Tissue Distribution, Histopathological and Genotoxic Effects of Magnetite Nanoparticles on Ehrlich Solid Carcinoma.

Nanoparticles can potentially cause adverse effects on cellular and molecular level. The present study aimed to investigate the histopathological changes and DNA damage effects of magnetite nanoparticles (MNPs) on female albino mice model with Ehrlich solid carcinoma (ESC). Magnetite nanoparticles coated with L-ascorbic acid (size ~ 25.0 nm) were synthesized and characterized. Mice were treated with MNPs day by day, intraperitoneally (IP), intramuscularly (IM), or intratumorally (IT). Autopsy samples were taken from the solid tumor, thigh muscle, liver, kidney, lung, spleen, and brain for assessment of iron content, histopathological examination, and genotoxicity using comet assay. The liver, spleen, lung, and heart had significantly higher iron content in groups treated IP. On the other hand, tumor, muscles, and the liver had significantly higher iron content in groups treated IT. MNPs induced a significant DNA damage in IT treated ESC. While a significant DNA damage was detected in the liver of the IP treated group, but no significant DNA damage could be detected in the brain. Histopathological findings in ESC revealed a marked tumor necrosis, 50% in group injected IT but 40% in group injected IP and 20% only in untreated tumors. Other findings include inflammatory cell infiltration, dilatation, and congestion of blood vessels of different organs of treated groups in addition to appearance of metastatic cancer cells in the liver of non-treated tumor group. MNPs could have an antitumor effect but it is recommended to be injected intratumorally to be directed to the tumor tissues and reduce its adverse effects on healthy tissues.

Suppression of macrophages- Induced inflammation via targeting RAS and PAR-4 signaling in breast cancer cell lines.

Tumor associated macrophages (TAMs) have a crucial role in cancer progression, metastasis and drug response. Piroxicam and sulindac sulfide are non-steroidal anti-inflammatory drugs (NSAID) that decrease the incidence and progression of several types of cancer. However, their role in suppressing the interactions between TAMs and cancer cells remain unclear. Herein, we studied the impact of human monocytes conditioned media (CM) on cellular proliferation of ER-dependent MCF-7 and ER-independent MDA-MB-231 cells, and the effects of piroxicam and sulindac sulfide on the expression levels of RAS, COX-2, IL-6, IL-1ß and PAR-4 (qRT-PCR), BCL-2 and BAX (western blot), Caspase-3, VEGF-a and PGE2 (ELISA), MMP-2 and -9 (zymography) in the stimulated cells. Our results showed that CM caused a significant increase in cells survival through significant increase in RAS expression which resulted in upregulation of COX-2, PGE2, BCL-2, IL-6, IL-1ß, VEGF-A and MMP-9 and down regulation of PAR-4. Treatment with one of the NSAIDs used in this study produced a time and concentration dependent growth inhibition of stimulated cells by inhibiting RAS expression. Suppression of RAS was accompanied by downregulation of its downstream signaling of IL-1ß, IL-6, COX-2 and PGE2, activation of apoptotic machinery through upregulation of PAR-4 and caspase-3, as well as, inhibition of BCL-2, VEGF-A, MMP-2 and MMP-9. In conclusion, our data support the role of piroxicam and sulindac sulfide in suppressing inflammation-driven breast cancer progression and identifies promising novel target in RAS and PAR-4 signaling.

Inflammatory breast cancer: Activation of the aryl hydrocarbon receptor and its target CYP1B1 correlates closely with Wnt5a/b-ß-catenin signalling, the stem cell phenotype and disease progression.

