HspA1A facilitates DNA repair in human bronchial epithelial cells exposed to Benzo[a]pyrene and interacts with casein kinase 2

Benzo[a]pyrene (BaP) is a ubiquitously distributed environmental pollutant that induces deoxyribonucleic acid (DNA) damage. The inducible heat shock protein (HspA1A) can function as a molecular chaperone; however, its role in DNA repair remains largely unknown. In the present study, human bronchial epithelial cells (16HBE) stably transfected with plasmids carrying HspA1A gene or shRNAs against HspA1A were treated with BaP. DNA damage levels of the cells were evaluated by comet assay. Results suggest that HspA1A could protect cells against DNA damage and facilitate the decrease of DNA damage levels during the first 2 h of DNA repair. DNA repair capacity (DRC) of Benzo(a)pyrene diol epoxide (BPDE)-DNA adducts was evaluated by host cell reactivation assay in the stable 16HBE cells transfected with luciferase reporter vector PCMVluc pretreated with BPDE. Compared with control cells, cells overexpressing HspA1A showed higher DRC (p < 0.01 at 10 μM BPDE and p < 0.05 at 20 μM BPDE, respectively), while knockdown of HspA1A inhibited DNA repair (p < 0.05 at 10 μM BPDE). Moreover, casein kinase 2 (CK2) was shown to interact with HspA1A by mass spectrometry and co-immunoprecipitation assays. The two proteins were co-localized in the cell nucleus and perinuclear region during DNA repair, and were identified by confocal laser scanning microscope. In addition, cells overexpressing HspA1A showed an increased CK2 activity after BaP treatment compared with control cells (p < 0.01). Our results suggest that HspA1A facilitates DNA repair after BaP treatment. HspA1A also interacts with CK2 and enhances the kinase activities of CK2 during DNA repair.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-013-0454-7) contains supplementary material, which is available to authorized users.     

Tumor-associated neutrophils (TAN) develop pro-tumorigenic properties during tumor progression

The role and characteristics of tumor-associated neutrophils (TAN) in cancer are poorly defined. We have recently shown that TAN can have anti-tumorigenic (N1) or pro-tumorigenic (N2) functions. An interesting unanswered question is how the phenotype of TAN is influenced by the ongoing evolvement of tumor microenvironment. We therefore studied the phenotype and effects of TAN at different time points during tumor progression. We used two models of murine tumor cancer cell lines—Lewis lung carcinoma (LLC) and AB12 (mesothelioma). Neutrophils were studied at early and late stages and compared to each other and to neutrophils from bone marrow/periphery of naïve mice. Although there was no difference in the number of neutrophils entering the tumor, we found that at early stages of tumor development, neutrophils were almost exclusively at the periphery of the tumor. Only at later stages, neutrophils were also found scattered among the tumor cells. We further found that TAN from early tumors are more cytotoxic toward tumor cells and produce higher levels of TNF-α, NO and H₂O₂. In established tumors, these functions are down-regulated and TAN acquire a more pro-tumorigenic phenotype. In line with this phenotype, only depletion of neutrophils at later stages of tumor development inhibited tumor growth, possibly due to their central location in the tumor. Our work adds another important layer to the understanding of neutrophils in cancer by further characterizing the changes in TAN during time. Additional research on the functional role of TAN and differences between subsets of TAN is currently underway.     

Modulation of polyglutamine inclusion formation by the Hsp70 chaperone machine

Components of the Hsp70 chaperone machine have been implied in protection against polyglutamine (poly-Q) pathologies. Yet, little is known about specific mechanisms and the rate-limiting components that account for this protective effect. Here, we examined the effects of an Hsp70 chaperone family member (HspA1A) and its cofactors Hsp40 (DnaJB1), Bag-1 and CHIP on poly-Q protein inclusion formation and SDS-insolubilization. Overexpression of HspA1A alone did not suppress inclusion formation, while overexpression of DnaJB1 reduced poly-Q inclusion formation and insolubilization. The reducing effect of DnaJB1 on inclusion formation was enhanced by coexpressing HspA1A, and was dependent on the interaction of DnaJB1 with Hsp70/Hsc70 chaperones. Additionally, two factors connecting Hsp70 activity with protein degradation by the ubiquitin-proteasome system Bag-1 and CHIP slightly decreased the levels of soluble poly-Q protein, but the amount of aggregated protein and fraction of cells with inclusions remained unaltered. Our data suggest that the HspA1A chaperone machine can modulate poly-Q inclusion formation depending on the ratio of its components and that DnaJB1 is the rate-limiting step.     

