Nuclear Factor-κB Clinical Significance in Breast Cancer: An Immunohistochemical Study.

OBJECTIVES: Nuclear factor κB (NF-κB) is a superfamily of transcription factors that plays a key role in cancer genesis and progression. The present study aimed to examine the expression of NF-κB/p65 in breast cancer and its relationship with prognostic markers such as tumour grade, tumour size, hormone receptors, and HER-2. METHODS: Ninety-nine unselected formalin-fixed paraffin-embedded invasive ductal and lobular tissue sections were evaluated by immunohistochemistry methods to measure the expression of NF-κB/p65, oestrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor-2 (HER-2), and Ki-67. We assessed the correlation between NF-κB/p65 and clinicopathological parameters. RESULTS: NF-κB/p65 was found only in the cytoplasm and positively correlated with large tumours (≥2 cm) and high-grade tumours (p < 0.001 and p = 0.018, respectively). Other breast cancer markers, such as histological type (p = 0.766), HER-2 (p = 0.416), PR (p = 0.356), and ER (p = 0.606), had no significant link with the expression of NF-κB/p65. Furthermore, no significant relation with the Ki-67 marker was detected (p = 0.117). CONCLUSIONS: The current study is indicative of a link between overexpression of NF-κB/p65 and both large tumour size and higher grade. This suggests that the expression of NF-κB/p65 is associated with aggressive biological activity in breast cancer; elucidating the mechanisms that lead to NF-κB/p65 cytoplasmic accumulation could lead to the development of novel therapeutic methods.

DUSP4 Silencing Enhances the Sensitivity of Breast Cancer Cells to Doxorubicin through the Activation of the JNK/c-Jun Signalling Pathway.

Doxorubicin (Dox) has limited efficiency in breast cancer (BC) due to drug-acquired resistance. The epithelial-mesenchymal transition (EMT) plays a major role in the survival and drug resistance of cancer cells. It was suggested that the JNK pathway was implicated in the response to Dox by regulating EMT. DUSP4/or MKP-2 is a well-known regulator of the JNK pathway and was found to be highly expressed in BC. However, its functional significance is not yet fully understood. In the present study, the possible involvement of MKP-2 in Dox-induced EMT was investigated in breast cancer cells. Immunohistochemistry for tissues obtained from BC patients (n = 108) revealed 71.3% of tissues stained positively for MKP-2 while only 28.7% stained negatively. However, MKP-2 protein expression exhibited no significant relationship between BC prognostic factors, such as histological grade, histological type, hormonal status, and Ki-67 marker, its expression was significantly correlated with age 40 or below. In MDA-MB-231 cells, Dox-induced phosphorylation of JNK was sufficiently enhanced in MKP-2 silenced cells. This resulted in the attenuation of Dox-induced EMT, cell cycle arrest, and ultimately accelerated apoptosis. It was confirmed that the acquisition of Dox sensitivity by MKP-2 silencing largely depends on the stimulation of the JNK pathway. Indeed, results showed that overexpressing MKP-2 in non-tumorigenic MCF-12A cells dramatically inhibited Dox-induced JNK activation and, subsequently, cell death. The present study, to our knowledge, is the first to provide evidence for the potential role of MKP-2 in chemoresistance to Dox through modulating the JNK pathway and enhancing EMT.

Characterization and Roles of Membrane Lipids in Fatty Liver Disease.

Obesity has reached global epidemic proportions and it affects the development of insulin resistance, type 2 diabetes, fatty liver disease and other metabolic diseases. Membrane lipids are important structural and signaling components of the cell membrane. Recent studies highlight their importance in lipid homeostasis and are implicated in the pathogenesis of fatty liver disease. Here, we discuss the numerous membrane lipid species and their metabolites including, phospholipids, sphingolipids and cholesterol, and how dysregulation of their composition and physiology contribute to the development of fatty liver disease. The development of new genetic and pharmacological mouse models has shed light on the role of lipid species on various mechanisms/pathways; these lipids impact many aspects of the pathophysiology of fatty liver disease and could potentially be targeted for the treatment of fatty liver disease.

Camel Urine Promotes Sensitization to Doxorubicin by Inhibiting Epithelial-Mesenchymal Transition and Modulating NF-κB-Snail Signaling Pathway in Breast Cancer Cells.