The aim of the present study was to evaluate the expression levels of the aryl hydrocarbon receptor (AHR) and its target gene CYP1B1 and to correlate their expression with Wnt5a/b-ß-catenin, the CD44+/CD24(-/low) cancer stem cell (CSC) subset and factors associated with poor prognosis in inflammatory breast cancer (IBC) and non-IBC patients. The methods of analysis used were quantitative real-time PCR, western blotting, immunohistochemistry and flow cytometry. Compared to non-IBC tissues, IBC tissues exhibited the overexpression of AHR and its target gene/protein CYP1B1. AHR and CYP1B1 mRNA levels were associated with the poor clinical prognosis markers tumour grade, lymphovascular invasion, cell proliferation and lymph node metastasis. Furthermore, AHR expression correlated with the expression of Wnt5a/b and ß-catenin signalling molecules, and Wnt5a mRNA expression was downregulated in the SUM149 human IBC cell line and the MDA-MB-231 non-IBC cell line upon inhibition of AHR. AHR gene knockout (CRISPR-Cas9) inhibits CYP1B1 and Wnt5a expression in the IBC cell line. The CD44+/CD24(-/low) subset was significantly correlated with the expression of AHR, CYP1B1, Wnt5a/b and ß-catenin in IBC tissues. The overexpression of AHR and its target CYP1B1 correlated with the expression of Wnt5a/b and ß-catenin, CSCs, and poor clinical prognostic factors of IBC. Thus, targeting AHR and/or its downstream target molecules CYP1B1 and Wnt5a/b may represent a therapeutic approach for IBC.

International Consensus on the Clinical Management of Inflammatory Breast Cancer from the Morgan Welch Inflammatory Breast Cancer Research Program 10th Anniversary Conference.

National and international experts in inflammatory breast cancer (IBC) from high-volume centers treating IBC recently convened at the 10th Anniversary Conference of the Morgan Welch Inflammatory Breast Cancer Research Program at The University of Texas MD Anderson Cancer Center in Houston Texas. A consensus on the clinical management of patients with IBC was discussed, summarized, and subsequently reviewed. All participants at the conference (patients, advocates, researchers, trainees, and clinicians) were queried using the MDRing electronic survey on key management issues. A summary of the expert consensus and participant voting is presented. Bilateral breast and nodal evaluation, breast magnetic resonance imaging, positron emission tomography/computed tomography, and medical photographs were endorsed as optimal. Neoadjuvant systemic therapy, modified radical mastectomy and level I and II ipsilateral axillary node dissection, post-mastectomy radiotherapy, adjuvant targeted therapy and hormonal therapy as indicated, and delayed reconstruction were agreed-upon fundamental premises of standard non-protocol-based treatment for IBC. Consideration for local-regional therapy in de novo stage IV IBC was endorsed to provide local control whenever feasible. Variation across centers and special circumstances were discussed.

Inflammatory breast cancer: High incidence of GCC haplotypes (-1082A/G, -819T/C, and -592A/C) in the interleukin-10 gene promoter correlates with over-expression of interleukin-10 in patients' carcinoma tissues.

Interleukin-10 is involved in carcinogenesis by supporting tumor escape from the immune response. The aim of this study was to assess the single nucleotide polymorphisms, -1082A/G, -819T/C and -592A/C, in interleukin-10 gene promoter in inflammatory breast cancer compared to non-inflammatory breast cancer and association of these polymorphisms with interleukin-10 gene expression. We enrolled 105 breast cancer tissue (72 non-inflammatory breast cancer and 33 inflammatory breast cancer) patients and we determined the three studied single nucleotide polymorphisms in all samples by polymerase chain reaction restriction fragment length polymorphism and investigated their association with the disease and with various prognostic factors. In addition, we assessed the expression of interleukin-10 gene by real-time quantitative reverse transcription polymerase chain reaction and the correlation between studied single nucleotide polymorphisms and interleukin-10 messenger RNA expression. We found co-dominant effect as the best inheritance model (in the three studied single nucleotide polymorphisms in non-inflammatory breast cancer and inflammatory breast cancer samples), and we didn't identify any association between single nucleotide polymorphisms genotypes and breast cancer prognostic factors. However, GCC haplotype was found highly associated with inflammatory breast cancer risk (p < 0.001, odds ratio = 43.05). Moreover, the expression of interleukin-10 messenger RNA was significantly higher (p < 0.001) by 5.28-fold and 8.95-fold than non-inflammatory breast cancer and healthy control, respectively, where GCC haplotype significantly increased interleukin-10 gene expression (r = 0.9, p < 0.001).

Leptin is overexpressed in the tumor microenvironment of obese patients with estrogen receptor positive breast cancer.

