The MAZ transcription factor is a downstream target of the oncoprotein Cyr61/CCN1 and promotes pancreatic cancer cell invasion via CRAF-ERK signaling.

Myc-associated zinc-finger protein (MAZ) is a transcription factor with dual roles in transcription initiation and termination. Deregulation of MAZ expression is associated with the progression of pancreatic ductal adenocarcinoma (PDAC). However, the mechanism of action of MAZ in PDAC progression is largely unknown. Here, we present evidence that MAZ mRNA expression and protein levels are increased in human PDAC cell lines, tissue samples, a subcutaneous tumor xenograft in a nude mouse model, and spontaneous cancer in the genetically engineered PDAC mouse model. We also found that MAZ is predominantly expressed in pancreatic cancer stem cells. Functional analysis indicated that MAZ depletion in PDAC cells inhibits invasive phenotypes such as the epithelial-to-mesenchymal transition, migration, invasion, and the sphere-forming ability of PDAC cells. Mechanistically, we detected no direct effects of MAZ on the expression of K-Ras mutants, but MAZ increased the activity of CRAF-ERK signaling, a downstream signaling target of K-Ras. The MAZ-induced activation of CRAF-ERK signaling was mediated via p21-activated protein kinase (PAK) and protein kinase B (AKT/PKB) signaling cascades and promoted PDAC cell invasiveness. Moreover, we found that the matricellular oncoprotein cysteine-rich angiogenic inducer 61 (Cyr61/CCN1) regulates MAZ expression via Notch-1-sonic hedgehog signaling in PDAC cells. We propose that Cyr61/CCN1-induced expression of MAZ promotes invasive phenotypes of PDAC cells not through direct K-Ras activation but instead through the activation of CRAF-ERK signaling. Collectively, these results highlight key molecular players in PDAC invasiveness and may help inform therapeutic strategies to improve clinical management and outcomes of PDAC.

Aspirin blocks growth of breast tumor cells and tumor-initiating cells and induces reprogramming factors of mesenchymal to epithelial transition.

Acetylsalicylic acid (ASA), also known as aspirin, a classic, nonsteroidal, anti-inflammatory drug (NSAID), is widely used to relieve minor aches and pains and to reduce fever. Epidemiological studies and other experimental studies suggest that ASA use reduces the risk of different cancers including breast cancer (BC) and may be used as a chemopreventive agent against BC and other cancers. These studies have raised the tempting possibility that ASA could serve as a preventive medicine for BC. However, lack of in-depth knowledge of the mechanism of action of ASA reshapes the debate of risk and benefit of using ASA in prevention of BC. Our studies, using in vitro and in vivo tumor xenograft models, show a strong beneficial effect of ASA in the prevention of breast carcinogenesis. We find that ASA not only prevents breast tumor cell growth in vitro and tumor growth in nude mice xenograft model through the induction of apoptosis, but also significantly reduces the self-renewal capacity and growth of breast tumor-initiating cells (BTICs)/breast cancer stem cells (BCSCs) and delays the formation of a palpable tumor. Moreover, ASA regulates other pathophysiological events in breast carcinogenesis, such as reprogramming the mesenchymal to epithelial transition (MET) and delaying in vitro migration in BC cells. The tumor growth-inhibitory and reprogramming roles of ASA could be mediated through inhibition of TGF-ß/SMAD4 signaling pathway that is associated with growth, motility, invasion, and metastasis in advanced BCs. Collectively, ASA has a therapeutic or preventive potential by attacking possible target such as TGF-ß in breast carcinogenesis.

Remdesivir, dexamethasone and angiotensin-converting enzyme inhibitors use and mortality outcomes in COVID-19 patients with concomitant troponin elevation.

