Snail transcription factors - Characteristics, regulation and molecular targets relevant in vital cellular activities of ovarian cancer cells.

Snail transcription factors play essential roles in embryonic development and participate in many physiological processes. However, these genes have been implicated in the development and progression of various types of cancer. In epithelial ovarian cancer, high expression of these transcription factors is usually associated with the acquisition of a more aggressive phenotype and thus, considered to be a poor prognostic factor. Numerous molecular signals create a complex network of signaling pathways regulating the expression and stability of Snails, which in turn control genes involved in vital cellular functions of ovarian cancer cells, such as invasion, survival, proliferation and chemoresistance.

Snail transcription factors as key regulators of chemoresistance, stemness and metastasis of ovarian cancer cells.

Ovarian cancer is one of the deadliest gynecological malignancies among women. The reason for this outcome is the frequent acquisition of cancer cell resistance to platinum-based drugs and unresponsiveness to standard therapy. It has been increasingly recognized that the ability of ovarian cancer cells to adopt more aggressive behavior (mainly through the epithelial-to-mesenchymal transition, EMT), as well as dedifferentiation into cancer stem cells, significantly affects drug resistance acquisition. Transcription factors in the Snail family have been implicated in ovarian cancer chemoresistance and metastasis. In this article, we summarize published data that reveal Snail proteins not only as key inducers of the EMT in ovarian cancer but also as crucial links between the acquisition of ovarian cancer stem properties and spheroid formation. These Snail-related characteristics significantly affect the ovarian cancer cell response to treatment and are related to the acquisition of chemoresistance.

Neuromedin U secreted by colorectal cancer cells promotes a tumour-supporting microenvironment.

BACKGROUND: Neuromedin U (NMU) was identified as one of the hub genes closely related to colorectal cancer (CRC) progression and was recently shown to be a motility inducer in CRC cells. Its autocrine signalling through specific receptors increases cancer cell migration and invasiveness. Because of insufficient knowledge concerning NMU accessibility and action in the tumour microenvironment, its role in CRC remains poorly understood and its potential as a therapeutic target is still difficult to define. METHODS: NMU expression in CRC tissue was detected by IHC. Data from The Cancer Genome Atlas were used to analyse gene expression in CRC. mRNA and protein expression was detected by real-time PCR, immunoblotting or immunofluorescence staining and analysed using confocal microscopy or flow cytometry. Proteome Profiler was used to detect changes in the profiles of cytokines released by cells constituting tumour microenvironment after NMU treatment. NMU receptor activity was monitored by detecting ERK1/2 activation. Transwell cell migration, wound healing assay and microtube formation assay were used to evaluate the effects of NMU on the migration of cancer cells, human macrophages and endothelial cells. RESULTS: Our current study showed increased NMU levels in human CRC when compared to normal adjacent tissue. We detected a correlation between high NMUR1 expression and shorter overall survival of patients with CRC. We identified NMUR1 expression on macrophages, endothelial cells, platelets, and NMUR1 presence in platelet microparticles. We confirmed ERK1/2 activation by treatment of macrophages and endothelial cells with NMU, which induced pro-metastatic phenotypes of analysed cells and changed their secretome. Finally, we showed that NMU-stimulated macrophages increased the migratory potential of CRC cells. CONCLUSIONS: We propose that NMU is involved in the modulation and promotion of the pro-metastatic tumour microenvironment in CRC through the activation of cancer cells and other tumour niche cells, macrophages and endothelial cells. Video abstract.

E-Cadherin Expression in Relation to Clinicopathological Parameters and Survival of Patients with Epithelial Ovarian Cancer.

It is generally accepted that loss/reduction of E-cadherin expression on tumor cells promotes their migration, invasiveness, and metastasis. It is also an indicator of cancer cells' aggressiveness. The aim of this study was to assess how the expression of E-cadherin varies in primary ovarian cancer tissue in regard to overall survival of patients; FIGO stage; grade; histopathological type of tumor; and potential factors discriminating malignant and nonmalignant ovarian tumors. Our analysis was based on literature research (1 January 2000-8 November 2021) conducted according to the PRISMA guidelines. Most studies support the assumption that loss/reduced expression of E-cadherin results in shorter overall survival of EOC patients. Moreover, most research has shown that there is a correlation between the low level of E-cadherin and the advancement stage of disease, especially in high-grade serous ovarian carcinoma type. However, E-cadherin expression seems to not be helpful to distinguish malignant and nonmalignant tumors. In conclusion, reduced E-cadherin expression in primary ovarian cancer tissue may indicate a less favorable disease outcome and is associated with high advancement of the disease.