MicroRNA-574-5p Was Pivotal for TLR9 Signaling Enhanced Tumor Progression via Down-Regulating Checkpoint Suppressor 1 in Human Lung Cancer

Accumulating data suggested that functional expression of Toll-like receptors (TLRs) in tumor cells was involved in tumor progression. Our previous study demonstrated that TLR9 signaling could enhance the tumor progression of human lung cancer cells in vitro and in vivo. We further showed that miR-574-5p was the mostly up-regulated miRNA in human lung cancer cells under TLR9 signaling by miRNA array analysis. Here we characterized the potential role of miRNA-574-5p in enhanced tumor progression induced by TLR9 signaling in human lung cancer. We confirmed that TLR9 signaling effectively elevated the expression of miR-574-5p in human lung cancer cells. Notably, we found that down-regulation of miRNA-574-5p using miR-574-5p inhibitor in vitro or miR-574-5p sponge in vivo significantly abrogated the enhanced tumor progression induced by TLR9 signaling. Further studies showed that miR-574-5p was an important player associated with enhanced tumor progression of human lung cancer cells. Notably, we identified checkpoint suppressor 1 (Ches1) as the dominant direct target for miRNA-574-5p to confer the TLR9 signaling enhanced tumor progression. We revealed that over-expression of Ches1 significantly inhibited the cell cycle entry of human lung cancer cells. Finally, we revealed that the expression of miR-574-5p was positively correlated with TLR9 and reversely correlated with Ches1 in lung cancer patients. Our findings not only facilitated the further understanding of the crosstalk between miRNAs and TLRs in tumor biology, but also provided novel potential candidates for treatment of cancer.     

Ultrastructural stability under high temperature or intensive light stress conferred by a small heat shock protein in cyanobacteria

The role and sub-cellular localization of the small heat shock protein HspA under stress conditions was investigated comparing the cyanobacterium Synechococcus strain ECT16-1, which constitutively expresses HspA, with the reference strain ECT. The ultrastructure of ECT cells under elevated temperature or intensive light stress exhibited severe damage including aggregation of cytosol and disordered thylakoid membranes, but in ECT16-1 cells these ultrastructural changes were much less conspicuous. Immunocytochemical studies showed that the main localization of HspA in the ECT16-1 cells shifted from the thylakoid area to the cytoplasm, then back to thylakoid area during the heat stress. Expression of HspA stabilized the morphology of nucleoids. The results are discussed, in particular with respect to the unique property of HspA to associate with thylakoid membranes.     

IL-8 reduced tumorigenicity of human ovarian cancer in vivo due to neutrophil infiltration

Paclitaxel is a frontline therapy for ovarian cancer. Our laboratory has shown that paclitaxel induces IL-8, a member of the C-X-C family of chemokines, in subsets of human ovarian cancer cells. However, the critical issue concerns the biological significance of this chemokine in human ovarian cancer. To study the influence of IL-8 on tumor growth, human ovarian cancer cell lines were transfected with an expression vector for human IL-8 and tested for their ability to form tumors in nude mice. IL-8 expression by the transfected cells did not alter their growth properties in vitro. In contrast, tumor growth in vivo was significantly attenuated in animals receiving IL-8-expressing cells when compared with mice injected with control cells. As additional evidence that IL-8 is a crucial factor in tumor growth, it was noted that ovarian cell lines in which constitutive IL-8 expression is elevated did not form tumors. Injection of neutralizing Ab to IL-8 reverted the phenotype and caused tumor growth in vivo. Examination of tissue from the inoculation site revealed a dramatically elevated cellularity, containing neutrophils and macrophages, in mice receiving IL-8-expressing tumor cells. These results suggest that IL-8 production by human ovarian tumor cells can play a role in reducing the rate of tumor growth; this effect may be mediated by the increased targeting of neutrophil and other mononuclear cells to the tumor injection site. These studies indicate a role for IL-8 in ovarian cancer control and suggest that chemotherapy-induced IL-8 may have a positive role in controlling tumor growth.     

