Inhibition Of Motility Research Articles

Abstract 6319: Mechanism of anti-EGFR antibody endostatin fusion protein action on inhibition of vasculogenic mimicry and tumor cell motility in triple negative breast cancer

Abstract Triple Negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer with high metastatic potential and increased morbidity and mortality. Although expression of and signaling by the epidermal growth factor receptor (EGFR) is commonly seen in TNBC, anti-EGFR antibodies such as Cetuximab have had limited therapeutic efficacy, used alone or in combination with chemotherapy. Primary TNBC tumor growth and metastases require supporting vasculature, which develops through a combination of endothelial angiogenesis and vasculogenic mimicry (VM). VM is frequently seen in TNBC and is associated with aggressive metastatic behavior. We previously developed αEGFR-E-P125A, an antibody-endostatin fusion protein, linking an anti-EGFR antibody to a mutated version of the anti-angiogenic protein endostatin (E-P125A). αEGFR-E-P125A delivers a dimeric E-P125A payload which inhibits TNBC angiogenesis and VM in vitro and in vivo, and markedly decreases metastasis in multiple TNBC xenograft models. To study changes in the transcriptome following induction of VM and inhibition of VM with αEGFR-E-P125A, RNA-seq was conducted on MDA-MB-231-4175 TNBC cells grown in a 2D monolayer and compared to TNBC cells undergoing VM when plated in matrigel. A second comparison was made between TNBC cells undergoing VM and TNBC cells in which VM was inhibited by treatment with αEGFR-E-P125A. Differential gene expression analysis and gene set enrichment analysis (GSEA), were used to define pathways which were inversely regulated during the induction of VM on matrigel and inhibition of VM with αEGFR-E-P125A. Gene set enrichment analysis demonstrated that αEGFR-E-P125A treatment significantly downregulated genes in the JAK-STAT and angiogenesis signaling pathways. Phospho-array analysis demonstrated decreased EGFR phosphorylation at Y1069 in addition to decreased phosphorylation of FAK Y397 and STAT3 Y705 sites downstream of α5β1 integrin. Since inhibition of EGFR signaling alone did not inhibit VM, and endostatin is a known ligand of α5β1 integrin, we focused on effects of αEGFR-E-P125A on inhibition of α5β1 integrin/FAK signaling pathway. Independent siRNA knockdowns of α5 integrin and FAK in MDA-MB-231-4175 cells confirmed that downregulation of α5 integrin/FAK signaling inhibited VM in vitro. αEGFR-E-P125A additionally prevented EGF-induced EGFR phosphorylation and fibronectin-induced FAK phosphorylation by α5β1 integrin. Treatment of TNBC cells with αEGFR-E-P125A reduced total α5 integrin protein levels and decreased co-localization of EGFR and α5β1 integrin receptors. These results indicate that αEGFR-E-P125A suppressed both EGFR and α5β1 integrin signaling. Simultaneous inhibition of EGFR and α5β1integrin signaling by αEGFR-E-P125A fusion is a promising approach to inhibition of TNBC growth and metastases. Citation Format: Ankita Sankar, Hava Gil Henn, Hyun Mi Cho, Dania Nassar, Sundaram Ramakrishnan, Yu Zhang, Christian Elledge, Seung Uon Shin, Joseph Rosenblatt. Mechanism of anti-EGFR antibody endostatin fusion protein action on inhibition of vasculogenic mimicry and tumor cell motility in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6319.

Abstract 1299: Transcriptomic analysis to unveil alveolar rhabdomyosarcoma metastatic process

