Ribonuclease binase inhibits primary tumor growth and metastases via apoptosis induction in tumor cells

Exogenous ribonucleases are known to inhibit tumor growth via apoptosis induction in tumor cells, allowing to consider them as promising anticancer drugs for clinical application. In this work the antitumor potential of binase was evaluated in vivo and the mechanism of cytotoxic effect of binase on tumor cells was comprehensively studied in vitro. We investigated tumoricidal activity of binase using three murine tumor models of Lewis lung carcinoma (LLC), lymphosarcoma RLS₄₀ and melanoma B-16. We show for the first time that intraperitoneal injection of binase at a dose range 0.1–5 mg/kg results in retardation of primary tumor growth up to 45% in LLC and RLS₄₀ and inhibits metastasis up to 50% in LLC and RLS₄₀ and up to 70% in B-16 melanoma. Binase does not exhibit overall toxic effect and displays a general systemic and immunomodulatory effects. Treatment of RLS₄₀-bearing animals with binase together with polychemotherapy revealed that binase decreases the hepatotoxicity of polychemotherapy while maintaining its antitumor effect. It was demonstrated that the cytotoxic effect of binase is realized via the induction of the intrinsic and extrinsic apoptotic pathways. Activation of intrinsic apoptotic pathway is manifested by a drop of mitochondrial potential, increase in calcium concentration and inhibition of respiratory activity. Subsequent synthesis of TNF-α in the cells under the action of binase triggers extrinsic apoptotic pathway through the binding of TNF with cell-death receptors and activation of caspase 8. Thus binase is a potential anticancer therapeutics inducing apoptosis in cancer cells.     

Late stage inhibition of hematogenous melanoma metastasis by cystatin C over-expression

Background  

Tumor metastasis is a frequent cause of treatment failure for cancer patients. A key feature of metastatic cancer cells is their invasive ability. Cysteine proteases contribute to invasive properties of many cancer cell types. To analyze the contribution of cysteine proteases to metastasis we have over-expressed in B16 melanoma cells the natural cysteine protease inhibitor, cystatin C. We measured in vitro invasion of cystatin over-expression clones with Boyden chamber type assays. Tail-vein injections of cells were used to compare lung tumor colonization. Subcutaneous tumor growth and tumor cell metastasis from primary tumors were also analyzed. Apoptosis of tumor cells was measured in lung tissues following melanoma cell injection.     

Induction of Apoptosis in Tumor Cells by Binase

The possibility of inducing apoptosis in K562 myelogenic erythroleukemia cells, A549 lung carcinoma cells, and normal human lymphocytes was studied for Bacillus intermedius RNase (binase) and its mutants Lys²⁶ Ala and His¹⁰¹ Glu with impaired catalytic activity. Selective induction of apoptosis in leukemic blood cells by binase was demonstrated for the first time. Binase did not exert an antiproliferative or proapoptotic effect on peripheral blood lymphocytes of healthy donors. Low-molecular-weight (less than 50 kb in size) oligonucleosomal DNA fragments, which are early markers of apoptosis, were observed in human solid-tumor cells treated with binase. Studies with the binase mutants showed that a decrease in catalytic activity to 2.5% of the level characteristic of the wild-type enzyme deprives binase of its proapoptotic effect. The selective proapoptotic effect of binase on malignant cells provides evidence that bacterial RNases are promising for designing alternative antitumor drugs.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 3, 2005, pp. 457–463.Original Russian Text Copyright © 2005 by Zelenikhin, Kolpakov, Cherepnev, Ilinskaya.     

Binase penetration into alveolar epithelial cells does not induce cell death

Microbial ribonucleases possess a broad spectrum of biological activities, which demonstrate stimulating properties at low concentrations and cytotoxicity and genotoxicity at high concentrations. Mechanisms of their penetration into the cells still remain unclear. In this study penetration of Bacillus intermedius RNase (binase) in alveolar lung epithelial cells, type II (ATII) pneumocytes, has been investigated. Using immunofluorescence analysis we have shown for the first time internalization of binase by primary non-differentiated pneumocytes ATII. The enzyme did not penetrate in MLE-12 (Murine Lung Epithelial-12 cells). However, binase was cytotoxic towards tumor MLE-12 cells, but not ATII cells. These results clearly indicate higher sensitivity of tumor cells to binase compared to normal cells; they also demonstrate that penetration of the enzyme into alveolar epithelial cells is not directly associated with their death.     