Role of neutrophil extracellular traps in regulation of lung cancer invasion and metastasis: Structural insights from a computational model

Lung cancer is one of the leading causes of cancer-related deaths worldwide and is characterized by hijacking immune system for active growth and aggressive metastasis. Neutrophils, which in their original form should establish immune activities to the tumor as a first line of defense, are undermined by tumor cells to promote tumor invasion in several ways. In this study, we investigate the mutual interactions between the tumor cells and the neutrophils that facilitate tumor invasion by developing a mathematical model that involves taxis-reaction-diffusion equations for the critical components in the interaction. These include the densities of tumor and neutrophils, and the concentrations of signaling molecules and structure such as neutrophil extracellular traps (NETs). We apply the mathematical model to a Boyden invasion assay used in the experiments to demonstrate that the tumor-associated neutrophils can enhance tumor cell invasion by secreting the neutrophil elastase. We show that the model can both reproduce the major experimental observation on NET-mediated cancer invasion and make several important predictions to guide future experiments with the goal of the development of new anti-tumor strategies. Moreover, using this model, we investigate the fundamental mechanism of NET-mediated invasion of cancer cells and the impact of internal and external heterogeneity on the migration patterning of tumour cells and their response to different treatment schedules.     

Prostate cancer cells modulate the differentiation of THP‐1 cells in response to etoposide and TLR agonists treatments

The aim of this study was to determine the polarization of macrophages in the tumor microenvironment, as well as the effect of soluble factors secreted from these polarized macrophages on etoposide‐induced cancer cell apoptosis. We investigated the effect of soluble factors secreted from the supernatant of PC3 cells treated with TLR4 and TLR8 agonists, and etoposide on macrophage polarization at the protein level through flow cytometry and enzyme‐linked immunosorbent assay. We further explored the cell cycle distribution and phagocytic activity of THP‐1 cells by flow cytometry. To imitate the relationship between cancer cells and tumor‐associated macrophages (TAMs), we cocultured macrophages with etoposide‐treated PC3 cells. After the incubation, the apoptosis in cancer cells was assessed through FACS analysis and by annexin V and PI staining. Our results demonstrate that protein expression of M1 and M2 markers confirmed the upregulation of M1 markers upon etoposide treatment, and mixed M1/M2 phenotype upon treatment with TLR agonists‐treated PC3 supernatant. In coculture methods, our results demonstrate that the apoptosis of etoposide‐treated cancer cells increases in the presence of M0 macrophages and THP‐1 cells incubated with the supernatant of TLR4 agonists‐treated PC3 cells. These results indicate clear protective effects of M0 macrophages and THP‐1 cells incubated with the supernatant of PC3 cells treated with TLR4 agonists (THP‐1 + SUP + TLR4a) on etoposide‐induced cancer cell apoptosis.     

