Microbiome Research and Multi-Omics Integration for Personalized Medicine in Asthma.

Asthma is a multifactorial inflammatory disorder of the respiratory system characterized by high diversity in clinical manifestations, underlying pathological mechanisms and response to treatment. It is generally established that human microbiota plays an essential role in shaping a healthy immune response, while its perturbation can cause chronic inflammation related to a wide range of diseases, including asthma. Systems biology approaches encompassing microbiome analysis can offer valuable platforms towards a global understanding of asthma complexity and improving patients' classification, status monitoring and therapeutic choices. In the present review, we summarize recent studies exploring the contribution of microbiota dysbiosis to asthma pathogenesis and heterogeneity in the context of asthma phenotypes-endotypes and administered medication. We subsequently focus on emerging efforts to gain deeper insights into microbiota-host interactions driving asthma complexity by integrating microbiome and host multi-omics data. One of the most prominent achievements of these research efforts is the association of refractory neutrophilic asthma with certain microbial signatures, including predominant pathogenic bacterial taxa (such as Proteobacteria phyla, Gammaproteobacteria class, especially species from Haemophilus and Moraxella genera). Overall, despite existing challenges, large-scale multi-omics endeavors may provide promising biomarkers and therapeutic targets for future development of novel microbe-based personalized strategies for diagnosis, prevention and/or treatment of uncontrollable asthma.

Computational Analysis of Transcriptomic and Proteomic Data for Deciphering Molecular Heterogeneity and Drug Responsiveness in Model Human Hepatocellular Carcinoma Cell Lines.

Hepatocellular carcinoma (HCC) is associated with high mortality due to its inherent heterogeneity, aggressiveness, and limited therapeutic regimes. Herein, we analyzed 21 human HCC cell lines (HCC lines) to explore intertumor molecular diversity and pertinent drug sensitivity. We used an integrative computational approach based on exploratory and single-sample gene-set enrichment analysis of transcriptome and proteome data from the Cancer Cell Line Encyclopedia, followed by correlation analysis of drug-screening data from the Cancer Therapeutics Response Portal with curated gene-set enrichment scores. Acquired results classified HCC lines into two groups, a poorly and a well-differentiated group, displaying lower/higher enrichment scores in a "Specifically Upregulated in Liver" gene-set, respectively. Hierarchical clustering based on a published epithelial-mesenchymal transition gene expression signature further supported this stratification. Between-group comparisons of gene and protein expression unveiled distinctive patterns, whereas downstream functional analysis significantly associated differentially expressed genes with crucial cancer-related biological processes/pathways and revealed concrete driver-gene signatures. Finally, correlation analysis highlighted a diverse effectiveness of specific drugs against poorly compared to well-differentiated HCC lines, possibly applicable in clinical research with patients with analogous characteristics. Overall, this study expanded the knowledge on the molecular profiles, differentiation status, and drug responsiveness of HCC lines, and proposes a cost-effective computational approach to precision anti-HCC therapies.

MTH1 favors mesothelioma progression and mediates paracrine rescue of bystander endothelium from oxidative damage.

Oxidative stress and inadequate redox homeostasis is crucial for tumor initiation and progression. MTH1 (NUDT1) enzyme prevents incorporation of oxidized dNTPs by sanitizing the deoxynucleoside triphosphate (dNTP) pool and is therefore vital for the survival of tumor cells. MTH1 inhibition has been found to inhibit the growth of several experimental tumors, but its role in mesothelioma progression remained elusive. Moreover, although MTH1 is nonessential to normal cells, its role in survival of host cells in tumor milieu, especially tumor endothelium, is unclear. We validated a clinically relevant MTH1 inhibitor (Karonudib) in mesothelioma treatment using human xenografts and syngeneic murine models. We show that MTH1 inhibition impedes mesothelioma progression and that inherent tumoral MTH1 levels are associated with a tumor's response. We also identified tumor endothelial cells as selective targets of Karonudib and propose a model of intercellular signaling among tumor cells and bystander tumor endothelium. We finally determined the major biological processes associated with elevated MTH1 gene expression in human mesotheliomas.