Abstract Background: Rhabdomyosarcoma (RMS) is the most prevalent soft tissue sarcoma in children and adolescents. It is divided in two major histological subtypes, being the Alveolar (ARMS) the one associated to a poorer prognosis1. The cure rate for these tumors is up to 70% for children with localized disease, but this numbers decrease notably to a 20% 5-year survival rate for patients that show metastatic spreading. However, mechanisms leading to metastatic disease remain poorly understood. Hence, there is an urgent need to better understand this process, which will hopefully lead to novel targeted therapies and improved survival rates for these patients. Methods: ARMS cells (RH4) were injected in the gastrocnemius muscle of Athymic Nude-Foxn1nu mice2. Primary tumors (800mm3) and peritoneal metastases (visible by IVIS®) were collected, and RNA extracted. Xenograft-derived metastatic cell lines were obtained directly as a primary culture from mice metastases. Characterization of paired tumors and metastases was performed using Clariom™ D microarray (Applied Biosystems). After data normalization and comparative analyses (R; RMA and limma), a list of differentially expressed transcripts between sample groups was obtained. These are being validated by qPCR and Western Blot. Results: Transcriptomic profiling of tumors (n=3), paired metastases (n=3), xenograft-derived metastatic cell lines (n=3) and RH4 cell line (n=3) revealed 3420 differentially expressed transcripts (2747 increased in metastases, 673 in tumors). Several transcripts of interest are overexpressed in metastases samples compared to primary tumors as showed by microarray analysis and validated by qPCR. These targets are related in literature to metastasis in other cancers, as well as lymph nodes metastasis, which occurs to happen in our model and in ARMS clinics3. Additionally, some transcripts are overexpressed in tumor samples, which are linked to inhibition of proliferation and motility. Conclusions/Future Work: In vitro phenotypic characterization of the obtained targets will involve proliferation (MTT), migration (Transwell®) and clonogenic assays, including loss-of-function (siRNA and commercial inhibitors). Using stable knock-out and knock-in models of the genes of interest, their role in ARMS metastasis will be further studied in vitro and in vivo. The clinical relevance of these targets will be explored in several cohorts of patients by immunohistochemistry on Tissue Microarrays. Studying the profile of paired tumors and metastases provides a unique tool to decipher the mechanisms of tumor progression. Fully understanding the metastatic process will ultimately lead to the development of better targeted therapies and thus to improved outcomes for ARMS patients. Also, these same steps are being followed in our laboratory for Ewing Sarcoma, another developmental tumor with discouraging survival rates for metastatic patients (<30%)4. Citation Format: Sara Sanchez-Serra, Mariona Chicon-Bosch, Paola Monaco, Laura Santana-Viera, Susana Maqueda-Marcos, Manuel Gris-Lorente, Roser Lopez-Alemany, Oscar M. Tirado. Transcriptomic analysis to unveil alveolar rhabdomyosarcoma metastatic process [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1299.

Abstract 2028: Using bioinformatic analysis of proteomic data to probe novel drug mechanism of action

Abstract Introduction: Metastasis is a major cause of death for patients with solid tumors. Our group has discovered a selective inhibitor of cell motility, KBU2046, that blocks the activation of Raf1 by inhibiting the phosphorylation of ser338 on its activation motif (Nature Communications 2018). To better understand the effect of KBU2046 on human prostate cancer (PCa) cells, we undertook a proteomic analysis. Experimental Procedures: After treatment of PC3 cells with KBU2046 or vehicle (N=3/group), protein expression of whole cell lysate was quantified by Liquid Chromatography/Tandem Mass Spectrometry analysis (LC-MS/MS). In separate experiments (N=4), membrane fractions were isolated, and LC-MS/MS was performed after Tandem Mass Tag (TMT) labeling. Resultant data were analyzed by several complementary bioinformatics tools, including: Gene Set Enrichment Analysis (GSEA), Gene Ontology (GO) term analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, Ingenuity Pathway Analysis (IPA), and protein-protein interaction (PPI) network analysis to characterize the proteins that were significantly differentially expressed upon KBU2046 treatment. Results: Independent bioinformatic analyses of differentially expressed proteins affected by KBU2046 treatment identify significantly enriched processes related to cell motility, inclusive of effects on structural machinery. This constitutes orthogonal confirmation of KBU2046’s known effects on cell motility. Of importance, major effects on cellular energy-generating processes were also identified. This is consistent with prior studies that found changes in motility induce obligatorye changes in cellular energy requirements. Our focused analysis of membrane fractions identified changes in membranous compartments, providing a relevant positive control for bioinformatic analysis, which we confirmed by Western blot. Analytics also pointed to a major role for adhesion proteins in response to KBU2046, consistent with our prior identification of primary effects on cell migration. Conclusions: These findings provide orthogonal mechanistically relevant support for a novel acting agent. They highlight the ability of proteomics and bioinformatic tools to efficiently probe the mechanism of action and global impact on PCa in response to treatment. Citation Format: Weining Chen, Nicholas Woods, Fangfang Qiao, Henry Chun Hin Law, Sankarasubramanian Jagadesan, Chittibabu Guda, Raymond C. Bergan. Using bioinformatic analysis of proteomic data to probe novel drug mechanism of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2028.

