and not in mice treated with unmodified B16F10 cells with or without concurrent injection of rGM-CSF. In addition, histological studies also revealed enhanced neutrophil and CD4+ T cell infiltration, as well as the presence of apoptotic cells, at the injection site of mice treated with GM-CSF-secreting tumor cells. To evaluate the scope of the immune response generated by GM-CSF-secreting cancer immunotherapies, several related B16 melanoma tumor cell subclones that exist as a result of genetic drift in the original cell line were used to challenge mice previously immunized with GM-CSF-secreting B16F10 cells. These studies revealed that GM-CSF-secreting cancer immunotherapies elicit T cell responses that effectively control growth of related but antigenically distinct tumors. Taken together, these studies provide important new insights into the mechanism of action of this promising novel cancer immunotherapy. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Melanoma-induced suppression of tumor antigen-specific T cell expansion is comparable to suppression of global T cell expansion

We have observed that in vivo interaction between melanoma and resting T cells promotes suppression of antigen-driven proliferative T cell expansion. We hypothesized that this suppression would affect tumor antigen-specific T cell populations more potently than tumor-unrelated T cell populations. A B16F10 cell line was stably transfected to express low levels of the lymphocytic choriomeningitis virus (LCMV) glycoprotein GP33 (B16GP33). Mice bearing B16F10 or B16GP33 tumors were infected with LCMV, and proliferative expansion of LCMV epitope-specific T cell populations was quantified. In vitro and in vivo assays confirmed low levels of antigenic GP33 expression by B16GP33 tumors. Suppressed expansion of GP33-specific T cells was equivalent between mice bearing B16F10 and B16GP33 tumors. These observations suggest that the ability of growing melanoma tumors to impair antigen-driven proliferative expansion of activated T cells is global and not antigen-specific, and provide further insight into the influence of cancer on activated T cell homeostasis.     

Inhibition of metastasis of syngeneic murine melanoma in vivo and vasculogenesis in vitro by monoclonal antibody C11C1 targeted to domain 5 of high molecular weight kininogen

Metastasis of malignant tumors is a major cause of morbidity and mortality. Inhibition of tumor growth in distant organs is of clinical importance. We have demonstrated that C11C1, a murine monoclonal antibody to the light chain region of high molecular weight kininogen (HK), reduces growth of murine multiple myeloma in normal mice and human colon cancer in nude mice. C11C1 inhibits angiogenesis by reducing tumor microvascular density by blocking binding of HK to endothelial cells. We now evaluate the anti-metastatic effect of C11C1 on C57BL/6 mouse lung metastatic model using B16F10 melanoma cells. The tail veins of mice were injected with 0.5 × 10⁶ cells of melanoma B16F10. One group received C11C1 and the other received saline (control) intraperitoneally. When mice were killed at 28 days, 6 of 10 control mice had detectable metastatic pulmonary nodules which stained positive with an antibody against S-100 protein, a tumor antigen present in malignant melanoma cells. In the C11C1 groups, none of the mice showed metastatic foci in their lungs. We showed that C11C1 inhibits endothelial cell tube formation in a 3-D collagen fibrinogen gel model by inhibiting the rate of cleavage of HK by plasma kallikrein without changing the binding affinity for HK. These studies demonstrate that a monoclonal antibody to HK has the potential to prevent metastasis with minimal side effects.     

NADPH oxidase subunit p22phox-mediated reactive oxygen species contribute to angiogenesis and tumor growth through AKT and ERK1/2 signaling pathways in prostate cancer

Excessive generation of reactive oxygen species (ROS) in cancer cells is associated with cancer development, but the underlying mechanisms and therapeutic significance remain elusive. In this study, we reported that levels of ROS and p22^phox expression are greatly increased in human prostate cancer tissues, and knockdown of p22^phox by specific small interfering RNA (siRNA) decreased ROS levels in prostate cancer cells. We also showed that stable downregulation of p22^phox in prostate cancer cells inhibited cell proliferation and colony formation, which was mediated by AKT and extracellular signal-regulated kinase (ERK)1/2 signaling pathways and their downstream molecules hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF). The NADPH oxidase subunit NOX1 was also elevated in prostate cancer cells, and was involved in activation of AKT/ERK/HIF-1/VEGF pathway and regulation of cell proliferation. Knockdown of p22^phox resulted in inhibition of tumor angiogenesis and tumor growth in nude mice. These findings reveal a new function of p22^phox in tumor angiogenesis and tumor growth, and suggest that p22^phox is a potential novel target for prostate cancer treatment.