BACKGROUND: Camel urine (CU) has been used as traditional treatment in the Arabian Peninsula for centuries. Although, researchers have reported CU anti-cancer effects, the exact mechanism(s) of action involved has not been fully elucidated. The epithelial-mesenchymal transition EMT is a phenotypic switch that promotes the acquisition of a fibroblastoid-like morphology by epithelial tumor cells, resulting in enhanced tumor cell motility and invasiveness. EMT has been shown to contribute to metastasis and chemoresistance of carcinomas. For that, in the present study, we have assessed the potential mechanism (s) by which CU exert its anti-cancer effects and its possible synergistic therapeutic effect with Doxorubicin (DOX) in breast cancer cells. METHODS: Determination of anti-proliferative and apoptosis validation of CU was performed by 3-(4,5-Dimethylthiazol-2-yl)-2,5,-diphenyltetrazolium bromide (MTT), annexin-V-fluorescein isothiocyanate assays, and Western blot. EMT protein markers, migration and invasion of cells were determined by Western blot or immunofluorescent staining, Scratch assay, Transwell invasion assay, respectively. RESULTS: CU applied a significant anti-cancer effect on breast cancer cells via induction of DNA damage and apoptosis in a concentration- and time-dependent manner. Also, CU remarkably reversed the EMT by downregulating N-cadherin and Vimentin expression and upregulating E-cadherin expression. As a result, the stemness, migration and invasion of breast cancer cells were also inhibited, which was likely mediated by NF-κB-Snail signalling pathway and its downstream inflammatory effectors. CU successfully enhanced DOX cytotoxicity by reversing EMT which possibly through inhibition of NF-κB-Snail signalling and subsequently inflammation.  Thus, our study provides new mechanistic bases for the therapeutic application of CU that may improve the outcomes of anti-cancer chemotherapy.

Appropriateness of Using Vitamin K for the Correction of INR Elevation Secondary to Hepatic Disease in Critically ill Patients: An Observational Study.

BACKGROUND: Using vitamin K for correction of coagulopathy in critically ill patients is controversial with limited evidence. This study aims to evaluate the efficacy and safety of vitamin K in the correction of international normalized ratio (INR) elevation secondary to liver disease in critically ill patients. METHOD: A retrospective study of critically ill patients with coagulopathy secondary to liver disease. The primary outcome was to evaluate the association between vitamin K administration and the incidence of new bleeding events in critically ill patients with INR elevation; other outcomes were considered secondary. Patients were categorized into two groups based on vitamin K administration to correct INR elevation. The propensity score was generated based on disease severity scores and the use of pharmacological DVT prophylaxis. RESULTS: A total of 98 patients were included in the study. Forty-seven patients (48%) received vitamin K during the study period. The odds of the new bleeding event was not statistically different between groups (OR 2.4, 95% CI 0.28-21.67, P = .42). Delta of INR reduction was observed with a median of 0.63 when the first dose is given (P-value: <.0001). However the INR reduction with other subsequent doses of vitamin K was not statistically significant. CONCLUSION: The administration of vitamin K for INR correction in critically ill patients with coagulopathy secondary to liver disease was not associated with a lower odds of new bleeding events. Further studies are needed to assess the value of vitamin K administration in critically ill patients with liver diseases related coagulopathy.

Clinical features and outcomes of critically ill patients with coronavirus disease 2019 (COVID-19): A multicenter cohort study.

BACKGROUND: Coronavirus disease-19 (COVID-19) manifested by a broad spectrum of symptoms, ranging from asymptomatic manifestations to severe illness and death. The purpose of the study was to extensively describe the clinical features and outcomes in critically ill patients with COVID-19 in Saudi Arabia. METHOD: This was a multicenter, non-interventional cohort study for all critically ill patients aged 18 years or older, admitted to intensive care units (ICUs) between March 1 to August 31, 2020, with an objectively confirmed diagnosis of COVID-19. The diagnosis of COVID-19 was confirmed by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) on nasopharyngeal and/or throat swabs. Multivariate logistic regression and generalized linear regression were used. We considered a P value of <0.05 statistically significant. RESULTS: A total of 560 patients met the inclusion criteria. An extensive list of clinical features was associated with higher 30-day ICU mortality rates, such as requiring mechanical ventilation (MV) or developing acute kidney injury within 24 hours of ICU admission, higher body temperature, white blood cells, blood glucose level, serum creatinine, fibrinogen, procalcitonin, creatine phosphokinase, aspartate aminotransferase, and total iron-binding capacity. During ICU stay, the most common complication was respiratory failure that required MV (71.4%), followed by acute kidney injury (AKI) and thrombosis with a proportion of 46.8% and 11.4%, respectively. CONCLUSION: Among patients with COVID-19 who were admitted to the ICU, several variables were associated with an increased risk of ICU mortality at 30 days. Respiratory failure that required MV, AKI, and thrombosis were the most common complications during ICU stay.

Influence of MX1 promoter rs2071430 G/T polymorphism on susceptibility to systemic lupus erythematosus.