The present study aimed to investigate the potential role of leptin in the progression of breast cancer and the associated cell proliferation signalling pathway(s). A total of 44 female patients diagnosed with breast cancer and 24 healthy donors from Ain Shams University Hospitals (Cairo, Egypt) were enrolled in the present study. The present study assessed leptin expression in breast cancer tissues at the gene and protein level using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry. The results demonstrate that the expression of leptin was significantly higher in tissue of breast cancer samples from obese patients than overweight and control samples (P<0.001). ELISA results indicated a significant increase (P<0.001) of leptin expression in obese patients. To investigate whether there is any difference in leptin expression between the peripheral and tumor microenvironment blood of patients with breast cancer, the concentration of leptin was assessed in plasma from both using ELISA assays. The results demonstrated a statistically significant increase in the level of leptin in plasma samples from the tumor microenvironment of obese patients with estrogen receptor positive (ER+) breast cancer, compared with peripheral plasma samples. Furthermore, the leptin gene was overexpressed in obese ER+ breast cancer tissue. RT-qPCR was also performed to assess the expression of genes involved in proliferation pathways including leptin receptor (LEPR), aromatase, mitogen activated protein kinase (MAPK) and signal transducer and activator of transcription-3 (STAT3). A positive association between leptin expression, LEPR, aromatase, MAPK and STAT3 was detected in tissue samples of patients with breast cancer. The current study concluded that leptin may enhance breast cancer progression by inducing the expression of JAK/STAT3, ERK1/2 and estrogen pathways in obese patients breast cancer.

Hormonal-receptor positive breast cancer: IL-6 augments invasion and lymph node metastasis via stimulating cathepsin B expression.

Hormonal-receptor positive (HRP) breast cancer patients with positive metastatic axillary lymph nodes are characterized by poor prognosis and increased mortality rate. The mechanisms by which cancer cells invade lymph nodes have not yet been fully explored. Several studies have shown that expression of IL-6 and the proteolytic enzyme cathepsin B (CTSB) was associated with breast cancer poor prognosis. In the present study, the effect of different concentrations of recombinant human IL-6 on the invasiveness capacity of HRP breast cancer cell line MCF-7 was tested using an in vitro invasion chamber assay. The impact of IL-6 on expression and activity of CTSB was also investigated. IL-6 treatment promoted the invasiveness potential of MCF-7 cells in a dose-dependent manner. Furthermore, MCF-7 cells displayed elevated CTSB expression and activity associated with loss of E-cadherin and upregulation of vimentin protein levels upon IL-6 stimulation. To validate these results in vivo, the level of expression of IL-6 and CTSB in the carcinoma tissues of HRP-breast cancer patients with positive and negative axillary metastatic lymph nodes (pLNs and nLNs) was assessed. Western blot and immunohistochemical staining data showed that expression of IL-6 and CTSB was higher in carcinoma tissues in HRP-breast cancer with pLNs than those with nLNs patients. ELISA results showed carcinoma tissues of HRP-breast cancer with pLNs exhibited significantly elevated IL-6 protein levels by approximately 2.8-fold compared with those with nLNs patients (P < 0.05). Interestingly, a significantly positive correlation between IL-6 and CTSB expression was detected in clinical samples of HRP-breast cancer patients with pLNs (r = 0.78, P < 0.01). Collectively, this study suggests that IL-6-induced CTSB may play a role in lymph node metastasis, and that may possess future therapeutic implications for HRP-breast cancer patients with pLNs. Further studies are necessary to fully identify IL-6/CTSB axis in different molecular subtypes of breast cancer.

Positive lymph-node breast cancer patients ­ activation of NF-κB in tumor-associated leukocytes stimulates cytokine secretion that promotes metastasis via C-C chemokine receptor CCR7.