Tumor-Associated Macrophages: Reasons to Be Cheerful, Reasons to Be Fearful.

Tumor microenvironment (TME) is a complex and constantly evolving entity that consists not only of cancer cells, but also of resident host cells and immune-infiltrating cells, among which macrophages are significant components, due to their diversity of functions through which they can influence the immune response against tumor cells. Macrophages present in tumor environment are termed as tumor-associated macrophages (TAMs). They are strongly plastic cells, and depending on the TME stimuli (i.e., cytokines, chemokines), TAMs polarize to antitumoral (M1-like TAMs) or protumoral (M2-like TAMs) phenotype. Both types of TAMs differ in the surface receptors' expression, activation of intracellular signaling pathways, and ability of production and various metabolites release. At the early stage of tumor formation, TAMs are M1-like phenotype, and they are able to eliminate tumor cells, i.e., by reactive oxygen species formation or by presentation of cancer antigens to other effector immune cells. However, during tumor progression, TAMs M2-like phenotype is dominating. They mainly contribute to angiogenesis, stromal remodeling, enhancement of tumor cells migration and invasion, and immunosuppression. This wide variety of TAMs' functions makes them an excellent subject for use in developing antitumor therapies which mainly is based on three strategies: TAMs' elimination, reprograming, or recruitment inhibition.

The implication of IL-6 in the invasiveness and chemoresistance of ovarian cancer cells. Systematic review of its potential role as a biomarker in ovarian cancer patients.

Interleukin 6 (IL-6) is a pleiotropic cytokine that is strongly implicated in the development and progression of ovarian cancer. The most recognized actions of IL-6 in ovarian cancer (OC) cells are the induction of cell proliferation and inhibition of cell apoptosis. Equally important is its ability to enhance the migratory and invasive potential of OC cells. Moreover, the increased expression and secretion of this cytokine positively correlates with OC cell chemoresistance. Elevated concentrations of IL-6 are observed in the serum and ascites of ovarian cancer patients. Thus, its level is discussed in the literature as a potential biomarker that can help to discriminate malignant and nonmalignant ovarian tumors and allow for the prediction of the chemotherapy response. The importance of IL-6 in ovarian cancer is proved by the fact that this cytokine is a potential target to anti-cancer therapy. This review is divided into two parts. The first summarizes the general biological activity of IL-6, and overviews its impact on OC cells, as well as discusses the current proposition of IL-6 inclusion in combination of anti-OC therapy. The second part is a systematic review of IL-6 as a possible biomarker in ovarian cancer patients.

Impact of Selected Signaling Proteins on SNAIL 1 and SNAIL 2 Expression in Ovarian Cancer Cell Lines in Relation to Cells' Cisplatin Resistance and EMT Markers Level.

It has been increasingly recognized that SNAIL1 and SNAIL2, as major EMT-inducers, might also be involved in drug resistance of cancer cells. We sought to determine a relation between SNAIL1/2, E-cadherin and N-cadherin expression, as well as ovarian cancer cells' resistance to cisplatin and EMT markers' level. Thus, four ovarian cancer cell lines, were used: A2780, A2780cis, SK-OV-3 and OVCAR-3. We assessed the impact of ERK1/2, AKT and STAT3 proteins (chosen by the profiling activity of over 40 signaling proteins) on SNAIL1/2 expression, along with E-cadherin and N-cadherin levels. We showed that expression of SNAIL1 and N-cadherin are the highest in cisplatin-resistant A2780cis and SK-OV-3 cells, while high SNAIL2 and E-cadherin levels were observed in cisplatin-sensitive A2780 cells. The highest E-cadherin level was noticed in OVCAR-3 cells. SNAIL1/2 expression was dependent on ERK1/2 activity in cisplatin-resistant and potentially invasive SK-OV-3 and OVCAR-3 cells. STAT-3 regulates expression of SNAIL1/2 and leads to the so-called "cadherin switch" in cancer cells, independently of their chemoresistance. In conclusion, SNAIL1, but not SNAIL2, seems to be involved in ovarian cancer cells' cisplatin resistance. STAT3 is a universal factor determining the expression of SNAIL1/2 in ovarian cancer cells regardless of their chemoresitance or invasive capabilities.