Lipopeptide surfactant killing of Ichthyophthirius multifiliis: Mode of action

The lipopeptide SPH6 isolated from the bacterial strain Pseudomonas H6 has surfactant properties and shows a lethal effect on all external stages of the problematic gill parasitic ciliate Ichthyopthirius multifiliis. The mode of action has remained enigmatic, but in the present study we have elucidated the mechanisms. I. multifiliis tomonts were exposed to a range of SPH6 concentrations (from 2.5 to 160 µg/mL), until the total motility inhibition of cells, cytoplasm and cilia. The parasites were subsequently examined by histology, light and scanning electron microscopy and enzyme activity assays (19 enzymes). The surfactant did not interfere with enzyme reactivity at any concentration, but at SPH6 concentrations 40 µg/mL and above the parasite cells became de-ciliated. Perforations were observed in the plasma membrane, and their origin and function may be associated with surfactant damage of the cell membrane or areas for extrusome release. Additional killing mechanisms may be involved, because lower concentrations were lethal as well, although the effect on density of cilia was not as pronounced. The study suggests that one mode of action mechanism is the surfactant attachment to and penetration of the parasite plasma membrane followed by detachment of cilia.

Clinostomum complanatum: Anthelmintic potential of curcumin on the infective progenetic metacercarial stage

The emerging resistance against commonly used antiparasitic drugs has driven investigators to explore alternative approaches using plant-derived active ingredients. These compounds have been tested for antiviral, antibacterial, and anthelmintic properties, particularly against adult worms. However, their effects on larval forms have been neglected. Curcumin is a polyphenol that is a significant constituent of the rhizome of Curcuma longa and possesses various biological activities, including antioxidant, anti-inflammatory, anti-infectious, and anti-carcinogenic. In the present study, the anthelmintic potential of curcumin was tested in vitro for its efficacy against the zoonotically important larval form, the progenetic metacercariae of Clinostomum complanatum, which were procured from the forage fish, Trichogaster fasciatus. Curcumin produced time and concentration-dependent inhibition in the motility of treated metacercarial worms, with the maximum inhibition of motility reported at 60 μM along with a significant increase of (36–92%) in ROS and (57–112%) in GSH levels at the end of a period of 6 h. In contrast, curcumin at the highest concentration significantly inhibited the activities of the antioxidant and detoxification enzymes SOD (36%) and GST (16%), respectively, in addition to altering the polypeptide profile and inhibiting cysteine proteases. The tegumental surface appeared to be highly disrupted in curcumin-treated worms, exhibiting severe blebbing, shearing of the tegument, and spine erosion. Such changes would affect the tegumental functions and survival of worms in the hostile microenvironment. This would render worms more susceptible to host-mediated rejection responses. Based on the results of the present study, it is inferred that C. complanatum could serve as an excellent model for screening novel anthelmintic drugs against larval trematodes of great economic significance. Furthermore, we conclude that curcumin could be exploited as an excellent phytotherapeutic agent against the virulent larval form under investigation.

In vitro Anthelmintic Activity of Vitis vinifera Leaf Extract on Dicrocoelium dendriticum

Background: Helminth parasites of ruminants are a major factor limiting animal production in many parts of the world. Dicrocoelium dendriticum, causes liver fluke disease in ruminants and is of zoonotic and economic importance. D. dendriticum lives in the adult stage, in the bile ducts and gallbladders of cattle, sheep, goats and pigs. Dicrocoeliasis causes severe pathological changes in the liver and bile system such as abscesses, granulomas and fibrosis. The objective of this study was to identify the anthelmintic activity of Vitis vinifera leaf extract against D. dendriticum and comparison with some drugs used against internal parasites. Methods: A study was performed to evaluate the anthelmintic activity of the methanolic extract of V. vinifera leaf against D. dendriticum using an adult worm motility test. Six graduated concentrations of extract (25, 50, 75, 100, a125 and 150 mg/ml), were tested at different periods and changes over time in the viability of worms were registered for 2, 4 and 6 hr. Normal RPMI-1640 medium and Albendazole were used as negative and positive control, respectively. Result: A significant elevation in mean inhibition of adult worm motility beginning 2 h post-exposure with 150 mg/ml of V. vinifera leaf extract and Albendazole. After 4 h of the exposure time, Albendazole and concentrations of 125 and 150 mg/ml of the extract resulted in significantly higher inhibited motility compared to the remaining concentrations below 125 mg/ml. The lower concentration (25 mg/ml) was significantly more lethal than the negative control (RPMI-1640 medium) at 6 h of exposure. Within 6 h after exposure, concentrations of 100, 125 and 150 mg/ml of V. vinifera leaf extract were more effective against adult worms than the remaining concentrations below 100 mg/ml. As the concentration and duration of exposure increased, the mortality of adult worms and also increased death for all parasites (P less than 0.05). Each concentration damages the tegument and muscles of D. dendriticum. The increase in concentration is proportional to the increase in damage to the integument. The results of this investigation demonstrated the anthelmintic action of V. vinifera leaf extract.