Antimetastatic effect of recombinant human macrophage-colony-stimulating factor against lung and liver metastatic B16 melanoma

 We studied the effect of recombinant human macrophage-colony-stimulating factor (rhM-CSF) on the formation of lung and liver metastases following the i.v. injection of the B16 melanoma subline (B16 LiLu) into mice. When rhM-CSF was administered before the B16 inoculation, the number of tumor metastases decreased in the lung and liver. However, the administration of rhM-CSF after B16 inoculation did not produce an antimetastatic effect in the lung, but did in the liver. B16 cells labeled with 5-[¹²⁵I]-iodo-2′-deoxyuridine (¹²⁵I-dUrd) were injected and the arrest of tumor cell emboli was examined in the capillary beds of the lung and liver of mice treated with either vehicle or rhM-CSF. In both groups, there were the same numbers of B16 cells in both the lung and the liver 3 minutes after the B16 injection, and almost all tumor cells died within 24 h. However, the number of cells surviving in the lung was decreased in mice injected with rhM-CSF (37%). There was no difference in the number of cells in the livers of mice treated either with vehicle or rhM-CSF in the first 24 h after tumor cell injection. The administration of rhM-CSF increased NK 1.1⁺ cells in the mouse spleen and facilitated NK activity in vivo. At the same time, the administration of an anti-NK 1.1 antibody blocked the antimetastatic effect of rhM-CSF in the lung but not in the liver. The antibody was effective only when it was injected before the B16 inoculation. These results suggest that the antimetastatic effect of rhM-CSF in the lung was mediated by NK 1.1⁺ cells within 24 h of B16 injection. In contrast, the antimetastatic effect of rhM-CSF in the liver was mediated not only by NK 1.1⁺ cells but also by other antimetastatic systems such as macrophages. Received: 8 April 1996 / Accepted: 26 November 1996     

Increased prevalence of regulatory T cells in the lung cancer microenvironment: a role of thymic stromal lymphopoietin

Expansion of CD4+CD25+ regulatory T cells (Tregs) in tumor microenvironment was one of the mechanisms by which cancer cells escaped host defense. Thymic stromal lymphopoietin (TSLP) contributes to the generation of natural Tregs in thymus. Therefore, the purpose of this report was to investigate the role of TSLP in the increasing prevalence of Tregs in lung cancer microenvironment. The expression ratio of TSLP protein in tumor tissues was significantly increased compared with that in benign lesion and non-cancer lung tissue. The prevalence of Tregs in tumor microenvironment was correlated with the expression of TSLP in lung cancer. Dendritic cells (DCs) were induced from peripheral blood mononuclear cells (PBMCs) collected from lung cancer patients and left unstimulated (imDCs) or exposed to hTSLP (TSLP-DCs) or LPS (LPS-DCs). TSLP-DCs expressed intermediate levels of CD83 and high levels of CD86, CD11C, and HLA-DR, which showed a characteristic of less mature DCs. TSLP-DCs secreted low levels of IL-6, IL-12, IL-10, TNF-α and IFN-γ, and high levels of TGF-β and MDC. The percentage of Tregs in CD4+CD25− T cells cocultured with TSLP-DCs group was statistically higher than that of LPS-DCs and imDCs. Transwell assays showed that TSLP-DCs exhibited increased ability to attract the migration of CD4+CD25− Tregs, when compared with imDCs. These results indicated that TSLP proteins were expressed in lung tumor tissue and correlated with the prevalence of Tregs. TSLP-DCs could induce CD4+CD25− T cells to differentiate into CD4+CD25+foxp3+ T cells and the migration of CD4+CD25+ T cells.     

Antimetastatic activity of pinosylvin, a natural stilbenoid, is associated with the suppression of matrix metalloproteinases

Metastasis is a major cause of death in cancer patients. Our previous studies showed that pinosylvin, a naturally occurring trans-stilbenoid mainly found in Pinus species, exhibited a potential cancer chemopreventive activity and also inhibited the growth of various human cancer cell lines via the regulation of cell cycle progression. In this study, we further evaluated the potential antimetastatic activity of pinosylvin in in vitro and in vivo models. Pinosylvin suppressed the expression of matrix metalloproteinase (MMP)-2, MMP-9 and membrane type 1-MMP in cultured human fibrosarcoma HT1080 cells. We also found that pinosylvin inhibited the migration of HT1080 cells in colony dispersion and wound healing assay systems. In in vivo spontaneous pulmonary metastasis model employing intravenously injected CT26 mouse colon cancer cells in Balb/c mice, pinosylvin (10 mg/kg body weight, intraperitoneal administration) significantly inhibited the formation of tumor nodules and tumor weight in lung tissues. The analysis of tumor in lung tissues indicated that the antimetastatic effect of pinosylvin coincided with the down-regulation of MMP-9 and cyclooxygenase-2 expression, and phosphorylation of ERK1/2 and Akt. These data suggest that pinosylvin might be an effective inhibitor of tumor cell metastasis via modulation of MMPs.     