Calreticulin-Multifunctional Chaperone in Immunogenic Cell Death: Potential Significance as a Prognostic Biomarker in Ovarian Cancer Patients.

Immunogenic cell death (ICD) is a type of death, which has the hallmarks of necroptosis and apoptosis, and is best characterized in malignant diseases. Chemotherapeutics, radiotherapy and photodynamic therapy induce intracellular stress response pathways in tumor cells, leading to a secretion of various factors belonging to a family of damage-associated molecular patterns molecules, capable of inducing the adaptive immune response. One of them is calreticulin (CRT), an endoplasmic reticulum-associated chaperone. Its presence on the surface of dying tumor cells serves as an "eat me" signal for antigen presenting cells (APC). Engulfment of tumor cells by APCs results in the presentation of tumor's antigens to cytotoxic T-cells and production of cytokines/chemokines, which activate immune cells responsible for tumor cells killing. Thus, the development of ICD and the expression of CRT can help standard therapy to eradicate tumor cells. Here, we review the physiological functions of CRT and its involvement in the ICD appearance in malignant disease. Moreover, we also focus on the ability of various anti-cancer drugs to induce expression of surface CRT on ovarian cancer cells. The second aim of this work is to discuss and summarize the prognostic/predictive value of CRT in ovarian cancer patients.

Determination of in vitro and in vivo immune response to recombinant cholesterol oxidase from Mycobacterium tuberculosis.

Cholesterol oxidase (ChoD) is an enzyme that is involved but is dispensable in the process of cholesterol degradation by Mycobacterium tuberculosis (Mtb). Interestingly, ChoD is a virulence factor of Mtb, and it strongly modulates the function of human macrophages in vitro, allowing the intracellular survival of bacteria. Here, we determined the immunogenic activity of recombinant ChoD from Mtb in a mouse model. We found that peritoneal exudate cells obtained from mice injected i.p. with ChoD but not those from mice injected with PBS responded in vitro with highly spontaneous, as well as phorbol 12-myristate 13-acetate (PMA)-stimulated, production of reactive oxygen species (ROS). However, ChoD significantly reduced the ROS response to PMA in re-stimulated cells in vitro. The cytokine secretion pattern in mice immunized s.c. with ChoD emulsified with incomplete Freund's adjuvant (IFA) showed evidence of Th2-induced or proinflammatory immune responses. The main cytokines detected in sera were interleukin (IL) 6 and 5, tumour necrosis factor α (TNF-α) and monocyte chemoattractant protein 1, while IL-2 and IL-12 as well as interferon γ were undetectable. Similarly, ChoD protein alone activated THP-1-derived macrophages to release proinflammatory IL-6, IL-8 and TNF-α, in vitro. Moreover, a statistically significant predominance of the IgG1 isotype over that of IgG2a in the sera of mice immunized with ChoD/IFA was observed. In conclusion, we demonstrated here that ChoD of Mtb is an active protein, which is able to induce the immune response both in vivo and in vitro.

MiRNA-103/107 in Primary High-Grade Serous Ovarian Cancer and Its Clinical Significance.