Flagella-driven motility is a target of human Paneth cell defensin activity.

In the mammalian intestine, flagellar motility can provide microbes competitive advantage, but also threatens the spatial segregation established by the host at the epithelial surface. Unlike microbicidal defensins, previous studies indicated that the protective activities of human α-defensin 6 (HD6), a peptide secreted by Paneth cells of the small intestine, resides in its remarkable ability to bind microbial surface proteins and self-assemble into protective fibers and nets. Given its ability to bind flagellin, we proposed that HD6 might be an effective inhibitor of bacterial motility. Here, we utilized advanced automated live cell fluorescence imaging to assess the effects of HD6 on actively swimming Salmonella enterica in real time. We found that HD6 was able to effectively restrict flagellar motility of individual bacteria. Flagellin-specific antibody, a classic inhibitor of flagellar motility that utilizes a mechanism of agglutination, lost its activity at low bacterial densities, whereas HD6 activity was not diminished. A single amino acid variant of HD6 that was able to bind flagellin, but not self-assemble, lost ability to inhibit flagellar motility. Together, these results suggest a specialized role of HD6 self-assembly into polymers in targeting and restricting flagellar motility.

Sustainable Protocols for Cellulose Nanocrystals Synthesis from Tomato Waste and Their Antimicrobial Properties against Pseudomonas syringae pv. tomato

Nanotechnology is rapidly gaining ground in crop protection, with the growing quest for sustainable nanopesticides and nanocarriers for plant pathogen management. Among them, cellulose nanocrystals (CNC) are emerging as innovative agrofood-waste-derived antimicrobial materials. In this work, new chemical and enzymatic CNC extraction methods from tomato harvest residues were evaluated. The obtained nanomaterials were characterized and tested for their antimicrobial properties on Pseudomonas syringae pv. tomato (Pto), the causal agent of bacterial speck disease on tomato. Both protocols were efficient. The enzymatic extraction method was greener, producing purer CNC at slightly lower yield. The obtained CNC, although they weakly inhibited cell growth and did not promote reactive oxygen species (ROS) formation, provoked bacterial aggregation and the inhibition of biofilm production and swimming motility. Both protocols produced CNC with similar morpho-chemical features, as well as promising antimicrobial activity against plant bacterial pathogens, suggesting their potential role in sustainable crop protection strategies. The new protocols could be a valuable alternative to conventional methods.

Caldesmon enhances motility inhibition by myosin dephosphorylation.

Some studies have suggested that smooth muscle caldesmon (hCaD) participates in force maintenance at low myosin light chain (LC20) phosphorylation. To elucidate the effects of hCaD on the molecular mechanics of dephosphorylating smooth muscle myosin, we injected myosin light chain phosphatase (MLCP) during in vitro motility measurements performed in the presence of hCaD (400 nM) or its absence. Because hCaD increases the binding force of unphosphorylated smooth muscle myosin to actin, we hypothesized that, upon LC20 dephosphorylation, the fraction (fmot) and the average velocity (vavg) of moving actin filaments would decrease at a greater rate in the presence of hCaD. We fitted sigmoidal curves to fmot and vavg and estimated the rate constant (k) and the midpoint (t0) of the decrease. k was larger in the presence of hCaD (3.6×10−1±6.4×10−2 s−1) than without (1.8×10−1±1.4×10−2 s−1), p = 0.039, n = 5. Additionally, t0 was smaller in the presence of hCaD (32±3 s) than without (44±2 s), p = 0.008, n = 5. These results can be explained either by an increased dephosphorylation rate in the presence of hCaD or by an increase of the reattachment of dephosphorylated myosin to actin. In line with previous experiments that demonstrated that hCaD inhibits the actomyosin ATPase, our sigmoidal fits also show a lower vavg in the presence of hCaD (0.45±0.04 µm/s) than without (0.63±0.06 µm/s), p=0.04, before the addition of MLCP. However, hCaD had no effect on k or t0 of vavg during MLCP injection. The lack of concordance between the fmot and vavg data may be due to the fact that the vavg data are noisier after dephosphorylation. Our results agree with previous data obtained at the whole muscle level that showed that hCaD increases the rate of relaxation of arterial smooth muscle and accelerates the decay in tension of skinned smooth muscle strips.