Tumor metastasis to lymph nodes is most deadly complication among breast cancer patients. Herein, we investigated the molecular mechanism by which tumor-associated leukocytes (TALs) mediate lymph node metastasis. The density of different leukocyte subtypes infiltrating the tumor microenvironment of negative and positive lymph nodes (nLNs, pLNs) in breast cancer patients was measured using immunohistochemistry. In addition, we isolated TALs from blood drained from the axillary tributaries of nLN and pLN patients during breast surgery. Secretions of TALs were subjected to cytokine profiling using a cytokine antibody array. Our results showed an increase in the number of infiltrated CD45+ cells in the carcinoma tissues of pLN patients with the major proportion being myeloid subsets compared with nLN patients. Furthermore, TALs of pLN patients show a significant fivefold increase in the secretion of interleukin (IL)-1α, interferon-γ, IL-5, IL-3 and tumor necrosis factor-ß, and are characterized by enhanced constitutive NF-κB/p65 signaling compared with TALs isolated from nLN patients. Using an invasion assay, cytokines secreted by TALs of pLN patients were shown to augment the invasive phenotype of breast cancer MCF-7 and SKBR3 cells compared with nLN patients. Using flow cytometry, we found that C-C chemokine receptor 7 (CCR7) is significantly overexpressed in breast carcinoma of pLN patients compared with nLNs patients. Intriguingly, CCR7, a mechanistic clue for metastasis, is upregulated in MCF-7 cells upon stimulation with TAL-conditioned media of pLN patients. Our findings show that the molecular cues secreted by TALs alone or in combination with CCR7 may emerge as future therapeutic targets for lymph node metastasis in breast cancer patients.

Magnetite nanoparticles inhibit tumor growth and upregulate the expression of p53/p16 in Ehrlich solid carcinoma bearing mice.

BACKGROUND: Magnetite nanoparticles (MNPs) have been widely used as contrast agents and have promising approaches in cancer treatment. In the present study we used Ehrlich solid carcinoma (ESC) bearing mice as a model to investigate MNPs antitumor activity, their effect on expression of p53 and p16 genes as an indicator for apoptotic induction in tumor tissues. METHOD: MNPs coated with ascorbic acid (size: 25.0±5.0 nm) were synthesized by co-precipitation method and characterized. Ehrlich mice model were treated with MNPs using 60 mg/Kg day by day for 14 injections; intratumorally (IT) or intraperitoneally (IP). Tumor size, pathological changes and iron content in tumor and normal muscle tissues were assessed. We also assessed changes in expression levels of p53 and p16 genes in addition to p53 protein level by immunohistochemistry. RESULTS: Our results revealed that tumor growth was significantly reduced by IT and IP MNPs injection compared to untreated tumor. A significant increase in p53 and p16 mRNA expression was detected in Ehrlich solid tumors of IT and IP treated groups compared to untreated Ehrlich solid tumor. This increase was accompanied with increase in p53 protein expression. It is worth mentioning that no significant difference in expression of p53 and p16 could be detected between IT ESC and control group. CONCLUSION: MNPs might be more effective in breast cancer treatment if injected intratumorally to be directed to the tumor tissues.

Promoter hypermethylation and suppression of glutathione peroxidase 3 are associated with inflammatory breast carcinogenesis.

Reactive oxygen species (ROS) play a crucial role in breast cancer initiation, promotion, and progression. Inhibition of antioxidant enzymes that remove ROS was found to accelerate cancer growth. Studies showed that inhibition of glutathione peroxidase-3 (GPX3) was associated with cancer progression. Although the role of GPX3 has been studied in different cancer types, its role in breast cancer and its epigenetic regulation have not yet been investigated. The aim of the present study was to investigate GPX3 expression and epigenetic regulation in carcinoma tissues of breast cancer patients' in comparison to normal breast tissues. Furthermore, we compared GPX3 level of expression and methylation status in aggressive phenotype inflammatory breast cancer (IBC) versus non-IBC invasive ductal carcinoma (IDC). We found that GPX3 mRNA and protein expression levels were downregulated in the carcinoma tissues of IBC compared to non-IBC. However, we did not detect significant correlation between GPX3 and patients' clinical-pathological prosperities. Promoter hypermethylation of GPX3 gene was detected in carcinoma tissues not normal breast tissues. In addition, IBC carcinoma tissues showed a significant increase in the promoter hypermethylation of GPX3 gene compared to non-IBC. Our results propose that downregulation of GPX3 in IBC may play a role in the disease progression.

Inflammatory breast cancer: New factors contribute to disease etiology: A review.

