Synthesis and Antitumor Activity of Brominated-Ormeloxifene (Br-ORM) against Cervical Cancer

Aberrant regulation of β-catenin signaling is strongly linked with cancer proliferation, invasion, migration, and metastasis, thus, small molecules that can inhibit this pathway might have great clinical significance. Our molecular modeling studies suggest that ormeloxifene (ORM), a triphenylethylene molecule that docks with β-catenin, and its brominated analogue (Br-ORM) bind more effectively with relatively less energy (−7.6 kcal/mol) to the active site of β-catenin as compared to parent ORM. Herein, we report the synthesis and characterization of a Br-ORM by NMR and FTIR, as well as its anticancer activity in cervical cancer models. Br-ORM treatment effectively inhibited tumorigenic features (cell proliferation and colony-forming ability, etc.) and induced apoptotic death, as evident by pronounced PARP cleavage. Furthermore, Br-ORM treatment caused cell cycle arrest at the G1-S phase. Mechanistic investigation revealed that Br-ORM targets the key proteins involved in promoting epithelial–mesenchymal transition (EMT), as demonstrated by upregulation of E-cadherin and repression of N-cadherin, Vimentin, Snail, MMP-2, and MMP-9 expression. Br-ORM also represses the expression and nuclear subcellular localization of β-catenin. Consequently, Br-ORM treatment effectively inhibited tumor growth in an orthotopic cervical cancer xenograft mouse model along with EMT associated changes as compared to vehicle control-treated mice. Altogether, experimental findings suggest that Br-ORM is a novel, promising β-catenin inhibitor and therefore can be harnessed as a potent anticancer small molecule for cervical cancer treatment

Role of Occludin in the Regulation of Epithelial Tight Junctions

Tight junctions (TJ) constitute the primary component of epithelial barrier function, a disruption of which is involved in the pathogenesis of many gastrointestinal, pulmonary and renal diseases. Occludin is the major transmembrane protein of TJ, a deletion of which leads to a complex phenotype including chronic inflammation in several epithelial tissues of occludin deficient mice and poor TJ integrity in epithelial cell lines. Its down regulation was seen in Crohn’s disease, tumors of the colon, brain, endometrium and breast cancer. Occludin is also known to be a target that enables Hepatitis C Virus infection and bacterial pathogenesis. But the specific function of occludin in TJ remains unknown. Numerous studies implicated that occludin phosphorylation plays a crucial role in TJ regulation. Based on our previous studies, we have identified a conserved phosphorylation hotspot in the C-terminal domain of occludin, called occludin regulatory motif (ORM), which has a potential to confer dynamic properties to occludin. The present study was designed to determine the function of ORM in TJ regulation. Stable, occludin-deficient MDCK cell line (OD-MDCK) that expresses wild type occludin (OclWT) or a deletion mutant occludin (OclDM) that lacked ORM were developed. Confocal imaging and immunoprecipitation analyses showed that absence of ORM does not prevent occludin localization and interaction with ZO-1 at TJ. However, FRAP analysis showed that mobile fraction of occludin is reduced by the absence of ORM, suggesting the role of ORM in occludin dynamics. Also, Ca2+ depletion-induced disruption of TJ, adherens junctions and cytoskeleton and barrier dysfunction were attenuated in the absence of ORM. Mutation anlaysis of ORM phosphosites Y 398, Y 402, T403 and T404, revealed that ORM-mediated phosphorylation events determine the dynamic properties of TJ. Then the role of ORM-mediated protein interactions in TJ modulation was investigated. A synthetic peptide analogous to occludin regulatory region (ORP), which enhanced TJ assembly and attenuated radiation-induced barrier dysfunction in Caco-2 cells, was used as bait to pull down proteins that interact with ORM. Proteomic analyses and pull down assays using ORP and GST tagged occludin C-terminus identified that MAP7 (an epithelial specific microtubule associated protein) interacts with ORM in a tyrosine phosphorylation-dependent mechanism. Furthermore, MAP7 binding to occludin in Caco-2 cells was elevated by treatment with osmotic stress or dextran sodium sulfate, both known to increase occludin Tyr-phosphorylation. These data demonstrated that MAP7 interacts with ORM and mediates occludin redistribution during TJ disruption. In conclusion ORM confers dynamic properties to TJ via its phosphorylation dependent protein-protein interactions. The findings in my project establish the functional significance of ORM in TJ dynamics and regulatory mechanisms associated with TJ modulation. Identification of proteins that interact with ORM provides new therapeutic targets in the treatment of diseases associated with occludin depletion and epithelial barrier dysfunction