Ethoxysanguinarine Induces Apoptosis, Inhibits Metastasis and Sensitizes cells to Docetaxel in Breast Cancer Cells through Inhibition of Hakai.

Ethoxysanguinarine (ESG) is a benzophenanthridine alkaloid extracted from plants of Papaveraceae family, such as Macleaya cordata (Willd) R. Br. The anti-cancer activity of ESG has been rarely reported. In this study, we investigated the anti-breast cancer effect of ESG and its underlying mechanism. MTT assay and flow cytometry analysis showed that ESG inhibited the viability and induced apoptosis in MCF7 and MDA-MB-231 human breast cancer cells. Western blot revealed that ESG triggered intrinsic and extrinsic apoptotic pathways, as evidenced by the activation of caspase-8, caspase-9 and caspase-3. ESG attenuated breast cancer cell migration and invasion through Hakai/E-cadherin/N-cadherin. Moreover, Hakai knockdown sensitized ESG-triggered viability and motility inhibition, suggesting that Hakai mediated the anti-breast cancer effect of ESG. In addition, ESG potentiated the anti-cancer activity of docetaxel (DTX) in breast cancer cells. Overall, our findings demonstrate that ESG exhibits outstanding pro-apoptosis and anti-metastasis effects on breast cancer via a mechanism related to Hakai-related signaling pathway.

Influence of cyclic di-GMP metabolism to T3SS expression, biofilm formation and symbiosis efficiency in Mesorhizobium japonicum MAFF303099.

A link between the T3SS and inhibition of swimming motility by the transcriptional regulator TtsI in Mesorhizobium japonicum MAFF303099 has been previously reported. Here, we show that mutants in T3SS components display impaired biofilm formation capacity, indicating that a functional T3SS, or at least pili formation, is required for this process. As a first approach to the cdiG regulation network in this bacterium, we started a study of the second messenger cdiG by overexpressing or by deleting some genes encoding cdiG metabolizing enzymes. Overexpression of two putative PDEs as well as deletion of various DGCs led to reduced biofilm formation on glass tubes. Mutation of dgc9509 also affected negatively the nodulation and symbiosis efficiency on Lotus plants, which can be related to the observed reduction in adhesion to plant roots. Results from transcriptional nopX- and ttsI-promoter-lacZ fusions suggested that cdiG negatively regulates T3SS expression in M. japonicum MAFF303099.

Invasiveness of endometrial cancer cell lines is potentiated by estradiol and blocked by a traditional medicine Guizhi Fuling at clinically relevant doses.

The Traditional Chinese medicine, Guizhi Fuling (here called Fuling), has been confirmed in meta-analysis studies to reduce recurrence of endometriosis and improve pregnancy outcomes; however, the possible use of Fuling as a fertility-preserving treatment in endometrial cancer has not previously been tested. Results here are the first to demonstrate dose-dependent inhibition of cell motility by Fuling in two endometrial cancer cell lines, classified as Grade I which is responsive to progesterone treatment, and Grade III (MFE-280) which is resistant. The major outcome of this study was the novel demonstration that Fuling (30-80 µg/ml) significantly inhibits invasiveness in both high and low grades of EC cells, achieving 70-80% block of trans-barrier migration without cytotoxicity. This effective dose range is estimated to be comparable to that used in human clinical trials and traditional practice. Results here further show that clinically relevant doses of Fuling override the motility-promoting effects of estradiol in endometrial cancer cell lines. Medroxyprogesterone acetate has to date been the standard therapy to treat metastatic or inoperable endometrial cancers; however, success rates are low with high rates of recurrence, due in part to acquired resistance to medroxyprogesterone acetate therapy. The discovery here that Fuling appears to control the spread of treatment-resistant advanced cancers is an exciting prospect.