BACKGROUND: There are indications that viral myocarditis, demand ischemia, and renin-angiotensin-aldosterone system pathway activation play essential roles in troponin elevation in coronavirus disease 2019 (COVID-19) patients. Antiviral medications and steroids are used to treat viral myocarditis, but their effect in patients with elevated troponin, possibly from myocarditis, has not been studied. AIM: To evaluate the effect of dexamethasone, remdesivir, and angiotensin-converting enzyme (ACE) inhibitors (ACEI) on mortality in COVID-19 patients with elevated troponin. METHODS: Our retrospective observational study involved 1788 COVID-19 patients at seven hospitals in Southern California, United States. We did a backward selection Cox multivariate regression analysis to determine predictors of mortality in our study population. Additionally, we did a Kaplan Meier survival analysis in the subset of patients with elevated troponin, comparing survival in patients that received dexamethasone, remdesivir, and ACEI with those that did not. RESULTS: The mean age was 66 years (range 20-110), troponin elevation was noted in 11.5% of the patients, and 29.9% expired. The patients' age [hazard ratio (HR) = 1.02, P < 0.001], intensive care unit admission (HR = 5.07, P < 0.001), and ventilator use (HR = 0.68, P = 0.02) were significantly associated with mortality. In the subset of patients with elevated troponin, there was no statistically significant difference in survival in those that received remdesivir (0.07), dexamethasone (P = 0.63), or ACEI (P = 0.8) and those that did not. CONCLUSION: Although elevated troponin in COVID-19 patients has been associated with viral myocarditis and ACE II receptors, conventional viral myocarditis treatment, including antiviral and steroids, and ACEI did not show any effect on mortality in these patients.

Pulmonary Artery Aneurysm in Behcet Disease: Medical, Endovascular or Surgical Intervention.

Behçet's disease is a chronic inflammatory condition that predominantly affects the body's blood vessels, exhibiting various clinical manifestations and complications. The exact cause remains unclear, but genetic predisposition, immune responses, and vascular activation are believed to contribute to its development. This disease is more prevalent in certain geographic regions and primarily affects young adults, particularly males. Pulmonary aneurysm, a complication of Behçet's disease, is the leading cause of mortality in Behcet disease. In this review, we summarize the complications of Behcet disease with a focus on pulmonary artery aneurysms. We discussed the medical, endovascular, and surgical management of pulmonary aneurysms in Behcet disease and the indications and outcomes of the different treatment options. Corticosteroids and cyclophosphamide remain the preferred first-line therapy. However, clinical improvement with infliximab or adalimumab, tumor necrosis factor-alpha (TNFα) blocking agents, have been reported after treatment failure with recommended first-line agents. In patients who fail medical therapy or those with life-threatening hemoptysis, endovascular or surgical intervention is the next option. Endovascular interventions include pulmonary artery embolization with coils or acrylic glue and using plugs, occluders, or stents. Endovascular interventions usually have fewer adverse effects than surgery. Although the risk of surgical procedures is high in pulmonary artery aneurysms, it could be a life-saving procedure in patients with life-threatening hemoptysis. Surgical options, including pulmonary artery ligation, aneurysmorrhaphy, segmentectomy, lobectomy, or pneumonectomy are available. However, the results of surgical therapy for Behçet aneurysms are often disappointing.

Culture of human breast cancer cell line (MDA-MB-231) on fibronectin-coated surface induces pro-matrix metalloproteinase-9 expression and activity.

Interaction between cell surface integrin receptors with extracellular matrix (ECM) plays an important role in cell survival, proliferation, and migration including tumor development and invasion. Binding of ECM to integrins initiates intracellular signaling cascades, modulating expression and activity of different matrix metalloproteinases (MMPs) which is important in ECM degradation. The present study investigates fibronectin-integrin-mediated signaling and thereby modulation of MMPs expression and activity in human breast cancer cell line, MDA-MB-231. Culture of MDA-MB-231 cells on fibronectin (FN) induced expression and activity of pro-matrixmetalloproteinase-9 (MMP-9). Appreciable reduction of FN-induced pro-MMP-9 activity was observed in anti-α5 antibody treated cells. Inhibitor studies revealed that inhibitors of phosphatidyl inositiol-3-kinase (PI-3K), and nuclear factor kappa B (NF-κB) inhibited FN-induced pro-MMP-9 activity. FN increased tyrosine phosphorylation of focal adhesion kinase (FAK), integrin linked kinase (ILK), and PI-3K in MDA-MB-231 cells. FN-induced the transactivation of MMP-9 promoter by enhancing DNA binding activity of NF-κB and Sp1. Wound healing assay showed faster migration of MDA-MB-231cells grown on fibronectin-coated as surface as compared to control. Our findings indicated that culture of MDA-MB-231 on fibronectin perhaps send signals via fibronectin-integrin-mediated signaling pathways recruiting FAK, PI-3K, ILK, NF-κB, and modulate expression and activation of pro-MMP-9. These observations may enrich fundamental aspects of cancer biology especially role of α5ß1 integrin in regulation of MMPs expression and activity.