Inflammatory breast cancer (IBC) is a highly metastatic and fatal form of breast cancer. In fact, IBC is characterized by specific morphological, phenotypic, and biological properties that distinguish it from non-IBC. The aggressive behavior of IBC being more common among young women and the low survival rate alarmed researchers to explore the disease biology. Despite the basic and translational studies needed to understand IBC disease biology and identify specific biomarkers, studies are limited by few available IBC cell lines, experimental models, and paucity of patient samples. Above all, in the last decade, researchers were able to identify new factors that may play a crucial role in IBC progression. Among identified factors are cytokines, chemokines, growth factors, and proteases. In addition, viral infection was also suggested to participate in the etiology of IBC disease. In this review, we present novel factors suggested by different studies to contribute to the etiology of IBC and the proposed new therapeutic insights.

Cytokines secreted by macrophages isolated from tumor microenvironment of inflammatory breast cancer patients possess chemotactic properties.

Although there is a growing literature describing the role of macrophages in breast cancer, the role of macrophages in inflammatory breast cancer (IBC) is unclear. The aim of present study was to isolate and characterize tumor associated macrophages of IBC and non-IBC patients and define their role in IBC. Tumor infiltrating monocytes/macrophages (CD14+ and CD68+) were measured by immunohistochemistry using specific monoclonal antibodies. Blood drained from axillary vein tributaries was collected during breast cancer surgery and the percentage of CD14+ in the total isolated leukocytes was assessed by flow cytometric analysis. CD14+ cells were separated from total leukocytes by immuno-magnetic beads technique and were cultured overnight. Media conditioned by CD14+ were collected and subjected to cytokine profiling using cytokine antibody array. Wound healing and invasion assays were used to test whether cytokines highly secreted by tumor drained macrophages induce motility and invasion of breast cancer cells. We found that macrophages highly infiltrate into carcinoma tissues of IBC patients. In addition blood collected from axillary tributaries of IBC patients is highly enriched with CD14+ cells as compared to blood collected from non-IBC patients. Cytokine profiling of CD14+ cells isolated from IBC patients revealed a significant increase in secretion of tumor necrosis factor-α; monocyte chemoattractant protein-1/CC-chemokine ligand 2; interleukin-8 and interleukin-10 as compared to CD14+ cells isolated from non-IBC patients. Tumor necrosis factor-α, interleukin-8 and interleukin-10 significantly increased motility and invasion of IBC cells in vitro. In conclusion, macrophages isolated from the tumor microenvironment of IBC patients secrete chemotactic cytokines that may augment dissemination and metastasis of IBC carcinoma cells.

Assessing the level of breast cancer awareness among recently diagnosed patients in Ain Shams University Hospital.