High levels of miRNA-103/107 are associated with poor outcomes in the case of breast cancer patients. MiRNA-103/107-DICER axis may be one of the key regulators of cancer aggressiveness. MiRNA-103/107 expression levels have never been related to patients' clinicopathological data in epithelial ovarian cancer. We aimed to assess miRNA-103/107 expression levels in high grade serous ovarian cancer tissues. Expression levels of both miRNAs were related to the clinicopathological features and survival. We also evaluated expression levels of miRNA-103/107 and DICER in selected ovarian cancer cell lines (A2780, A2780cis, SK-OV-3, OVCAR3). We assessed the relative expression of miRNA-103/107 (quantitative reverse transcription-polymerase chain reaction) in fifty archival formalin-fixed paraffin-embedded tissue samples of primary high grade serous ovarian cancer. Then, miRNA-103/107 and DICER expression levels were evaluated in selected ovarian cancer cell lines. Additionally, DICER, N-/E-cadherin protein levels were assessed with the use of western blot. We identified miRNA-107 up-regulation in ovarian cancer in comparison to healthy tissues (p = 0.0005). In the case of miRNA-103, we did not observe statistically significant differences between cancerous and healthy tissues (p = 0.07). We did not find any correlations between miRNA-103/107 expression levels and clinicopathological features. Kaplan-Meier survival (disease-free and overall survival) analysis revealed that both miRNAs could not be considered as prognostic factors. SK-OV-3 cancer cell lines were characterized by high expression of miRNA-103/107, relatively low expression of DICER (western-blot), and relatively high N-cadherin levels in comparison to other ovarian cancer cell lines. Clinical and prognostic significance of miRNA-103/107 was not confirmed in our study.

Mycobacterium tuberculosis Requires Cholesterol Oxidase to Disrupt TLR2 Signalling in Human Macrophages.

This study tested the hypothesis that Mycobacterium tuberculosis (Mtb) uses a cholesterol oxidase enzyme (ChoD) to suppress a toll-like receptor type 2- (TLR2-) dependent signalling pathway to modulate macrophages' immune response. We investigated the impact of Mtb possessing or lacking ChoD as well as TBChoD recombinant protein obtained from Mtb on the expression and activation of two key intracellular proteins involved in TLR2 signalling in human macrophages. Finally, the involvement of TLR2-related signalling proteins in an inflammatory/immunosuppressive response of macrophages to Mtb was evaluated. We demonstrate that wild-type Mtb but not the ∆choD mutant decreased the cytosolic IRAK4 and TRAF6 protein levels while strongly enhancing IRAK4 and TRAF6 mRNA levels in macrophages. Our data show that the TLR2 present on the surface of macrophages are involved in disturbing the signalling pathway by wild-type Mtb. Moreover, recombinant TBChoD effectively decreased the cytosolic level of TRAF6 and lowered the phosphorylation of IRAK4, which strongly confirm an involvement of cholesterol oxidase in affecting the TLR2-related pathway by Mtb. Wild-type Mtb induced an immunosuppressive response of macrophages in an IRAK4- and TRAF6-dependent manner as measured by interleukin 10 production. In conclusion, ChoD is a virulence factor that enables Mtb to disturb the TLR2-related signalling pathway in macrophages and modulate their response.

IRAK1 and IRAK4 signaling proteins are dispensable in the response of human neutrophils to Mycobacterium tuberculosis infection.

The involvement of neutrophils in the host response to Mycobacterium tuberculosis (Mtb) infection is not as well recognized as the involvement of macrophages and dendritic cells. Thus, this study gives more insight on the impact of the virulent Mtb H37Rv strain on proapoptotic and proinflammatory functions of human neutrophils in vitro. We found that neutrophils are not able to kill Mtb during the infection process, probably due to the lack of reactive oxygen species and nitric oxide production in response to bacteria. However, infected neutrophils effectively released cytokines, chemoattractant interleukin (IL) 8 and proinflammatory IL-1ß. Moreover, Mtb enhanced the early apoptosis of neutrophils at 2 h postinfection. Additionally, this proapoptotic and proinflammatory response of neutrophils to Mtb infection occurred in an IRAK1- and IRAK4-independent manner. We also found that Mtb did not affect the surface expression of Toll-like receptor (TLR) 2 and slightly enhanced the surface expression of TLR4, but did not influence mRNA levels of both TLRs during the infection process. In conclusion, we show that the inhibition of signaling proteins activated by MyD88-dependent pathway did not participate in the biological activity of neutrophils against Mtb.