Fibronectin-integrin mediated signaling in human cervical cancer cells (SiHa).

Interaction between cell surface integrin receptors and extracellular matrix (ECM) components plays an important role in cell survival, proliferation, and migration, including tumor development and invasion of tumor cells. Matrix metalloproteinases (MMPs) are a family of metalloproteinases capable of digesting ECM components and are important molecules for cell migration. Binding of ECM to integrins initiates cascades of cell signaling events modulating expression and activity of different MMPs. The aim of this study is to investigate fibronectin-integrin-mediated signaling and modulation of MMPs. Our findings indicated that culture of human cervical cancer cell (SiHa) on fibronectin-coated surface perhaps sends signals via fibronectin-integrin-mediated signaling pathways recruiting focal adhesion kinase (FAK) extracellular signal regulated kinase (ERK), phosphatidyl inositol 3 kinase (PI-3K), integrin-linked kinase (ILK), nuclear factor-kappa B (NF-kappaB), and modulates expression and activation of mainly pro-MMP-9, and moderately pro-MMP-2 in serum-free culture medium.

Aspirin suppresses tumor cell-induced angiogenesis and their incongruity.

Tumor neovascularization/tumor angiogenesis is a pathophysiological process in which new blood vessels are formed from existing blood vessels in the primary tumors to supply adequate oxygen and nutrition to cancer cells for their proliferation and metastatic growth to the distant organs. Therefore, controlling tumor angiogenesis is an attractive target for cancer therapy. Structural abnormalities of the vasculature (i.e., leakiness due to the abnormal lining of pericytes on the microvessels) are one of the critical features of tumor angiogenesis that sensitizes vascular cells to cytokines and helps circulating tumor cells to metastasize to distant organs. Our goal is to repurpose the drugs that may prevent tumor angiogenesis or normalize the vessels by repairing leakiness via recruiting pericytes or both. In this study, we tested whether aspirin (ASA), which could block primary tumor growth, regulates tumor angiogenesis. We investigated the effects of low (1 mM) and high (2.5 mM) doses of ASA (direct effect), and ASA-treated or untreated triple negative breast cancer (TNBC) cells' conditioned media (indirect effect) on endothelial cell physiology. These include in vitro migration using modified Boyden chamber assay, in vitro capillary-like structure formation on Matrigel, interactions of pericytes-endothelial cells and cell permeability using in vitro endothelial permeability assay. We also examined the effect of ASA on various molecular factors associated with tumor angiogenesis. Finally, we found the outcome of ASA treatment on in vivo tumor angiogenesis. We found that ASA-treatment (direct or indirect) significantly blocks in vitro migration and capillary-like structure formation by endothelial cells. Besides, we found that ASA recruits pericytes from multipotent stem cells and helps in binding with endothelial cells, which is a hallmark of normalization of blood vessels, and decreases in vitro permeability through endothelial cell layer. The antiangiogenic effect of ASA was also documented in vivo assays. Mechanistically, ASA treatment blocks several angiogenic factors that are associated with tumor angiogenesis, and suggesting ASA blocks paracrine-autocrine signaling network between tumor cells and endothelial cells. Collectively, these studies implicate aspirin with proper dose may provide potential therapeutic for breast cancer via blocking as well as normalizing tumor angiogenesis.