UNLABELLED: Breast cancer is the leading female malignancy among Egyptian women. The majority of Egyptian breast cancer patients present at late stages of the disease with a large tumor size compared to Western countries. Low breast cancer awareness, social and cultural factors were suggested to play crucial role in late presentation of breast cancer among Egyptians. The aim of our present study is to establish a questionnaire-based survey that can assess levels of breast cancer awareness among Egyptians. Patients enrolled were interviewed and answered 60 questions related to knowledge, symptoms, risk factors, prevention and management options of breast cancer. We evaluated our interactions with breast cancer patients and defined the level of awareness gained from education and culture of Egyptian women. Our results described that Egyptian breast cancer patients lack knowledge about their illness and condition. The lowest levels of awareness were related to age, education and culture. We concluded that breast cancer public awareness and women education programs covering factors identified in our study is warranted among Egyptian population. Overview OBJECTIVE: To assess breast cancer awareness among recently diagnosed breast cancer Egyptian patients. SUBJECTS AND METHODS: Among 289 interviewed breast cancer patients we enrolled 45 patients who fulfilled the study inclusion criteria. Participants were asked to answer a validated 60-item questionnaire that inquires about socio-demographic characteristics, knowledge of breast cancer symptoms, risk factors, symptoms, prevention, general management and willingness to participate in awareness campaigns. The average of interview time was about 45 min, depending on patient's age and education level. RESULTS: The mean age of included patients was 48.2 ± 10.19 years. Geographical distribution revealed that 66.7% patients were from Cairo and the rest were from other governorates, including Aswan, Sharqia, Mansora, Qena, Kalyobia, Elminya and Sohag. Among interviewed patients 85% were non-working housewives, 42.2% of them were illiterate. Questions about knowledge of breast cancer revealed that 53.33% of patients knew an acquaintance with breast cancer; however, they spent a median time of 3 months to seek medical advice after recognizing the first symptom with a delay range between a month and 72 months. We found that 73% of the participants presented to a physician with the same first recognized symptom and 75.6% didn't think of cancer then as a possible diagnosis. Total breast cancer knowledge scores had an average of 13.3 (out of 35 knowledge points), with 93% of the patients recognizing "painless breast mass" as a breast cancer symptom and 44% only recognized the concept of breast self examination. Interestingly, 61.4% identified breastfeeding as a risk factor for breast cancer, 60% did not recognize mammography as an early detection method, and 57.7% agreed that clinical breast examination (CBE) is important for early detection. Regarding management, 75% said breast cancer was potentially curable and 60% said medical care could be helpful regardless the age of presentation. CONCLUSION: Egyptian breast cancer patients knew little about their condition. Less awareness was related to age and education level. Low knowledge of risk factors, early detection and management of breast cancer should be addressed by designing patient education programs, where less educated patients are supported by health care professionals to participate in the management of breast cancer. Moreover, we found that 67% and 97% of enrolled breast cancer patients were willing as well to participate in spreading awareness among their community and among their own families, respectively.

Human leukocyte antigen class II alleles (DQB1 and DRB1) as predictors for response to interferon therapy in HCV genotype 4.

Human leukocyte antigens class II play an important role in immune response against HCV. We investigated whether HLA class II alleles influence susceptibility to HCV infection and response to interferon therapy. HLA-DRB1 and -DQB1 loci were genotyped using PCR-SSO Luminex technology. According to our regimen, 41 (66%) of patients achieved sustained virological response to combined treatment of IFN and ribavirin. Frequencies of DQB1*0313 allele and DRB1*04-DRB1*11, DQB1*0204-DQB1*0313, DQB1*0309-DQB1*0313, and DQB1*0313-DQB1*0319 haplotypes were significantly more frequent in nonresponders than in responders. In contrast, DQB1*02, DQB1*06, DRB1*13, and DRB1*15 alleles were significantly more frequent in responders than in nonresponders. Similarly, DRB1*1301, DRB1*1361, and DRB1*1369 alleles and DRB1*1301-DRB1*1328, DRB1*1301-DRB1*1361, DRB1*1301-DRB1*1369, DRB1*1328-DRB1*1361, and DRB1*1328-DRB1*1369 haplotypes were significantly found only in responders. Some alleles and linkages showed significantly different distributions between patient and healthy groups. These alleles may be used as predictors for response to treatment or to susceptibility to HCV infection in the Egyptian population.

Liver fibrosis staging through a stepwise analysis of non-invasive markers (FibroSteps) in patients with chronic hepatitis C infection.

BACKGROUND: Non-invasive fibrosis markers can distinguish between liver fibrosis stages in lieu of liver biopsy or imaging elastography. AIMS: To develop a sensitive, non-invasive, freely-available algorithm that differentiates between individual liver fibrosis stages in chronic hepatitis C virus (HCV) patients. METHODS: Chronic HCV patients (n = 355) at Cairo University Hospital, Egypt, with liver biopsy to determine fibrosis stage (METAVIR), were tested for preselected fibrosis markers. A novel multistage stepwise fibrosis classification algorithm (FibroSteps) was developed using random forest analysis for biomarker selection, and logistic regression for modelling. FibroSteps predicted fibrosis stage using four steps: Step 1 distinguished no(F0)/mild fibrosis(F1) vs. moderate(F2)/severe fibrosis(F3)/cirrhosis(F4); Step 2a distinguished F0 vs. F1; Step 2b distinguished F2 vs. F3/F4; and Step 3 distinguished F3 vs. F4. FibroSteps was developed using a randomly-selected training set (n = 234) and evaluated using the remaining patients (n = 118) as a validation set. RESULTS: Hyaluronic Acid, TGF-ß1, α2-macroglobulin, MMP-2, Apolipoprotein-A1, Urea, MMP-1, alpha-fetoprotein, haptoglobin, RBCs, haemoglobin and TIMP-1 were selected into the models, which had areas under the receiver operating curve (AUC) of 0.973, 0.923 (Step 1); 0.943, 0.872 (Step 2a); 0.916, 0.883 (Step 2b) and 0.944, 0.946 (Step 3), in the training and validation sets respectively. The final classification had accuracies of 94.9% (95% CI: 91.3-97.4%) and 89.8% (95% CI: 82.9-94.6%) for the training and validation sets respectively. CONCLUSIONS: FibroSteps, a freely available, non-invasive liver fibrosis classification, is accurate and can assist clinicians in making prognostic and therapeutic decisions. The statistical code for FibroSteps using R software is provided in the supplementary materials.