The Potential Role of iNOS in Ovarian Cancer Progression and Chemoresistance.

Inducible nitric oxide synthase (iNOS), the enzyme responsible for nitric oxide (NO) production, is not present in most cells under normal conditions. The expression of its mRNA, as well as its protein synthesis and full enzymatic activity, undergoes multilevel regulation including transcriptional and posttranscriptional mechanisms, the availability of iNOS substrate and cofactors and oxygen tension. However, in various malignant diseases, such as ovarian cancer, the intracellular mechanisms controlling iNOS are dysregulated, resulting in the permanent induction of iNOS expression and activation. The present review summarizes the multistaged processes occurring in normal cells that promote NO synthesis and focuses on factors regulating iNOS expression in ovarian cancer. The possible involvement of iNOS in the chemoresistance of ovarian cancer and its potential as a prognostic/predictive factor in the course of disease development are also reviewed. According to the available yet limited data, it is difficult to draw unequivocal conclusions on the pros and cons of iNOS in ovarian cancer. Most clinical data support the hypothesis that high levels of iNOS expression in ovarian tumors are associated with a greater risk of disease relapse and patient death. However, in vitro studies with various ovarian cancer cell lines indicate a correlation between a high level of iNOS expression and sensitivity to cisplatin.

Cisplatin-induced ERK1/2 activity promotes G1 to S phase progression which leads to chemoresistance of ovarian cancer cells.

The link between ERK1/2 activity and cisplatin cytotoxicity, in association with the cell cycle, in ovarian cancer cell lines resistant (A2780cis; SK-OV-3) and sensitive (A2780) to cisplatin was determined. We observed that cisplatin, at a low concentration enhanced the activation of ERK1/2 in A2780 cells and increased their accumulation in the S phase, resulting in low cytotoxicity. A high concentration of drug induced dephosphorylation and degradation of ERK1/2 and was extremely toxic, accumulating most of to these cells in the sub-G1 phase. The PD98059, pharmacological inhibitor of ERK1/2 activation, increased the cytotoxicity of cisplatin applied at a low concentration to A2780 cells (decreased ERK1/2 activity), causing shift of cell accumulation from the S to G1 phase. Surprisingly, PD98059 enhanced cell viability when a chemotherapeutic was used at high concentration, intensifying phosphorylation level of ERK1/2 and reversing cell cycle arrest in sub-G1 to promote the G1 and S phases. A2780cis cells demonstrated resistance to cisplatin with high ERK1/2 activity and accumulation of cells in the G1 and S phases. PD98059 sensitized resistant cells to drug toxicity during the first 24 hours of treatment, with blocked ERK1/2 phosphorylation and prevented progression from the G1 to S phase. SK-OV-3 resistant cells characterized with extremely high basal phosphorylation of ERK1/2, which wasn't changed after exposure to cisplatin. Administration of PD98059 didn't change the cytotoxicity of cisplatin in these cells. In conclusion, ERK1/2, activated by cisplatin, participates in the cell cycle progression from the G1 to S phase, enhancing cells' survival and drug resistance.

Severe inhibition of lipooligosaccharide synthesis induces TLR2-dependent elimination of Mycobacterium marinum from THP1-derived macrophages.

BACKGROUND: Although mycobacterial glycolipids are among the first-line molecules involved in host-pathogen interactions, their contribution in virulence remains incomplete. Mycobacterium marinum is a waterborne pathogen of fish and other ectotherms, closely related to Mycobacterium tuberculosis. Since it causes tuberculosis-like systemic infection it is widely used as a model organism for studying the pathogenesis of tuberculosis. It is also an occasional opportunistic human pathogen. The M. marinum surface-exposed lipooligosaccharides (LOS) are immunogenic molecules that participate in the early interactions with macrophages and modulate the host immune system. Four major LOS species, designated LOS-I to LOS-IV, have been identified and characterized in M. marinum. Herein, we investigated the interactions between a panel of defined M. marinum LOS mutants that exhibited various degrees of truncation in the LOS structure, and human-derived THP-1 macrophages to address the potential of LOSs to act as pro- or avirulence factors. RESULTS: A moderately truncated LOS structure did not interfere with M. marinum invasion. However, a deeper shortening of the LOS structure was associated with increased entry of M. marinum into host cells and increased elimination of the bacilli by the macrophages. These effects were dependent on Toll-like receptor 2. CONCLUSION: We provide the first evidence that LOSs inhibit the interaction between mycobacterial cell wall ligands and appropriate macrophage pattern recognition receptors, affecting uptake and elimination of the bacteria by host phagocytes.