Regulation of MMP-2 expression and activity by β-1,3-<Emphasis Type="Italic">N</Emphasis>-acetylglucosaminyltransferase-8 in AGS gastric cancer cells

β-1,3-N-acetylglucosaminyltransferase-8(β3Gn-T8) catalyzes the transfer of GlcNAc to the non-reducing terminus of the Galβ1-4GlcNAc of tetraantennary N-glycan in vitro. It has been reported to be involved in malignant tumors, but a comprehensive understanding of how the glycolsyltransferase correlates with the invasive potential of human gastric cancer is not currently available. Therefore, we investigated the ability and possible mechanism involved with β3Gn-T8 in modulating matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) in AGS gastric cancer cells. Here, we found out that siRNA-mediated suppression of the β3Gn-T8 could directly reduce the MMP-2 expression and activity as observed in RT-PCR, western blot and gelatin zymography analysis. Meanwhile, TIMP-2 expression had been increased. Cell invasion assay using matrigel matrix-coated transwell inserts showed that the invasive property was greatly suppressed in β3Gn-T8 siRNA transfected cells. Furthermore, cells overexpressing β3Gn-T8 gene (when transfected with pEGFP-C1 plasmid) also expressed MMP-2 gene, but TIMP-2 expression had been inhibited. The invasive ability of these cells was also enhanced. Protein–protein interaction analysis using STRING database showed that β3Gn-T8 and MMP-2 may have related signal pathway. In summary, our results reveal a new mechanism by which β3Gn-T8 can regulate MMP-2 and TIMP-2. We suggest that β3Gn-T8 can be used as a novel therapeutic target for human gastric treatment.     

Exogenous <Emphasis Type="Italic">Bacillus pumilus</Emphasis> RNase (binase) suppresses the reproduction of reovirus serotype 1

The experimental study identified the antiviral activity of Bacillus pumilus RNase (binase) against the reovirus of serotype 1/strain Lang. For the first time, it has been found that 50 μg/mL of binase effectively reduced the hemagglutinin and cytocidal activity of reovirus in Vero cell line. The preincubation of the enzyme with reovirus before infection of the cells inhibited the viral replication. To determine the stagedependent effect of reovirus reproduction upon binase inhibition, the infected cells were treated with binase or RNase A at different phases of the infectious cycle. The treatment of virus-infected cells has revealed that both enzymes have a maximal antiviral effect on the reovirus propagation during early phases of the reovirus reproduction cycle, with binase being more effective than RNase A. It has been hypothesized that the combined action of the oncolytic reovirus and binase is promising for the elimination of tumor cells carrying mutated RAS gene.     

Induction of apoptosis of tumor cells by binase

An induction of apoptosis by RNase from Bacillus intermedius (binase) and its mutants characterized with low catalytic activity (Lys26Ala and His101Glu) in human myelogenic erythroleukemia K562 cells, human lung carcinoma A549 cells and human peripheral blood mononuclear cells was studied. For the first time selective apoptogenic effects of binase toward leukemic blood cells was determined. Neither antiproliferative nor apoptotic effects of binase were detected in normal human peripheral blood mononuclear cells. Formation of low molecular weight oligonucleosomal DNA fragments (less than 50 Kb) which are an early marks of apoptosis was registered in solid tumor cells treated by binase. Using mutant RNases it was shown that decrease of catalytic activity to 2.5% of wild type enzyme activity leads to the loss of apoptogenic properties of enzyme. Selective apoptogenicity of binase found towards malignant cells confirmed that antitumor agents based on bacterial RNases could be considered as an alternative to standard chemotherapeutic drugs.     