The expression of interferon inducible genes are reported to be heightened in systemic lupus erythematosus (SLE); nevertheless, not much is known regarding the genetic variants underlying these genes and their role in the pathogenesis of disease. Herein, we aim to explore the potential association and contribution of polymorphisms in MX1 gene (i) promoter with part of exon 1 (ii) intron 6, and (iii) their resulting haplotypes, with susceptibility to SLE. A total of 306 subjects, 152 SLE and 154 healthy controls (HC), were screened by direct sequencing method. Statistical analysis was carried out using appropriate software. The screening region of interest in MX1 revealed the existence of promoter (-123C/A, -88G/T, -20 A/C) and intron 6 (+9862G/A, +10190G/A, +9901C/G, +9920C/A, +9959C/T, +10047A/G) variants in SLE and HC. A significant association was observed between MX1 -88G/T SNP and susceptibility to SLE (χ (2) = 4.18, p = 0.04, OR = 1.89, 95 % CI 1.03-3.5). Haplotype analysis also revealed increased risk of SLE among individuals carrying CTA haplotype (-123 C, -88 T, -20 A) (χ (2) = 5.74, p = 0.017, OR = 4.28, 95 % CI 1.30-14.06). None of the other tested variants showed any significant association with SLE. The present study is the first to reveal the influence of genetic variation in MX1 gene in susceptibility to SLE.

Hepatic mitogen-activated protein kinase phosphatase 1 selectively regulates glucose metabolism and energy homeostasis.

The liver plays a critical role in glucose metabolism and communicates with peripheral tissues to maintain energy homeostasis. Obesity and insulin resistance are highly associated with nonalcoholic fatty liver disease (NAFLD). However, the precise molecular details of NAFLD remain incomplete. The p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) regulate liver metabolism. However, the physiological contribution of MAPK phosphatase 1 (MKP-1) as a nuclear antagonist of both p38 MAPK and JNK in the liver is unknown. Here we show that hepatic MKP-1 becomes overexpressed following high-fat feeding. Liver-specific deletion of MKP-1 enhances gluconeogenesis and causes hepatic insulin resistance in chow-fed mice while selectively conferring protection from hepatosteatosis upon high-fat feeding. Further, hepatic MKP-1 regulates both interleukin-6 (IL-6) and fibroblast growth factor 21 (FGF21). Mice lacking hepatic MKP-1 exhibit reduced circulating IL-6 and FGF21 levels that were associated with impaired skeletal muscle mitochondrial oxidation and susceptibility to diet-induced obesity. Hence, hepatic MKP-1 serves as a selective regulator of MAPK-dependent signals that contributes to the maintenance of glucose homeostasis and peripheral tissue energy balance. These results also demonstrate that hepatic MKP-1 overexpression in obesity is causally linked to the promotion of hepatosteatosis.

The effect of UGT1A1 promoter polymorphism in the development of hyperbilirubinemia and cholelithiasis in hemoglobinopathy patients.

Present study was aimed to explore the effect of (TA)n UGT1A1 gene promoter polymorphism on bilirubin metabolism, bilirubinaemia, predisposition to cholelithiasis and subsequent cholecystectomy, in Sickle-Cell Anemia (SCA) and beta-Thalasemia major (bTH) in Kuwaiti subjects compared to other population. This polymorphism was analyzed and correlated to total bilirubin and cholelithiasis in 270 age, gender, ethnically matched subjects (92 bTH, 116 SCA and 62 Controls) using PCR, dHPLC, fragment analysis and direct sequencing. Four genotypes of UGT1A1 were detected in this study (TA6/6, TA6/7, TA6/8 and TA7/7). (TA)6/8 was found only in four individuals; hence it was not included in the analysis. There was a statistically significant association of genotypes with serum total bilirubin levels in both bTH and SCA groups (p<0.001). Subjects with (TA)7/7 had the highest total serum bilirubin level (178.7 ± 3.5 µmole/l). A significant association was observed between allele (TA)7 and cholelithiasis development (p = 0.0001). The 40%, 67.5% and 100% of SCA with (TA)6/6, (TA)6/7 and (TA)7/7 respectively developed cholelithiasis and were subsequently cholecystectomized. Our results confirm UGT1A1 (TA)7 allele as one of the factors accounting for the hyperbilirubinemia and cholelithiasis observed in SCA and bTH.

MAP kinase phosphatase-2 plays a critical role in response to infection by Leishmania mexicana.