CYR61/CCN1 Regulates dCK and CTGF and Causes Gemcitabine-resistant Phenotype in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) develops extrinsic- and intrinsic-resistant phenotypes to prevent chemotherapies from entering into the cells by promoting desmoplastic reactions (DR) and metabolic malfunctions of the drugs. It is well established that these responses are also associated with pancreatic cancer cells' gemcitabine resistance. However, the mechanism by which these resistant pathways function in the pancreatic cancer cells remains poorly understood. In these studies, we show that CYR61/CCN1 signaling plays a vital role in making pancreatic cancer cells resistant to gemcitabine in vitro and also in a tumor xenograft model. We proved that the catastrophic effect of gemcitabine could significantly be increased in gemcitabine-resistant PDAC cells when CYR61/CCN1 is depleted, while this effect can be suppressed in gemcitabine-sensitive neoplastic cells by treating them with CYR61/CCN1 recombinant protein. Ironically, nontransformed pancreatic cells, which are sensitive to gemcitabine, cannot be resistant to gemcitabine by CYR61/CCN1 protein treatment, showing a unique feature of CYR61/CCN signaling that only influences PDAC cells to become resistant. Furthermore, we demonstrated that CYR61/CCN1 suppresses the expression of the gemcitabine-activating enzyme deoxycytidine kinase (dCK) while it induces the expression of a DR-promoting factor CTGF (connective tissue growth factor) in pancreatic cancer cells in vitro and in vivo Thus, the previously described mechanisms (dCK and CTGF pathways) for gemcitabine resistance may be two novel targets for CYR61/CCN1 to protect pancreatic cancer cells from gemcitabine. Collectively, these studies reveal a novel paradigm in which CYR61/CCN1regulates both extrinsic and intrinsic gemcitabine resistance in PDAC cells by employing unique signaling pathways.

Purification and characterization of a low molecular weight multifunctional cytotoxic phospholipase A2 from Russell's viper venom.

A basic toxin from Russell's viper venom of 7.2 kDa (RVV-7) has been purified to homogeneity after partial unfolding by 4 M urea followed by filtration through Centricon-30 membrane. Its N-terminal sequence showed strong homology with snake venom cytotoxins. Cytotoxic activity of RVV-7 has been demonstrated with B16F10 melanoma cells. PLA2 activity was observed in cytotoxin (CX3) from Naja kauthia bearing sequence homology with RVV-7. Phospholipase A2 and trypsin inhibitory activities were also observed with RVV-7. Chemical modification and inhibition studies suggested independent functional sites for these activities. A qualitative assessment of tumor growth inhibition by RVV-7 has been made.

Detection of CCN1 and CCN5 mRNA in Human Cancer Samples Using a Modified In Situ Hybridization Technique.

In situ hybridization is an ideal tool for the detection and localization of mRNA expression of specific gene(s) in tissue sections and cell lines for prognosis, predictive markers, and highlighted potential therapeutic targets. Given the importance of CCN1 and CCN5 in breast and pancreatic cancer progression, these two secretory proteins could be novel therapeutic targets. Thus, evaluating the distribution of mRNA of these targets using in situ hybridization could be important preclinical tools. This chapter describes a detailed in situ hybridization technique for the detection of CCN1 and CCN5 in formalin-fixed, paraffin-embedded patient samples of breast and pancreatic cancers.

Deficiency of CCN5/WISP-2-Driven Program in breast cancer Promotes Cancer Epithelial cells to mesenchymal stem cells and Breast Cancer growth.

Breast cancer progression and relapse is conceivably due to tumor initiating cells (TICs)/cancer stem cells. EMT (epithelial-mesenchymal-transition)-signaling regulates TICs' turnover. However, the mechanisms associated with this episode are unclear. We show that, in triple-negative-breast cancer (TNBC) cells enriched with TICs, CCN5 significantly blocks cellular growth via apoptosis, reversing EMT-signaling and impairing mammosphere formation, thereby blocking the tumor-forming ability and invasive capacity of these cells. To corroborate these findings, we isolated tumor-initiating side populations (SP) and non-side population (NSP or main population) from MCF-7 cell line, and evaluated the impact of CCN5 on these subpopulations. CCN5 was overexpressed in the NSP but downregulated in the SP. Characteristically, NSP cells are ER-α positive and epithelial type with little tumorigenic potency, while SP cells are very similar to triple-negative ones that do not express ER-α- and Her-2 and are highly tumorigenic in xenograft models. The overexpression of CCN5 in SP results in EMT reversion, ER-α upregulation and delays in tumor growth in xenograft models. We reasoned that CCN5 distinguishes SP and NSP and could reprogram SP to NSP transition, thereby delaying tumor growth in the xenograft model. Collectively, we reveal how CCN5-signaling underlies the driving force to prevent TNBC growth and progression.