Pharmacological restoration of PKD1: A novel strategy for prostate cancer therapy

Background: Prostate cancer has poor prognosis owing to late diagnosis and ineffective multimodal clinical treatment. Extensive efforts are ongoing to establish methods that can resolve the expression of genes implicated in disease development and treatment. Previously, we reported that Protein Kinase D1 (PKD1), a serine threonine kinase, controls a number of tumor suppressor functions including cell aggregation, cell motility, cell proliferation, and cell invasion. Thus, PKD1 is considered as an emerging therapeutic target for prostate cancer treatment. Objective: To investigate the restoration of PKD1 by a pharmacological modulator ormeloxifene, which showed well-defined PK/PD and safety profiles in humans. Methods: Proliferation, clonogenicity, migration, invasion, western blotting and qPCR analysis were performed to investigate the anticancer effect of ORM, docetaxel and/or their combination on PKD1 and related signaling mechanisms in prostate cancer. Results: ORM treatment inhibited cell proliferation, invasion, migration and colony formation abilities of prostate cancer cells in a dose-dependent manner compared to vehicle treated group. ORM treatment selectively induces the expression of PKD1 both at mRNA and protein levels in C4-2 cells. Moreover, our results have also shown that ORM effectively attenuates MTA1 expression in prostate cancer cells. MTA1 physically interact and shown to have inverse relationship with PKD1. In addition, we observed that ORM treatment enhances the therapeutic efficacy of docetaxel in C4-2 cells. Our results also indicate that ORM treatment potentiate the effects of docetaxel as determined by MTS and colony formation assays. Conclusion: These results suggest that ORM exhibit potent anticancer activity via restoration of PKD1 in prostate cancer

Long‐term effects of Na+/Ca2+ exchanger inhibition with ORM‐11035 improves cardiac function and remodelling without lowering blood pressure in a model of heart failure with preserved ejection fraction

Aims: Heart failure with preserved ejection fraction (HFpEF) is increasingly common but there is currently no established pharmacological therapy. We hypothesized that ORM-11035, a novel specific Na+/Ca2+ exchanger (NCX) inhibitor, improves cardiac function and remodelling independent of effects on arterial blood pressure in a model of cardiorenal HFpEF. Methods and results: Rats were subjected to subtotal nephrectomy (NXT) or sham operation. Eight weeks after intervention, treatment for 16 weeks with ORM-11035 (1 mg/kg body weight) or vehicle was initiated. At 24 weeks, blood pressure measurements, echocardiography and pressure–volume loops were performed. Contractile function, Ca2+ transients and NCX-mediated Ca2+ extrusion were measured in isolated ventricular cardiomyocytes. NXT rats (untreated) showed a HFpEF phenotype with left ventricular (LV) hypertrophy, LV end-diastolic pressure (LVEDP) elevation, increased brain natriuretic peptide (BNP) levels, preserved ejection fraction and pulmonary congestion. In cardiomyocytes from untreated NXT rats, early relaxation was prolonged and NCX-mediated Ca2+ extrusion was decreased. Chronic treatment with ORM-11035 significantly reduced LV hypertrophy and cardiac remodelling without lowering systolic blood pressure. LVEDP [14 ± 3 vs. 9 ± 2 mmHg; NXT (n = 12) vs. NXT + ORM (n = 12); P = 0.0002] and BNP levels [71 ± 12 vs. 49 ± 11 pg/mL; NXT (n = 12) vs. NXT + ORM (n = 12); P < 0.0001] were reduced after ORM treatment. LV cardiomyocytes from ORM-treated rats showed improved active relaxation and diastolic cytosolic Ca2+ decay as well as restored NCX-mediated Ca2+ removal, indicating NCX modulation with ORM-11035 as a promising target in the treatment of HFpEF. Conclusion: Chronic inhibition of NCX with ORM-11035 significantly attenuated cardiac remodelling and diastolic dysfunction without lowering systemic blood pressure in this model of HFpEF. Therefore, long-term treatment with selective NCX inhibitors such as ORM-11035 should be evaluated further in the treatment of heart failure

Characterization of different aspects of selective NCX inhibition in the heart: from inotropy to arrhythmias