Dynamic analysis of modification of peripheral neutrophils functional activity and its regulation during tumor growth in vivo

Polymorphonuclear granulocytes (neutrophils) release the reactive oxygen species (ROS) for destruction of pathogens, providing quicker of an organism from infections and own defective of transformed cells. Reactive oxygen species are also potential carcinogens because they facilitate mutagenesis, tumor promotion and progression. Balance between these opposite influences is supported by coordinated interrelations in intracellular signaling systems. Tumor growth influence on the NADPH oxidase in peripheral innate immune cells is unclear. A solid cancer model was developed after an intramuscular injection of Ehrlich carcinoma cells into hind leg of NMRI strain mice. Intensity of the respiratory burst was estimated by luminol-dependent chemiluminescence technique. Transformation of inflammatory reaction was revealed during tumor growth: greater amounts of neutrophils were recruited into peritoneal cavity; sizes of the cells, their nuclei and granules were enlarged; the ratio of different cell types in peritoneal exudation was changed. The study revealed that tumor progression was accompanied by significant changes in functional activity of neutrophils. Dynamic increase in spontaneous level of ROS production and concentration-dependent change of intensity of the respiratory burst induced with chemotactic peptide N-formyl-Met-Leu-Phe (fMLF) was revealed in peripheral neutrophils under tumor growth conditions. It was found that effects of inhibitors of tyrosine protein kinases, protein kinase C, mitogen-activated protein kinase p38MAPK (p38MAPK) and phosphatidylinositol 3-kinase (PI3K) were altered in neutrophils from tumor-bearing mice in comparison with the cells of control mice. This indicates a change in the role of the enzymes in regulation of the neutrophil respiratory burst. Data obtained show that p38MAPK and PI3K entangle up- and down-regulation of NADPH oxidase in peripheral neutrophils during tumor growth.     

Neutrophil-Derived Proteases in the Microenvironment of Pancreatic Cancer -Active Players in Tumor Progression

A hallmark of pancreatic ductal adenocarcinoma (PDAC) is the fibro-inflammatory microenvironment, consisting of activated pancreatic stellate cells, extracellular matrix proteins, and a variety of inflammatory cells, such as T cells, macrophages, or neutrophils. Tumor-infiltrating immune cells, which are found in nearly all cancers, including PDAC, often fail to eliminate the tumor, but conversely can promote its progression by altering the tumor microenvironment. Pancreatic cancer cells are able to attract polymorphonuclear neutrophils (PMN) via tumor secreted chemokines and in human PDAC, PMN infiltrates can be observed in the vicinity of tumor cells and in the desmoplastic tumor stroma, which correlate with undifferentiated tumor growth and poor prognosis. The behavior of tumor-infiltrating neutrophils in the tumor micromilieu is not yet understood at a mechanistic level. It has been shown that PMN have the potential to kill tumor cells, either directly or by antibody-dependent cell-mediated cytotoxicity, but on the other side various adverse effects of PMN, such as promotion of aggressive tumor growth with epithelial-to-mesenchymal transition and increased metastatic potential, have been described. Recent therapeutic approaches for PDAC focus not only the tumor cell itself, but also elements of the tumor microenvironment. Therefore, the role of PMN and their derived products (e.g. cytokines, proteases) as a new vein for a therapeutic target should be critically evaluated in this context. This review summarizes the current understanding of the interplay between proteases of tumor-infiltrating neutrophils and pancreatic tumor cells and elements of the desmoplastic stroma.     

Oxidative burst and anticancer activities of rat neutrophils

It is assumed that oxidative damage caused by reactive oxygen species (ROS) from activated neutrophil granulocytes may contribute to pathology of tumors. ROS are crucial in neutrophil-mediated tumor cell lysis. The present study is focused on the oxidative burst and antitumorous activities of neutrophils when challenged with Walker carcinoma W256. Survival and tumor growth dynamics were monitored in vivo, while tumor cell proliferation when mixed with neutrophils was studied in vitro together with the generation/release of neutrophil respiratory burst products, primarily 1O2. Neutrophils were collected upon Sephadex injection. The survival of Sephadex injected animals was slightly improved, while their tumors grew less than in controls. The presence of tumor cells in vitro activated neutrophils to produce singlet oxygen similar to phorbol ester. Neutrophils from Sephadex-bearing animals diminished tumor cell proliferation in vitro (measured by 3H-TdR incorporation), while neutrophils from Sephadex and the tumor-bearing animals did not show such activity in vitro. Our results confirm that in the case of rapidly growing tumors such as murine W256 carcinoma neutrophils have antitumorous effects in the early phase of tumor development.     