The Beneficial Effects of Pine Nuts and Its Major Fatty Acid, Pinolenic Acid, on Inflammation and Metabolic Perturbations in Inflammatory Disorders.

Inflammatory disorders such as atherosclerosis, diabetes and rheumatoid arthritis are regulated by cytokines and other inflammatory mediators. Current treatments for these conditions are associated with significant side effects and do not completely suppress inflammation. The benefits of diet, especially the role of specific components, are poorly understood. Polyunsaturated fatty acids (PUFAs) have several beneficial health effects. The majority of studies on PUFAs have been on omega-3 fatty acids. This review will focus on a less studied fatty acid, pinolenic acid (PNLA) from pine nuts, which typically constitutes up to 20% of its total fatty acids. PNLA is emerging as a dietary PUFA and a promising supplement in the prevention of inflammatory disorders or as an alternative therapy. Some studies have shown the health implications of pine nuts oil (PNO) and PNLA in weight reduction, lipid-lowering and anti-diabetic actions as well as in suppression of cell invasiveness and motility in cancer. However, few reviews have specifically focused on the biological and anti-inflammatory effects of PNLA. Furthermore, in recent bioinformatic studies on human samples, the expression of many mRNAs and microRNAs was regulated by PNLA indicating potential transcriptional and post-transcriptional regulation of inflammatory and metabolic processes. The aim of this review is to summarize, highlight, and evaluate research findings on PNO and PNLA in relation to potential anti-inflammatory benefits and beneficial metabolic changes. In this context, the focus of the review is on the potential actions of PNLA on inflammation along with modulation of lipid metabolism and oxidative stress based on data from both in vitro and in vivo experiments, and human findings, including gene expression analysis.

KRT17 promotes endometrial cancer cell migration as well as angiogenesis by regulating HIF-1α/VEGF pathway

Endometrial cancer (EC) is a common type malignant tumors in women. To combat this type of cancer, more effective treatments are still needed. EC cell growth and metastasis depend on angiogenesis. Keratin (KRT) is a family of proteins which are essential for hair formation. KRT17 affected a variety of cancers. Here, we reported that KRT17 expression was high in human EC. The depletion of KRT17 suppressed EC cell growth. We further showed the ablation of KRT17 led to the suppression of cell motility. Also, its depletion resulted in the inhibition of angiogenesis. We further demonstrated that KRT17 contributed to the progression of EC via regulating the HIF-1α/VEGF axis through a tumor growth assay in mice. In conclusion, KRT17 could serve as an EC target.

Phlomoides molucelloides (Bunge) Salmaki'nin Antibakteriyel Etkileri

The insufficiency of existing antibiotics in the combat against antibiotics-resistant bacteria has necessitated the discovery of new and effective antibacterial drugs. The discovery that bacteria synthesize various virulence factors by the quorum sensing system has suggested that quorum sensing inhibitors may be used in the fight against infectious diseases. This study aimed to determine the antibacterial and anti-quorum sensing activities of methanol and water extracts of aerial and root parts of Phlomoides molucelloides (Bunge) Salmaki. The minimal inhibitory concentration (MIC) values of the extracts were investigated against reference bacterial strains using the broth microdilution method. Anti-quorum sensing activities were examined by violacein and pyocyanin pigments inhibition and swarming motility inhibition assays against Chromobacterium violaceum ATCC 12472 and Pseudomonas aeruginosa PAO1 bioreporter strains. The antibiofilm activities of the extracts were tested against P. aeruginosa PAO1 using the crystal violet staining method. The MIC value (> 2000 µg/mL) of all extracts against the tested bacteria could not be determined at the concentrations studied. All extracts partially inhibited the swarming motility of P. aeruginosa. Methanol extract of the aerial part inhibited pyocyanin production by 81.7% without interfering with P. aeruginosa growth. The extracts had no significant inhibitory activity on biofilm formation and violacein pigment production. These results showed that the extracts of P. molucelloides may be good anti-quorum sensing agents. Further research can be done to elucidate the mechanisms underlying these biological activities.

Calotropis Procera Induced Caspase-Dependent Apoptosis and Impaired Akt/mTOR Signaling in 4T1 Breast Cancer Cells.