Human pancreatic cancer progression: an anarchy among CCN-siblings.

Decades of basic and translational studies have identified the mechanisms by which pancreatic cancer cells use molecular pathways to hijack the normal homeostasis of the pancreas, promoting pancreatic cancer initiation, progression, and metastasis, as well as drug resistance. These molecular pathways were explored to develop targeted therapies to prevent or cure this fatal disease. Regrettably, the studies found that majority of the molecular events that dictate carcinogenic growth in the pancreas are non-actionable (potential non-responder groups of targeted therapy). In this review we discuss exciting discoveries on CCN-siblings that reveal how CCN-family members contribute to the different aspects of the development of pancreatic cancer with special emphasis on therapy.

Exosomes in carcinogenesis: molecular palkis carry signals for the regulation of cancer progression and metastasis.

Exosomes, which act as biological cargo vessels, are cell-released, phospholipid-enclosed vesicles. In eukaryotic cells, exosomes carry and exchange biological materials or signals for the benefit or detriment to the cells. Thereby, we consider exosomes to be molecular Palkis (carriers). Although exosomes are currently one of the most popularly researched cellular entities, they have remained largely enigmatic and warrant continued investigation into their structure and functions. These membraned vesicles are between 30 and 150 nm in diameter and are actively secreted by all cell types. While initially considered cellular "trash bags," recent years have revealed exosomes to be dynamic and multi-functional vesicles that may play a crucial role in cancer development, progression and metastasis. Thereby, they have the potential to be used in development of therapeutic modalities for cancer and other diseases. As more research studies emerge, it's becoming evident that exosomes are released by cells with a purpose and are representatives of certain cell types and disease conditions. Hence, they may also be used as biomarkers for the detection of cancer initiation, progression and organotropic metastatic growth of cancer cells. This review will focus on the recent developments achieved in identifying the role of exosomes in cancer development and progression as well as therapeutic implications. The review will also discuss the pitfalls of methodologies used for the extraction of exosomes.

Pancreatic tumor cell secreted CCN1/Cyr61 promotes endothelial cell migration and aberrant neovascularization.

The complex signaling networks between cancer cells and adjacent endothelial cells make it challenging to unravel how cancer cells send extracellular messages to promote aberrant vascularization or tumor angiogenesis. Here, in vitro and in vivo models show that pancreatic cancer cell generated unique microenvironments can underlie endothelial cell migration and tumor angiogenesis. Mechanistically, we find that pancreatic cancer cell secreted CCN1/Cyr61 matricellular protein rewires the microenvironment to promote endothelial cell migration and tumor angiogenesis. This event can be overcome by Sonic Hedgehog (SHh) antibody treatment. Collectively, these studies identify a novel CCN1 signaling program in pancreatic cancer cells which activates SHh through autocrine-paracrine circuits to promote endothelial cell migration and tumor angiogenesis and suggests that CCN1 signaling of pancreatic cancer cells is vital for the regulation of tumor angiogenesis. Thus CCN1 signaling could be an ideal target for tumor vascular disruption in pancreatic cancer.

Laminin induces matrix metalloproteinase-9 expression and activation in human cervical cancer cell line (SiHa).