Glutathione-dependent regulation of platelet aggregation with neutrophils and tumor cells

It is shown that in the presence of reduced glutathione at low concentrations (1-5 microM) the extent of platelet aggregation with neutrophils increases and the lag period of platelet aggregation induced by tumor cells decreases. At the same time in the presence of reduced glutathione at high concentration (3 mM) the extent of platelet aggregation with neutrophils decreases, and the lag period of platelet aggregation induced by tumor cells increases. It is established that glutathione-dependent regulation of the intercellular contact formation between platelets and neutrophils depends on the ratio of glutathione oxidized and reduced forms: at fixed total glutathione concentration of 5 microM, increase of glutathione redox potential from -175 mV to 0 mV led to reduction in platelet aggregation with neutrophils. Thus, it is shown for the first time, that GSH has priming effect on the platelet aggregation with neutrophils and tumor cells, which may contribute to the regulation of inflammatory diseases and cancer.     

Tumor-associated neutrophils and neutrophil-targeted cancer therapies

Neutrophils are the frontline cells in response to microbial infections and are involved in a range of inflammatory disorders in the body. In recent years, neutrophils have gained considerable attention in their involvement of complex roles in tumor development and progression. Tumor-associated neutrophils (TANs) that accumulate in local region could be triggered by external stimuli from tumor microenvironment (TME) and switch between anti- and pro-tumor phenotypes. The anti-tumor neutrophils kill tumor cells through direct cytotoxic effects as well as indirect effects by activating adaptive immune responses. In contrast, the pro-tumor phenotype of neutrophils might be associated with cell proliferation, angiogenesis, and immunosuppression in TME. More recently, neutrophils have been proposed as a potential target in cancer therapy for their ability to diminish the pro-tumor pathways, such as by immune checkpoint blockade. This review discusses the complex roles of neutrophils in TME and highlights the strategies in neutrophil targeting in cancer treatment with a particular focus on the progresses of ongoing clinical trials involving neutrophil-targeted therapies.     

Nickel binding and immunological properties of the C-terminal domain of the Helicobacter pylori GroES homologue (HspA)

Helicobacter pylori synthesizes a heat-shock protein of the GroES class. The gene encoding this protein (heat-shock protein A, HspA) was recently cloned and it was shown to be unique in structure. H. pylori HspA consists of two domains: the N-terminal domain (domain A) homologous with other GroES proteins, and a C-terminal domain (domain B) corresponding to 27 additional residues resembling a metal-binding domain. Various recombinant proteins consisting of the entire HspA polypeptide, the A domain, or the B domain were produced independently as proteins fused to maltose-binding protein (MBP). Comparison of the divalent cation binding properties of the various MBP and MBP-fused proteins allowed us to conclude that HspA binds nickel ions by means of its C-terminal domain. HspA exhibited a high and specific affinity for nickel ions in comparison with its affinity for other divalent cations (copper, zinc, cobalt). Equilibrium dialysis experiments revealed that MBP–HspA binds nickel ions with an apparent dissociation constant (K_d) of 1.8 μM and a stoichiometry of 1.9 ions per molecule. The analysis of the deduced HspA amino acid sequences encoded by 35 independent clinical isolates demonstrated the existence of two molecular variants of HspA, i.e. a major and a minor variant present in 89% and 11% of strains, respectively. The two variants differed from each other by the simultaneous substitution of seven amino acids within the B domain, whilst the A domain was highly conserved amongst all the HspA proteins (99–100% identity). On the basis of serological studies, the highly conserved A domain of HspA was found to be the immunodominant domain. Functional complementation experiments were performed to test the properties of the two HspA variants. When co-expressed together with the H. pylori urease gene cluster in Escherichia coli cells, the two HspA variant-encoding genes led to a fourfold increase in urease activity, demonstrating that HspA in H. pylori has a specialized function with regard to the nickel metalloenzyme urease.     