Calotropis procera (Aiton) Dryand (Apocynaceae) is an herb that has been commonly used in folk medicine to treat various diseases for more than 1500 years. Our goal was to investigate the anti-metastatic effects of phenolics extracted from C. procera (CphE) against 4T1 breast cancer cells and in BALB/c mice. 4T1 cells were treated with CphE and quercetin (positive control) at concentrations that inhibited cell viability by 50% (IC50). Levels of reactive oxygen species (ROS), wound healing, and protein expressions were determined following standard protocols. For the in vivo pilot study, the syngeneic BALB/c mouse model was used. 4T1 cells were injected into mammary fat pads. Tumors were allowed to grow for 9 days before gavage treatment with CphE (150 mg GAE/kg/day) or PBS (controls) for one week. Excised tumors, liver, and lungs were analyzed for gene and protein expression and histology. In vitro results showed that CphE suppressed cell viability through apoptosis induction, via caspase-3 cleavage and total PARP reduction. CphE also scavenged ROS and suppressed Akt, mTOR, ERK1/2, CREB, and Src activation contributing to cell motility inhibition. CphE reduced IR, PTEN, TSC2, p70S6, and RPS6, protein levels, which are proteins involved in the PI3K/Akt/mTOR pathway, suggesting this pathway as CphE primary target. In vivo results showed downregulation of ERK1/2 activation by phosphorylation in tumor tissues, accompanied by angiogenesis reduction in tumor and lung tissues. A reduction of Cenpf mRNA levels in liver and lung tissues strongly suggested anti-invasive cancer activity of CphE. CphE inhibited 4T1 cell signal pathways that play a key role in cell growth and invasion. The potential for in vitro results to be translated in vivo was confirmed. A complete animal study is a guarantee to confirm the CphE anticancer and antimetastatic activity in vivo.

High-Throughput Transcriptomic Profiling Reveals the Inhibitory Effect of Hydroquinine on Virulence Factors in Pseudomonas aeruginosa.

Hydroquinine is an organic alkaloid compound that exhibits antimicrobial activity against several bacterial strains including strains of both drug-sensitive and multidrug-resistant P. aeruginosa. Despite this, the effects of hydroquinine on virulence factors in P. aeruginosa have not yet been characterized. We therefore aimed to uncover the mechanism of P. aeruginosa hydroquinine-sensitivity using high-throughput transcriptomic analysis. We further confirmed whether hydroquinine inhibits specific virulence factors using RT-qPCR and phenotypic analysis. At half the minimum inhibitory concentration (MIC) of hydroquinine (1.250 mg/mL), 254 genes were differentially expressed (97 downregulated and 157 upregulated). We found that flagellar-related genes were downregulated by between −2.93 and −2.18 Log2-fold change. These genes were consistent with the analysis of gene ontology and KEGG pathway. Further validation by RT-qPCR showed that hydroquinine significantly suppressed expression of the flagellar-related genes. By analyzing cellular phenotypes, P. aeruginosa treated with ½MIC of hydroquinine exhibited inhibition of motility (30–54% reduction) and pyocyanin production (~25–27% reduction) and impaired biofilm formation (~57–87% reduction). These findings suggest that hydroquinine possesses anti-virulence factors, through diminishing flagellar, pyocyanin and biofilm formation.

Elucidation of a Novel Mechanism of Perioperative and Postoperative Ileus: Microvascular Leak of Serum Immunoglobulins Directly Activates Inhibitory Nitrergic Neuromuscular Transmission, Causing Ileus

INTRODUCTION: Postoperative ileus delays enteral feeding, increases morbidity, prolongs hospitalization, and increases cost. Prevailing hypotheses are that anesthesia plays a major role in perioperative ileus and inflammation causes postoperative ileus. However, the direct causative mechanism of ileus is still unknown. Our goal was to delineate the components of an abdominal operative procedure on ileus and elucidate a major causative mechanism. METHODS: Five groups of C57Bl/6 were used: control, anesthesia, incision, laparotomy, and intestinal manipulation (IM), (N = 4 each). Gastrointestinal transit (GIT) with geometric center (GC) calculation was assessed immediately after resuscitation and after 24 hours. IM consisted of “running the bowel”. Circular muscle contractility assessed using an organ bath. Jejunal and colonic segments with fluorophore illuminated microvasculature were imaged by confocal microscopy. RESULTS: GIT for each component of the immediate perioperative ileus response was: anesthesia 25.7%, incision 13.6%, laparotomy 15.2% and IM 45.5%. Anesthesia decreased GIT, which rapidly waned. Only IM 24-hour postoperative GIT remained delayed (GC = 4.2 ± 0.52). Contractility was only significantly suppressed in IM 24-hour group. Imaging of control jejunal and colonic segments demonstrated illuminated microvasculature fluorophore containment. Vascular leak was prominent in IM immediate and IM 24-hour groups. Intraperitoneal serum injection (250µl) caused an immediate transit delay. Jejunal and colonic muscle spontaneous contraction was significantly inhibited by 1% serum, which was blocked with L-nitro-arginine (100µM). L-protein Ig elution and exogenous IgG abolished contractions. CONCLUSION: The data demonstrate that perioperative and postoperative ileus is mechanistically caused by the microvascular leak of serum immunoglobulin triggering a nitrergic neuromuscular suppression of motility.