Superoxide dismutases from larvae of the camel tick Hyalomma dromedarii.

Three superoxide dismutases (EC 1.15.1.1) (TLSOD1, TLSOD2 and TLSOD3) were purified from larvae of the camel tick Hyalomma dromedarii by ammonium sulfate precipitation, ion exchange and gel filtration columns. SDS-PAGE revealed that the subunit molecular masses of the SODs are 40±2 kDa, 67±1.5 kDa and 45±2.6 kDa for TLSOD1, TLSOD2 and TLSOD3, respectively. TLSOD1 and TLSOD2 are monomeric proteins, while TLSOD3 isoenzyme exhibits dimeric structure with native molecular mass of 90 kDa. The pI values are estimated at pH 8.0, pH 7.2 and pH 6.6 for the three SODs which displayed pH optima at 7.6, 8.0 and 7.8, respectively. CuCl(2) and ZnCl(2) increase the activity of TLSOD2 and TLSOD3, while MnCl(2) increases the activity of TLSOD1. KCN inhibits the activity of TLSOD2 and TLSOD3, while a remarkable resistance of TLSOD1 isoenzyme was detected. TLSOD1 is suggested to be a manganese containing isoenzyme while TLSOD2 and TLSOD3 are suggested to be copper/zinc-containing isoenzymes. These results indicate the presence of three different forms of SODs in the larval stage of camel tick. This finding will contribute to our understanding of the physiology of these ectoparasites and the development of non-traditional methods to control them.

Monocytes conditioned media stimulate fibronectin expression and spreading of inflammatory breast cancer cells in three-dimensional culture: A mechanism mediated by IL-8 signaling pathway.

BACKGROUND: Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer characterized by invasion of carcinoma cells into dermal lymphatic vessels where they form tumor emboli over expressing adhesion molecule E-cadherin. Although invasion and metastasis are dynamic processes controlled by complex interaction between tumor cells and microenvironment the mechanisms by which soluble mediators may regulate motility and invasion of IBC cells are poorly understood. The present study investigated the effect of media conditioned by human monocytes U937 secreted cytokines, chemokines and growth factors on the expression of adhesion molecules E-cadherin and fibronectin of human IBC cell line SUM149. Furthermore, cytokines signaling pathway involved were also identified. RESULTS: U937 secreted cytokines, chemokines and growth factors were characterized by cytokine antibody array. The major U937 secreted cytokines/chemokines were interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1/CCL2). When SUM149 cells were seeded in three dimensional (3D) models with media conditioned by U937 secreted cytokines, chemokines and growth factors; results showed: 1) changes in the morphology of IBC cells from epithelial to migratory spindle shape branched like structures; 2) Over-expression of adhesion molecule fibronectin and not E-cadherin. Further analysis revealed that over-expression of fibronectin may be mediated by IL-8 via PI3K/Akt signaling pathway. CONCLUSION: The present results suggested that cytokines secreted by human monocytes may promote chemotactic migration and spreading of IBC cell lines. Results also indicated that IL-8 the major secreted cytokine by U937 cells may play essential role in fibronectin expression by SUM149 cells via interaction with IL-8 specific receptors and stimulation of PI3K/Akt signaling pathway.