TLR1/TLR2 agonist induces tumor regression by reciprocal modulation of effector and regulatory T cells

Using TLR agonists in cancer treatment can have either beneficial or detrimental effects. Therefore, it is important to determine their effect on the tumor growth and understand the underlying mechanisms in animal tumor models. In this study, we report a general immunotherapeutic activity of a synthetic bacterial lipoprotein (BLP), a TLR1/TLR2 agonist, on established lung carcinoma, leukemia, and melanoma in mice. Systemic treatment of 3LL tumor-bearing mice with BLP, but not LPS, led to a dose-dependent tumor regression and a long-lasting protective response against tumor rechallenge. The BLP-mediated tumor remission was neither mediated by a direct tumoricidal activity nor by innate immune cells, because it lacked therapeutic effect in immunodeficient SCID mice. Instead, BLP treatment reduced the suppressive function of Foxp3(+) regulatory T cells (Tregs) and enhanced the cytotoxicity of tumor-specific CTL in vitro and in vivo. Furthermore, adoptive cotransfer of BLP-pretreated but not untreated CTL and Tregs from wild-type but not from TLR2(-/-) mice was sufficient to restore antitumor immunity in SCID mice by reciprocally modulating Treg and CTL function. These results demonstrate that the TLR1/TLR2 agonist BLP may have a general tumor therapeutic property involving reciprocal downregulation of Treg and upregulation of CTL function. This property may play an important role in the development of novel antitumor strategies.     

Glutathione-dependent regulation of platelet aggregation with neutrophils and tumor cells

It is shown that in the presence of reduced glutathione at low concentrations (1–5 μM) the extent of platelet aggregation with neutrophils increases and the lag period of platelet aggregation induced by tumor cells decreases. At the same time in the presence of reduced glutathione at high concentration (3 mM) the extent of platelet aggregation with neutrophils decreases, and the lag period of platelet aggregation induced by tumor cells increases. It is established that glutathione-dependent regulation of the intercellular contact formation between platelets and neutrophils depends on the ratio of glutathione oxidized and reduced forms: at fixed total glutathione concentration of 5 μM, increase of glutathione redox potential from −175 mV to 0 mV led to reduction in platelet aggregation with neutrophils. Thus, it is shown for the first time, that GSH has priming effect on the platelet aggregation with neutrophils and tumor cells, which may contribute to the regulation of inflammatory diseases and cancer.     

Beta-glucuronidase activity in neutrophils of patients with malignancies.

The human neutrophils induce cytotoxic effects on mammalian tumor cells. Hence it may be expected that intracellular enzymatic deficiencies of neutrophils may represent another cancer risk factor. In 205 patients with various malignancies the neutrophil beta-glucuronidase activity has been determined using a semiquantitative cytochemical method. A statistically significant deficiency of this enzyme in neutrophils has been observed in patients with precancerous states of the larynx, cancer of the larynx after radiotherapy and patients with cancer of large intestine. Patients with cancer of the lung and cancer of the stomach showed no changes with that regard whereas those with cancer of the breast demonstrated an increased enzyme activity.     

Inhibition of lung cancer cell proliferation mediated by human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are mostly studied for their potential clinical use. Recently, much attention in the field of cancer research has been paid to hMSCs. In this study, we investigated the influence of hMSCs on the proliferation of lung cancer cell lines SK-MES-1 and A549 in vitro and in vivo by using a co-culture system and the hMSCs-conditioned medium. Our results demonstrated that hMSCs could inhibit the proliferation of SK-MES-1 and A549 cells, and induce the apoptosis of tumor cells in vitro via some soluble factors. Animal study showed that these soluble factors from hMSCs could suppress tumorigenesis and tumor angiogenesis by treating preliminarily tumor cells with the hMSCs-conditioned medium. The downregulated expression of vascular endothelial growth factor in tumor cells might be the mechanism of interference in tumor angiogenesis, which was verified by western blot analysis and immunohistochemistry assay. Taken together, our results suggested that the hMSCs could inhibit tumor cell growth by secreting some soluble factors.