The cardiac sodium-calcium exchanger (NCX) has a pivotal role in the Ca2+ homeostasis as well as in several types of arrhythmias; therefore the potential therapeutic application of its modification was intensively studied in the past decade. However, the inotropic and antiarrhythmic effect of its inhibition has not yet been fully clarified due to the lack of appropriately selective inhibitor. The recently developed novel selective NCX inhibitors, ORM-10103 and especially ORM-10962, provided new insights in the understanding of NCX physiology as well as the possible therapeutic implications of these new potential drugs as novel antiarrhythmic agents. This thesis summarizes: 1) The pharmacological profile of the novel NCX inhibitor ORM-10962, compared its selectivity with that of the previously described compound, ORM-10103. ORM-10962 proved to be even more selective and exerts improved effectiveness on NCX current having EC50 values in the nanomolar range, without any influence on ICaL or other currents. 2) In contrast to previous inhibitors like SEA0400 or ORM-10103, we found marginal positive inotropic effect of the ORM-10962 without major influence on the action potential under normal condition. 3) Marginal but statistically significant reduction by ORM-10962 in the spontaneous firing rate of the atrial and Purkinje AP may indicate important role of NCX in the spontaneous pacemaker mechanism, which is in the line with the previously described coupled clock hypothesis. 4) We suggest that NCX inhibition could equally lead to positive and negative inotropy, depending on the actual value of NCX reversal potential. Selective NCX inhibition may have negative inotropic effect when Ca2+ load is coupled with marked increase of intracellular Na+ facilitating the reverse mode activity. This hypothesis was tested in dog Purkinje fibres, where digoxin induced DADs amplitude and incidence were significantly reduced by ORM-10962

Molecular Insights into Targeting PKD1 for Prostate Cancer Treatment

Background: Prostate cancer has a poor prognosis due to late diagnosis and ineffective multimodal clinical treatment. Efforts are underway to create strategies for resolving the abnormal expression of molecular targets implicated in disease development and progression. We previously reported that the serine threonine kinase Protein Kinase D1 (PKD1) regulates a multitude of tumor suppressor functions, including cell aggregation, motility, proliferation, and invasion in prostate cancer. Thus, PKD1 is regarded as a promising therapeutic target for the treatment of prostate cancer. Objective: The goal of this study was to investigate the therapeutic potential of ormeloxifene (ORM), a pharmacological modulator with well-defined PK/PD and safety profiles in humans, for PKD1 restoration in prostate cancer. Methods: The anticancer effect of ORM on PKD1 and associated signaling mechanisms in prostate cancer was investigated using proliferation, clonogenicity, migration, invasion, western blotting, and qPCR analysis. Results: In comparison to the vehicle-treated group, ORM treatment decreased prostate cancer cell proliferation, invasion, migration, and colony formation in a dose-dependent manner. In C4-2 cells, ORM treatment selectively induces PKD1 expression at both the mRNA and protein levels. Furthermore, our findings revealed that ORM efficiently suppresses MTA1 expression in prostate cancer cells. MTA1 physically interacts with PKD1 and has been shown to have an inverse correlation with it. Our results also showed that ORM treatment enhances the therapeutic efficacy of decetaxel. Conclusion: Taken together, these findings show that ORM has anticancer properties in prostate cancer via restoring PKD1

The effect of oncoplastic reduction on the incidence of post-operative lymphedema in breast cancer patients undergoing lumpectomy

Purpose: In breast cancer patients with macromastia, breast conservation surgery (BCS) followed by radiation therapy (RT) may be associated with a different complication profile than those without macromastia. Oncoplastic reduction mammoplasty (ORM) aims to reduce breast volume while excising the tumor bed and its margins. Since breast volume was found to be a risk factor for chronic breast lymphedema, this study was performed to determine the impact of ORM on chronic breast lymphedema as well as other complications compared to BCS without ORM. Material & Methods: We performed a retrospective chart review on patients who underwent lumpectomy with RT from 2014 to 2018. Chronic breast lymphedema (CBL) was defined as swelling that persisted \u3e1 year post-RT. Breast volumes (BV) were determined by contoured breast volumes or, if unavailable, estimated by the 95% isodose volumes from the RT treatment planning system. Univariate analysis was used to evaluate patient factors and treatment outcomes in women with BV ≥1300 cc compared to-Evaluate factors associated with ≥1 complication. Identify factors associated with the development of CBL. Results: The total population included 1173 patients: -1122 (95.7%) underwent BCS alone without ORM -51 (4.3%) underwent ORM -733 (62.5%) had a BVcc -440 (37.5%) had BV ≥1300 cc Multivariate regression analysis demonstrated that compared to patients with BV \u3c 1300 cc, patients with BV ≥1300 cc had: -Higher BMI (OR=1.200, P\u3c0.001) -Increased risk of CBL (OR=2.127, P=0.024) -Decreased risk of grade 2 radiation dermatitis (OR=0.457, P=0.002) Conclusion: Our data demonstrates that patients with breast volumes ≥1300 cc were two times more likely to develop CBL. Although patients with ORM had an increased risk for surgical site complications, the ORM procedure may have mitigated their risk for CBL. ORM should be considered at the time of BCS in women with macromastia to reduce their future risk of CBL as there is no cure for this disease.https://scholarlycommons.henryford.com/sarcd2021/1008/thumbnail.jp