Inhibition of cathepsin B activity attenuates extracellular matrix degradation and inflammatory breast cancer invasion.

INTRODUCTION: Inflammatory breast cancer (IBC) is an aggressive, metastatic and highly angiogenic form of locally advanced breast cancer with a relatively poor three-year survival rate. Breast cancer invasion has been linked to proteolytic activity at the tumor cell surface. Here we explored a role for active cathepsin B on the cell surface in the invasiveness of IBC. METHODS: We examined expression of the cysteine protease cathepsin B and the serine protease urokinase plasminogen activator (uPA), its receptor uPAR and caveolin-1 in two IBC cell lines: SUM149 and SUM190. We utilized a live cell proteolysis assay to localize in real time the degradation of type IV collagen by IBC cells. IBC patient biopsies were examined for expression of cathepsin B and caveolin-1. RESULTS: Both cell lines expressed comparable levels of cathepsin B and uPA. In contrast, levels of caveolin-1 and uPAR were greater in SUM149 cells. We observed that uPA, uPAR and enzymatically active cathepsin B were colocalized in caveolae fractions isolated from SUM149 cells. Using a live-cell proteolysis assay, we demonstrated that both IBC cell lines degrade type IV collagen. The SUM149 cells exhibit predominantly pericellular proteolysis, consistent with localization of proteolytic pathway constitutents to caveolar membrane microdomains. A functional role for cathepsin B was confirmed by the ability of CA074, a cell impermeable and highly selective cathepsin B inhibitor, to significantly reduce pericellular proteolysis and invasion by SUM149 cells. A statistically significant co-expression of cathepsin B and caveolin-1 was found in IBC patient biopsies, thus validating our in vitro data. CONCLUSION: Our study is the first to show that the proteolytic activity of cathepsin B and its co-expression with caveolin-1 contributes to the aggressiveness of IBC.

Cathepsin B: a potential prognostic marker for inflammatory breast cancer.

BACKGROUND: Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. In non-IBC, the cysteine protease cathepsin B (CTSB) is known to be involved in cancer progression and invasion; however, very little is known about its role in IBC. METHODS: In this study, we enrolled 23 IBC and 27 non-IBC patients. All patient tissues used for analysis were from untreated patients. Using immunohistochemistry and immunoblotting, we assessed the levels of expression of CTSB in IBC versus non-IBC patient tissues. Previously, we found that CTSB is localized to caveolar membrane microdomains in cancer cell lines including IBC, and therefore, we also examined the expression of caveolin-1 (cav-1), a structural protein of caveolae in IBC versus non-IBC tissues. In addition, we tested the correlation between the expression of CTSB and cav-1 and the number of positive metastatic lymph nodes in both patient groups. RESULTS: Our results revealed that CTSB and cav-1 were overexpressed in IBC as compared to non-IBC tissues. Moreover, there was a significant positive correlation between the expression of CTSB and the number of positive metastatic lymph nodes in IBC. CONCLUSIONS: CTSB may initiate proteolytic pathways crucial for IBC invasion. Thus, our data demonstrate that CTSB may be a potential prognostic marker for lymph node metastasis in IBC.

Interleukin-6 increases expression and secretion of cathepsin B by breast tumor-associated monocytes.

In the tumor microenvironment, monocytes respond to paracrine stimuli from breast cancer cells by secreting molecules that participate in breast cancer growth, invasion, intravasation and metastasis. Here we examined the effects of media conditioned by MDA-MB-231 human breast carcinoma cells (231-CM) on expression and secretion of proteases and secretion of cytokines by U937 human monocytes. We found that 231-CM increased U937: 1) proliferation; 2) expression, activity and secretion of the cysteine protease cathepsin B (CTSB); 3) secretion of matrix metalloproteinases (MMP)-2 and -9; and 4) secretion of interleukin-6 (IL-6) and insulin-like growth factor binding protein-1 (IGFBP-1). We further demonstrated by western blotting and enzymatic activity assays that the increases in CTSB secretion and activity induced by 231-CM could be reduced by neutralizing antibodies against IL-6. Our data suggest a role for IL-6 in increased monocyte expression and secretion of CTSB in response to soluble factors secreted by breast cancer cells.