Ferulic acid but not alpha-lipoic acid effectively protects THP-1-derived macrophages from oxidant and pro-inflammatory response to LPS.

OBJECTIVE: The diet supplementation with antioxidants-rich products is a way to protect people from free radical-induced diseases. In this study, we compare the anti-inflammatory/antioxidant activity of two compounds available as supplements: alpha lipoic acid (ALA) and ferulic acid (FA). MATERIALS AND METHODS: The free radical scavenging capacity of ALA and FA in the cell free system was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Anti-inflammatory activity of both compounds was determined, in vitro, on THP-1 derived macrophages model, both resting (not stimulated) and inflammatory (lipopolysaccharide- or tumor-necrosis factor α-stimulated). RESULTS: We have found that FA exhibits much higher radical scavenging activity than ALA, in cell free system. The functional assays demonstrated that although both ALA and FA limited the reactive oxygen species (ROS) formation in the presence of inflammatory macrophages, the latter acid was significantly more effective. Only FA reduced the release of pro-inflammatory interleukin 1ß and interleukin 6 by lipopolysaccharide-treated macrophages. Neither FA nor ALA affected the viability of macrophages. CONCLUSION: Among those two compounds only FA has significant free radical scavenging activity in cell free system and acts as anti-inflammatory and antioxidant agent on macrophages. It can be assumed that application of FA in a diet can protect the host from the development and/or progression of inflammation.

Secretion of cytokines and heat shock protein (HspA1A) by ovarian cancer cells depending on the tumor type and stage of disease.

Epithelial ovarian cancer is a heterogeneous disease comprising several tumor types that each have multiple histopathological features and different biological behaviors. Recent morphologic and molecular genetic studies have allowed for the categorization of various types of ovarian cancer into two groups: type I and type II. Type I tumors are low-grade and are genetically more stable, while type II tumors are high-grade and genetically unstable. The determination of the type of ovarian cancer may have implications in terms of the appropriate therapeutic strategy because different prognoses and responses to chemotherapeutic agents are observed. Therefore, the current challenge is better recognition of the features of cancer cells, which may result in more individualized therapy. The aim of the current studies was to compare the ability of ovarian cancer cells isolated from tumors, which were classified as type I or type II ovarian cancer, to release pro-inflammatory and immunosuppressive cytokines and heat shock protein (HspA1A). These factors are known to facilitate tumor cell survival, invasion and metastasis. Our studies demonstrated that ovarian cancer cells isolated from patients with type II tumors released high levels of immunosuppressive cytokines (i.e., interleukin 10 and transforming growth factor ß) and HspA1A in vitro. Conversely, ovarian cancer cells obtained from of type I tumors were significantly less active. We did not observe any difference in the ability of the isolated cancer cells to secrete pro-inflammatory cytokines, regardless of the type of ovarian cancer. In this study, we found that cancer cells from patients with type II tumors demonstrated more intense activity in regards to survival and metastasis, which should be considered during therapy.

Evaluation of nitric oxide donors impact on cisplatin resistance in various ovarian cancer cell lines.

Ovarian cancer chemoresistance, both intrinsic and acquired, is the main obstacle in improving the outcome of anticancer therapies. Therefore the development of new treatment strategies, including the use of new compounds that can support the standard therapeutics is required. Among many candidates, nitric oxide (NO) donors, agents with multivalent targeted activities in cancer cells, are worth considering. The aim of this study was evaluation of SPER/NO and DETA/NO ability to enhance cisplatin cytotoxicity against different ovarian cancer cell lines. Obtained data indicate that NO donors action varies between different cancer cell lines and is strongest in low aggressive and cisplatin sensitive cells. While statistically significant, the enhancement of cisplatin cytotoxicity by NO donors is of low magnitude. The rise in the percentage of late apoptotic/necrotic ovarian cancer cells may suggest that NO donors enhancement action might be based on the cellular ATP depletion. Nevertheless, no significant impact of the NO donors, cisplatin or their combination on the expressions of ABCB1, BIRC5 and PTEN genes has been found. Although our data puts the therapeutical potential of NO donors to aid cisplatin action in question it may also point out at the further approach to utilize these compounds in therapies.