Object-Relational Mapping on PHP Platform

Práce se zabývá problematikou objektově-relačního mapovaní na platformě PHP. První část obsahuje obecný popis technologie ORM včetně návrhových vzorů pro ni určených. V další části jsou popsány dva nejvýznamnější ORM frameworky, Doctrine 2 a Propel. Doctrine 2 se práce věnuje více podrobněji, protože právě tento framework je použit v ukázkové aplikaci, která znázorňuje, jak technologii ORM integrovat do svého projektu a jak s ní efektivně pracovat. Cílem této práce je seznámit čtenáře s technologii ORM a motivovat ho k použití ORM ve svých projektech.The thesis deals with an issue of object-relational mapping on the PHP platform. The first part contains general description of the ORM technology including design patterns meant for the technology. In the next part are described the two most important ORM frameworks - Doctrine 2 and Propel. The thesis is focused on Doctrine 2 as this framework is used in an exemplary application which represents how can be the ORM technology integrated to a project and how it can be effectively used. The aim of this thesis is to introduce the ORM technology to readers and to motivate them to use the ORM technology in their projects.

Urinary proteomics and the role of orosomucoid (ORM) in vascularization of bladder cancer

2-dimensional gel electrophoresis (2-DE) is currently the method of choice for separation of complex protein mixtures such as in cell and tissue extracts or body fluids including urine or serum. Human urine plays a central role in clinical diagnostics of diseases such as cancer and inflammation. Researchers and scientists are working on the development of a map of the human proteome but in the literature there are only a few investigations about urinary proteomics and their behaviour in the case of bladder tumor. The first aim of this study was to view the whole proteins present in urine and to predict their functions. For this aim, fourtyfive urine samples from patients with bladder cancer of different stages, patients on follow-up and healthy volunteers were analysed by 2-DE. At the beginning of this study the 2-DE protein pattern of human urine was almost unknown because of lack of appropriate preparation of urine samples prior to the 2-DE. Thus, the study focused on methods for preparation of urine samples providing the best preservation of urinary proteins. After experimental optimization of 2-DE, usable 2-D protein patterns of urine were analysed to identify proteins related to bladder cancer using subsequent mass spectrometric analyses and/or conventional immunoblotting and immunohistochemical methods. In comparison to other cancer types, the bladder carcinoma is the seventh causing death in man and ninth in women among malignant tumors. The growth and metastasis as well as the transformation of superficial noninvasive tumors to an invasive tumor phenotype are closely associated with activation of angiogenesis resulting in neovascularisation of tumor tissue. This process is regulated by a net balance between angiogenic activators and inhibitors. In the present study, orosomucoid (ORM) and human zinc-alpha-2-glycoprotein (ZAG) have been identified to be increased in 2-DE of urine samples of patients with bladder cancer in comparison to the urine samples of healthy volunteers. Immunohistochemical results let assume that in addition to cancer cells also a part of the tissue resident inflammatory cells and endothelial cells of tumor associated blood vessels may serve as source for this increase of ORM in urine samples of patient with bladder cancer. ORM is an acute phase protein and increased in acute infection, inflammation, and cancer. Recent studies show that ORM forms a complex with the active form of plasminogen activator inhibitor-1 (PAI-1) in thymosin β4 (Tβ4)- activated but not in quiescent endothelial cells. Therefore it was aimed to determine a potential role of ORM up-regulation in angiogenesis. The findings presented in this study and results reported by Sorensson showed, that, in addition to the cancer cells, human vascular endothelial cells (HDMECs) produce ORM endogenously. For functional characterization of ORM in vascular endothelial cells, ORM-gene overexpression and ORM-gene silencing were perfromed. Employing the supernatants of HDMECs-ORM and HDMECs-ORM-siRNA in in-vitro angiogenesis assays, it was found that ORM supports the VEGF-induced endothelial tube formation. This supportive effect of ORM was potentiated by co-treatment of HDMECs with VEGF, ORM and anti-PAI-1 antibody. This study demonstrates for the first time that ORM is increased in urine of patients with bladder cancer and acts pro-angiogenic supporting the tube forming effects of VEGF. This supportative effect was significantly increased by additive blockage of PAI-1. In summary, the interaction between ORM and PAI-1 and anti-PAI-1 system seems to be essentially involved in the VEGF-mediated angiogenesis and probably in the vascularisation of urinary bladder cancer