PURPOSE: Interaction between cell surface integrin receptors and extracellular matrix (ECM) components plays an important role in cell survival, proliferation and migration including tumor development and invasion. Matrix metalloproteinases (MMP) are a family of metalloproteinases capable of digesting ECM and facilitate cell migration. Binding of ECM to integrins initiates signaling cascades modulating expression and activity of different MMPs. The present study investigates whether laminin-mediated signaling modulates matrix metalloproteinases (MMP) expression and activity in human cervical cancer cell (SiHa). METHODS: Western blot, immunocytochemistry, ELISA, zymography, RT-PCR, EMSA and wound-healing assay were used. RESULTS: Culture of SiHa cells on laminin (LN)-coated surface induces MMP-9 expression and activation. Wound-healing assay showed that SiHa cells migrate much faster on laminin-coated surface than that of control. LN-induced MMP-9 expression and activation was appreciably reduced with treatment of extracellular signal-regulated kinase (ERK) inhibitor, phosphatidylinositol-3-kinase (PI-3K) inhibitor and anti-α2 antibody. Phosphorylation of focal adhesion kinase (FAK), ERK, and PI-3K was increased upon LN stimulation. LN induces nuclear translocation of PI-3K and nuclear factor kappa B (NF-κB). LN increases DNA-binding activity of NF-κB and activator protein-1 (AP-1) to MMP-9 promoter. CONCLUSIONS: Our findings indicate laminin-induced MMP-9 expression and activation possibly via α2ß1 integrin-mediated signaling involving FAK, PI-3K, ERK followed by transcriptional upregulation of MMP-9.

Thermal detoxification of the venom from Daboia russelli russelli of Eastern India with restoration of fibrinolytic activity.

The fibrinolytic components of venom have been evaluated for long in the enzymatic treatment of thrombosis. Russell's viper venom has fibrinolytic activity that is associated with hemorrhagic activity. Here it has been investigated whether the crude venom could be detoxified by thermal denaturation retaining fibrinolytic activity. The venom at 0.05 mg/ml in 20 mM K-phosphate, pH 7.5 when exposed to 100 °C for 5 min followed by cooling at 25 °C for 1 h led to its detoxification, while 80-85% of the fibrinolytic activity was recovered. Assessment of toxicity of the renatured venom in mice after injection at 5 fold excess of the lethal dose showed no lethality including hemorrhage, myotoxicity, cytotoxicity and liver toxicity. This simple method for preparation of fibrinolytic component for therapeutic use may be developed.

Fibronectin induces matrix metalloproteinase-9 (MMP-9) in human laryngeal carcinoma cells by involving multiple signaling pathways.

Cell adhesion to extracellular matrix initiates intracellular signaling cascade regulated by integrin family of receptors. Evidences show that cultured cells in presence of extracellular matrix adhesion molecule Fibronectin (FN) stimulates secretion of matrix metalloproteinases (MMPs), facilitating cancer cell invasion. Amongst all MMPs, MMP-9 is often reported to play crucial role in tumor cell growth and metastasis. The present study aims at examining the effects of FN on MMP-9 in laryngeal carcinoma cell line, HEp-2, and understand the molecular mechanism(s) involved. The study reports that FN induces the gelatinolytic activity, mRNA and protein expression of MMP-9 in HEp-2 cells. This effect appears to be mediated mainly by integrin receptor α5ß1, since, the blockade of α5 abrogated the FN-mediated stimulatory response on MMP-9. siRNA and inhibitor studies suggested involvement of Focal adhesion kinase (FAK), Phosphatidyl-inositol-3-kinase (PI-3K), Extracellular regulated kinase (ERK) and nuclear factor-kappaB (NFκB) in FN-mediated MMP-9 induction. Immunocytochemical analysis demonstrated the nuclear localization of ERK, PI-3K and NFκB; immunoblot showed enhanced expression of p-FAK, p-PI-3K, p-ERK and nuclear-NF-κB and indicated involvement of ILK in the FN-mediated response. FN-induced transactivation of MMP-9 gene by enhanced DNA binding activity of transcription factors NFκB, Activator protein-1 (AP-1) and Specificity protein-1 (Sp1) to the MMP-9 promoter. Thus, this study suggests that extracellular matrix protein FN induces MMP-9 in HEp-2 cells mainly by involving integrin receptor α5ß1 and involves activation of multiple signaling pathways which independently or in "cross-talk" to each other finally leads to the transactivation of the MMP-9 gene.