In this study we generated a novel dual specific phosphatase 4 (DUSP4) deletion mouse using a targeted deletion strategy in order to examine the role of MAP kinase phosphatase-2 (MKP-2) in immune responses. Lipopolysaccharide (LPS) induced a rapid, time and concentration-dependent increase in MKP-2 protein expression in bone marrow-derived macrophages from MKP-2(+/+) but not from MKP-2(-/-) mice. LPS-induced JNK and p38 MAP kinase phosphorylation was significantly increased and prolonged in MKP-2(-/-) macrophages whilst ERK phosphorylation was unaffected. MKP-2 deletion also potentiated LPS-stimulated induction of the inflammatory cytokines, IL-6, IL-12p40, TNF-α, and also COX-2 derived PGE(2) production. However surprisingly, in MKP-2(-/-) macrophages, there was a marked reduction in LPS or IFNγ-induced iNOS and nitric oxide release and enhanced basal expression of arginase-1, suggesting that MKP-2 may have an additional regulatory function significant in pathogen-mediated immunity. Indeed, following infection with the intracellular parasite Leishmania mexicana, MKP-2(-/-) mice displayed increased lesion size and parasite burden, and a significantly modified Th1/Th2 bias compared with wild-type counterparts. However, there was no intrinsic defect in MKP-2(-/-) T cell function as measured by anti-CD3 induced IFN-γ production. Rather, MKP-2(-/-) bone marrow-derived macrophages were found to be inherently more susceptible to infection with Leishmania mexicana, an effect reversed following treatment with the arginase inhibitor nor-NOHA. These findings show for the first time a role for MKP-2 in vivo and demonstrate that MKP-2 may be essential in orchestrating protection against intracellular infection at the level of the macrophage.

Over-expression of mitogen-activated protein kinase phosphatase-2 enhances adhesion molecule expression and protects against apoptosis in human endothelial cells.

BACKGROUND AND PURPOSE: We assessed the effects of over-expressing the dual-specific phosphatase, mitogen-activated protein (MAP) kinase phosphatase-2 (MKP-2), in human umbilical vein endothelial cells (HUVECs) on inflammatory protein expression and apoptosis, two key features of endothelial dysfunction in disease. EXPERIMENTAL APPROACHES: We infected HUVECs for 40 h with an adenoviral version of MKP-2 (Adv.MKP-2). Tumour necrosis factor (TNF)-α-stimulated phosphorylation of MAP kinase and protein expression was measured by Western blotting. Cellular apoptosis was assayed by FACS. KEY RESULTS: Infection with Adv.MKP-2 selectively abolished TNF-α-mediated c-Jun-N-terminal kinase (JNK) activation and had little effect upon extracellular signal-regulated kinase or p38 MAP kinase. Adv.MKP-2 abolished COX-2 expression, while induction of the endothelial cell adhesion molecules, intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM), two NFκB-dependent proteins, was not affected. However, when ICAM and VCAM expression was partly reduced by blockade of the NFκB pathway, Adv.MKP-2 was able to reverse this inhibition. This correlated with enhanced TNF-α-induced loss of the inhibitor of κB (IκB)α loss, a marker of NFκB activation. TNF-α in combination with NFκB blockade also increased HUVEC apoptosis; this was significantly reversed by Adv.MKP-2. Protein markers of cellular damage and apoptosis, H2AX phosphorylation and caspase-3 cleavage, were also reversed by MKP-2 over-expression. CONCLUSIONS AND IMPLICATIONS: Over-expression of MKP-2 had different effects upon the expression of inflammatory proteins due to a reciprocal effect upon JNK and NFκB signalling, and also prevented TNF-α-mediated endothelial cell death. These properties may make Adv.MKP-2 a potentially useful future therapy in cardiovascular diseases where endothelial dysfunction is a feature.

Differential regulation of MAP kinase activation by a novel splice variant of human MAP kinase phosphatase-2.

MAP kinase phosphatase-2 (MKP-2) is a member of the family of dual specificity phosphatases that functions to inactivate the ERK and JNK MAP kinase signalling pathways. Here, we identify a novel human MKP-2 variant (MKP-2-S) lacking the MAP kinase binding site but retaining the phosphatase catalytic domain. Endogenous MKP-2-S transcripts and proteins were found in PC3 prostate and MDA-MB-231 breast cancer cells and also human prostate biopsies. Cellular transfection of MKP-2-S gave rise to a nuclear protein of 33kDa which displayed phosphatase activity comparable to the formerly described long form of MKP-2 (MKP-2-L). Due to its lack of a kinase interacting motif (KIM), MKP-2-S did not bind to JNK or ERK; MKP-2-L bound ERK and to a lesser extent JNK. Protein turnover of adenoviral expressed MKP-2-S was accelerated relative to MKP-2-L, with a greater susceptibility to proteosomal-mediated degradation. MKP-2-S retained its ability to deactivate JNK in a similar manner as MKP-2-L and was an effective inhibitor of LPS-stimulated COX-2 induction. However, unlike MKP-2-L, MKP-2-S was unable to reverse serum-induced ERK activation or significantly inhibit endothelial cell proliferation. These findings reveal the occurrence of a novel splice variant of MKP-2 which is unable to bind ERK and may be significant in the dysregulation of MAP kinase activity in certain disease states, particularly in breast and prostate cancers.