The study of morphofunctional changes in the gastrointestinal tract after various operations on the colon (an experiment in rats)

Objective : modeling various types of surgical procedure in an experiment in rats, assessing the effect of surgery on the motility of the gastrointestinal tract (GIT). Materials and methods : the study was carried out on 30 female Wistar rats weighing 200–250 grams, comparable in age, divided into 3 groups (10 animals each) in random order. group 1 — rats underwented to laparotomy (LT), group 2 — laparotomy, creation of a loop colostomy (LT + C), group 3 — laparotomy, a colonic single-row anastomosis (LT + A). Gastrointestinal motility was assessed using the gastrointestinal transit index (GTI), calculated by the formula: (length of the stained area of the intestine / total length of the intestine) x100, as well as by the results of a morphological study. Results : surgery has a depressing effect on the motility of the gastrointestinal tract, while the maximum decrease in IHT was noted in the Colostomy group: 1.08±1.4% versus 2.3±3.3% in the Laparotomy group and versus 3.8±1.8% in the Anastomosis group. Comparison of the groups “Colostomy” and “Anastomosis” showed a significant difference in this feature — p=0.002. Conclusion : modeling of various surgical procedure in rats made it possible to prove the relationship between the formation of a stoma and the inhibition of gastrointestinal motility, further study of this pattern will allow us to develop ways to prevent the development of postoperative ileus.

The combination of hexanal and geraniol in sublethal concentrations synergistically inhibits quorum sensing in Pseudomonas fluorescens-In vitro and in silico approaches.

Hexanal and geraniol are essential oil components with anti-quorum sensing (QS) activity against Pseudomonas fluorescens. This study demonstrated that QS inhibition (QSI) efficacy of the hexanal and geraniol combination (HG) was significantly higher when compared to those of their mono-counterparts at the same concentration. Tests on P. fluorescens motility, biofilm formation, acyl-homoserine lactones' (AHLs) production, gene expression in vitro, and molecular docking in silico were conducted to evaluate the synergistic effect of hexanal and geraniol on QSI. HG mixture at 0.5 minimal inhibitory concentration (MIC) showed a strong synergistic inhibition of biofilm formation (51.8%), motility (60.13%), and extracellular protease activity (58.9%) of P. fluorescens. The synthesis of AHLs, e.g., C8 -HSL and C12 -HSL, was inhibited by hexanal, geraniol, and HG; both AHLs are responsible for regulating virulence factors in P. fluorescens. The expression of pcoI and gacA genes regulating AHL synthetase and sensor kinase was significantly down-regulated by HG (0.29 and 0.38-fold) at 0.5 MIC. Hexanal and HG showed significant inhibition of the expression of pcoR and gacS genes, which are responsible for AHL receptor protein and response regulation; however, geraniol failed to downregulate the two genes. Molecular docking in silico also supported these findings. Hexanal, which gets inserted into the minor groove of pcoI/pcoR DNA fragments, inhibits the expression of both the genes. Both hexanal (-31.487kcal/mol) and geraniol (-25.716kcal/mol) had a higher binding affinity with PcoI protein than the halogenated furanone C30 (-24.829kcal/mol), which is a known competitor of AHLs. Similarly, hexanal and geraniol strongly bind to the PcoR protein also. It was found that HG at 0.5 MIC could effectively inhibit QS by suppressing the expression of pcoR/gacS and gacA/gacS genes and therefore, could inhibit the motility and biofilm formation of P. fluorescens. The present study indicated that HG at sub-MIC as QS inhibitor could be further developed as a new preservative of agri-food products.