Mycobacterium tuberculosis RecA is indispensable for inhibition of the mitogen-activated protein kinase-dependent bactericidal activity of THP-1-derived macrophages in vitro.

Our knowledge about the mechanisms utilized by Mycobacterium tuberculosis to survive inside macrophages is still incomplete. One of the mechanism that protects M. tuberculosis from the host's microbicidal products and allows bacteria to survive involves DNA repair systems such as the homologous recombination (HR) and nonhomologous end-joining (NHEJ) pathways. It is accepted that any pathway that contributes to genome maintenance should be considered as potentially important virulence factor. In these studies, we investigated reactive oxygen species, nitric oxide and tumor necrosis factor-α production by macrophages infected with wild-type M. tuberculosis, with an HR-defective mutant (∆recA), with an NHEJ-defective mutant [∆(ku,ligD)], with a mutant defective for both HR and NHEJ [∆(ku,ligD,recA)], or with appropriate complemented strains. We also assessed the involvement of extracellular signal-regulated kinases (ERKs) 1 and 2 in the response of macrophages to infection with the above-mentioned strains, and ERK1/2 phosphorylation in M. tuberculosis-infected macrophages. We found that mutants lacking RecA induced a greater bactericidal response by macrophages than did the wild-type strain or an NHEJ-defective mutant, and activated ERK1/2 was involved only in the response of macrophages to recA deletion mutants [∆(ku,ligD,recA) and ∆recA]. We also demonstrated that only the triple mutant induced ERK1/2 phosphorylation in phorbol-12-myristate-13-acetate-stimulated macrophages. Moreover, HR-defective mutants induced lower amounts of tumor necrosis factor-α secretion than did the wild-type or ∆(ku,ligD). Our results indicate that RecA contributes to M. tuberculosis virulence, and also suggest that diminished ERK1/2 activation in macrophages infected with M. tuberculosis possessing recA may be an important mechanism by which wild-type mycobacteria escape intracellular killing.

Antitumoral Activity Of Nitric Oxide-Releasing Compounds.

BACKGROUND: Despite significant improvements in the conventional anti-ovarian cancer therapies, tumor cell resistance to various cytostatic drugs remains a relevant problem. Therefore, the new cancer treatment strategies are being developed. Among many agents that have been studied for their potential anti-cancer activity, the most promising are the nitric oxide (NO) donors-syntethic compounds that release NO in vivo and/or in vitro. AIM: We have evaluated the effect of NO donors on the SK-OV-3 and OVCAR-3 ovarian cancer cell lines. We assessed some of the cancer cells' specific features: the uncontrolled proliferation, over-activation of particular signaling proteins, high resistance to therapeutics and elevated expression and secretion of invasiveness/metastatic factors. METHODS: Two members of NONOates family were used: SPER/NO and DETA/NO. Cancer cell lines were cultured with different concentrations of NO donors. The cytotoxic, pro-apoptotic activity of NO donors and their impact on the phosphorylation status of STAT-3 and AKT in cells were determined. The expression of VEGF-A, MMPs and TGF-ß was also evaluated. RESULTS: NO donors inhibited ovarian cancer cells growth making them also more susceptible to the cisplatin cytotoxic activity. Moreover, both NO donors induced apoptosis of cells and decreased activity of signaling proteins (STAT3 and AKT). Similarly, SPER/NO and DETA/NO lowered the secretion of pro-metastatic factors, responsible for cancer cells invasiveness. CONCLUSIONS: The obtained results show that both NO donors demonstrated a wide range of action on both ovarian cancer cell lines. Therefore, they have a high potential of being a supporting compounds in the cancer therapies.