Antibodies to synthetic peptides for the detection of survivin in tumor tissues

Survivin, an endogenous protein, is a promising marker for the cancer diagnosis. The aim of the present work was to obtain antibodies specific to survivin and capable of detecting this protein in tumor tissues. Four peptides corresponding to fragments (1–22), (54–74), (80–88)–(153–165), and (118–144) of the survivin-2B sequence were selected and synthesized. Rabbits were immunized with the synthetic peptides. It has been shown that all peptides in a free state, without conjugation with a high-molecular-weight carrier, stimulate the production of antibodies capable of binding with recombinant survivin. Antipeptide antibodies were isolated from sera and their performance in the immunohistochemical detection of survivin in human tumor tissues was studied. It was shown that only antibodies to the (80–88)–(153–165) peptide bind to the survivin present in breast and bladder tumors. The ability of antibodies to this peptide to detect survivin in tumor tissue lysates was demonstrated by immunoblotting. The part of the sequence targeted by the antibodies against the (80–88)–(153–165) peptide was localized using truncated peptide fragments.     

Effect of Bacillus pumilus ribonuclease on the paramagnetic centers of microbial cells

The potential clinical application of Bacillus pumilus cytotoxic ribonuclease (binase) for selectively inducing the death of tumor cells makes it imperative to investigate its effect on the normal human microflora. Flow cytometry was used to determine that binase concentration causing the apoptosis of cancer cells had no effect of the viability of Escherichia coli K12. The changes in the paramagnetic centers of E. coli K12 cells in the presence of nontoxic binase concentrations revealed by EPR spectroscopy included higher EPR signals from iron-containing proteins (including those from the Fe-S clusters) and of the Mn(II) hyperfine structure. The TMTH spin probe (N-(1-hydroxy-2,2,6,6-tetramethylpiperidine-4-il)-2-methylpropanamide hydrochloride) was used to reveal a twofold increase in the levels of reactive oxygen species (ROS) in the cells, which induced oxidative stress in the enzyme-treated bacteria. Inductively coupled plasma mass spectrometry revealed elevated contents of alkaline (Li, Na, K), alkali earth (Mg, Ca), transition (Cr, Mn, Fe, Cu, Zn), and post-transition metals (Bi, Pb) in the cells. Elevated levels of Cu and Zn (which impair the activity of the respiratory chain enzymes) and of Mn, which is known as a superoxide dismutase cofactor, confirmed development of the oxidative stress in bacteria.     

Tumor-derived death receptor 6 modulates dendritic cell development

Studies in murine models of cancer as well as in cancer patients have demonstrated that the immune response to cancer is often compromised. This paradigm is viewed as one of the major mechanisms of tumor escape. Many therapies focus on employing the professional antigen presenting dendritic cells (DC) as a strategy to overcome immune inhibition in cancer patients. Death receptor 6 (DR6) is an orphan member of the tumor necrosis factor receptor superfamily (TNFRSF21). It is overexpressed on many tumor cells and DR6^−/− mice display altered immunity. We investigated whether DR6 plays a role in tumorigenesis by negatively affecting the generation of anti-tumor activity. We show that DR6 is uniquely cleaved from the cell surface of tumor cell lines by the membrane-associated matrix metalloproteinase (MMP)-14, which is often overexpressed on tumor cells and is associated with malignancy. We also demonstrate that >50% of monocytes differentiating into DC die when the extracellular domain of DR6 is present. In addition, DR6 affects the cell surface phenotype of the resulting immature DC and changes their cytokine production upon stimulation with LPS/IFN-γ. The effects of DR6 are mostly amended when these immature DC are matured with IL-1β/TNF-α, as measured by cell surface phenotype and their ability to present antigen. These results implicate MMP-14 and DR6 as a mechanism tumor cells can employ to actively escape detection by the immune system by affecting the generation of antigen presenting cells.     

A novel secreted ribonuclease from Bacillus intermedius: gene structure and regulatory control

A second secreted ribonuclease, designated binase II, has been detected in Bacillus intermedius 7P, and its structural gene was cloned and sequenced. Unlike the well-known binase I, a 109-amino acid guanyl-specific enzyme, the 292-residue binase II is closely related to the B. subtilis nuclease Bsn, in structure and in its enzymatic properties. Binase II is also insensitive to inactivation by barstar, an inhibitor protein that is specific for guanyl-specific ribonucleases. While both B. intermedius enzymes are induced upon phosphate starvation, only the gene for binase I belongs to the pho regulon system and carries pho-box elements adjacent to its promoter sequence. The gene for binase II is similar to that for Bsn in lacking such elements. The birB gene coding for binase II appears to be located next to the 3′-end of a ferric ion transport operon, with which it convergently overlaps. This would allow attenuator control over binase II expression under conditions of starvation for ferric ions. Received: 12 October 1999 / Accepted: 10 February 2000     

TGF-alpha as a candidate tumor antigen for renal cell carcinomas

Objectives  Patients with renal cell carcinomas (RCC) have few treatment options, underscoring the importance of developing new approaches such as immunotherapy. However, few tumor associated antigens (TAA), which can be targeted by immunotherapy, have been identified for this type of cancer. von Hippel-Lindau clear cell RCC (VHL^−/−RCC) are characterized by mutations in the VHL tumor suppressor gene. Loss of VHL function causes the overexpression of transforming growth factor (TGF)-α, leading us to hypothesize that TGF-α could be a potential TAA for immunotherapy of kidney cancer, which was evaluated in this study. Methods and results  We first confirmed the absent or weak expression of TGF-α in important normal tissues as well as its overexpression in 61% of renal tumors in comparison to autologous normal kidney tissues. In addition, we demonstrated the immunogenicity of TGF-α, by expanding many T cell lines specific for certain TGF-α peptides or the mature TGF-α protein, when presented by major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells. Interestingly, some of these TGF-α-specific T cells were polyfunctionals and secreted IFN-γ, TNF-α and IL-2. Conclusion  We have shown that TGF-α is a valid candidate TAA, which should allow the development of a targeted immunotherapy.     

Binase-Induced Changes of Tumor Cell Membranes

Exogenous ribonucleases of Bacilli can selectively induce apoptosis of malignant cells. The ability of Bacillus pumilus ribonuclease, binase, to induce processes leading to a dynamic disruption of the integrity of A549 human pulmonary adenocarcinoma cell membranes was analyzed. The influence of different enzyme concentrations on the state of the cytoplasmic membrane of cells and mitochondrial membranes was characterized. Using the methods of flow cytofluorometry and fluorescence microscopy, it has been established that binase leads to disruption in normal functioning of both types of membranes, with mitochondrial membranes affected first. The study made it possible to identify and visualize the effects of binase on the membrane structures of target cells and to confirm that bacterial RNase induces apoptosis of target cells mainly through the “internal” (mitochondrial) pathway.     

Spatial regulation of tumor cell protrusions by RhoC

Systemic metastasis is the dissemination of cancer cells from the primary tumor to distant organs and is the primary cause of death in cancer patients. How do cancer cells leave the primary tumor mass? The ability of the tumor cells to form different types of actin-rich protrusions including invasive protrusions (invadopodia) and locomotory protrusions (lamellipodia [2D] or pseudopodia [3D]), facilitate the invasion and dissemination of the tumor cells. Rho-family of p21 small GTPases plays a direct role in regulating the actin dynamics in these intracellular compartments. Recent studies have shown that the signaling molecules including RhoC/p190RhoGEF/p190RhoGAP acts as a “molecular compass” in order to direct the spatial and temporal dynamics of the formation of these invasive and locomotory protrusions leading to efficient invasion.     

肉毒碱棕榈酰基转移酶CPT2促进肝癌细胞迁移侵袭的作用研究

目的:探讨肉毒碱棕榈酰基转移酶2(CarnitinePalmitoyltransferase2, CPT2)在肝癌细胞迁移侵袭中的调控作用。方法:1).用免疫组化实验,检测62对肝癌癌组织与癌周组织中CPT2表达,以明确肝癌组织细胞中CPT2表达是否发生异常改变。2).细胞划痕实验,在人肝癌细胞HLE中分析干涉CPT2表达对肝癌细胞迁移能力的影响。3).Transwell侵袭实验,在人肝癌细胞HLE中分析干涉CPT2表达对肝癌细胞侵袭能力的影响。结果:1). CPT2主要为胞浆染色且染色呈线粒体蛋白染色典型的颗粒状分布;与癌周组织相比,肝癌组织中CPT2表达显著升高。2).细胞划痕实验证实,干涉人肝癌细胞HLE中CPT2表达后,细胞的相对迁移距离显著变短(si Ctrl VS si CPT2#1 VS si CPT2#2=1.00±0.8 VS 0.67±0.42 VS 0.64±0.31)。3).Transwell侵袭实验证实,干涉人肝癌细胞HLE中CPT2表达后,侵袭至小室底部的细胞数目显著变少(si Ctrl VS si CPT2#1 VS si CPT2#2=23.34±3.51 VS 8.00±2.00 VS8.67±1.53)。结论:CPT2在肝癌细胞中表达显著上调,CPT2表达上调促进了肝癌细胞的迁移与侵袭。