RNA-seq data analysis of stimulated hepatocellular carcinoma cells treated with epigallocatechin gallate and fisetin reveals target genes and action mechanisms.

Hepatocellular carcinoma (HCC) is an essentially incurable inflammation-related cancer. We have previously shown by network analysis of proteomic data that the flavonoids epigallocatechin gallate (EGCG) and fisetin (FIS) efficiently downregulated pro-tumor cytokines released by HCC through inhibition of Akt/mTOR/RPS6 phospho-signaling. However, their mode of action at the global transcriptome level remains unclear. Herein, we endeavor to compare gene expression alterations mediated by these compounds through a comprehensive transcriptome analysis based on RNA-seq in HEP3B, a responsive HCC cell line, upon perturbation with a mixture of prototypical stimuli mimicking conditions of tumor microenvironment or under constitutive state. Analysis of RNA-seq data revealed extended changes on HEP3B transcriptome imposed by test nutraceuticals. Under stimulated conditions, EGCG and FIS significantly modified, compared to the corresponding control, the expression of 922 and 973 genes, respectively, the large majority of which (695 genes), was affected by both compounds. Hierarchical clustering based on the expression data of shared genes demonstrated an almost identical profile in nutraceutical-treated stimulated cells which was virtually opposite in cells exposed to stimuli alone. Downstream enrichment analyses of the co-modified genes uncovered significant associations with cancer-related transcription factors as well as terms of Gene Ontology/Reactome Pathways and highlighted ECM dynamics as a nodal modulation point by nutraceuticals along with angiogenesis, inflammation, cell motility and growth. RNA-seq data for selected genes were independently confirmed by RT-qPCR. Overall, the present systems approach provides novel evidence stepping up the mechanistic understanding of test nutraceuticals, thus rationalizing their clinical exploitation in new preventive/therapeutic modalities against HCC.

Novel pyrazolopyridine derivatives as potential angiogenesis inhibitors: Synthesis, biological evaluation and transcriptome-based mechanistic analysis.

Modified purine derivatives exemplified by pyrazolopyrimidines have emerged as highly selective inhibitors of several angiogenic receptor tyrosine kinases. Herein, we designed and synthesized a new series of substituted pyrazolopyridines and explored their ability to influence crucial pro-angiogenic attributes of endothelial cells. Four of the synthesized compounds, possessing analogous substitution pattern, were found able to inhibit at low micromolar concentrations endothelial cell proliferation, migration and differentiation, constitutively or in response to Vascular Endothelial Growth Factor (VEGF) and to attenuate VEGF-induced phosphorylation of VEGF receptor-2 and downstream kinases AKT and ERK1/2. Administration of effective compounds in mice delayed the growth of syngeneic Lewis lung carcinoma transplants and reduced tumor microvessel density, without causing toxicity. Genome-wide microarray and gene ontology analyses of treated endothelial cells revealed derivative 18c as the most efficient modulator of gene expression and "mitotic cell cycle/cell division" along with "cholesterol biosynthesis" as the most significantly altered biological processes.

Biocatalytic synthesis and antitumor activities of novel silybin acylated derivatives with dicarboxylic acids.

Novel silybin acylated derivatives with dicarboxylic acids were prepared in various organic solvents using immobilized Candida antarctica lipase B (Novozym 435(®)). The reaction parameters affecting the silybin conversion, such as the nature of the organic solvent and the acyl donor used were investigated. The antiproliferative effects of silybin monoesters, and their ability to modulate the secretion of vascular endothelial growth factor (VEGF) were estimated using K562 human lymphoblastoma cells and compared to the parental compound. The synthesized esters retained the biological function of silybin and in some cases were more effective, indicating that target biotransformation may generate novel compounds with improved antitumor and antiangiogenic activities.

Mastic oil inhibits the metastatic phenotype of mouse lung adenocarcinoma cells.

Mastic oil from Pistacia lentiscus variation chia, a natural combination of bioactive terpenes, has been shown to exert anti-tumor growth effects against a broad spectrum of cancers including mouse Lewis lung adenocarcinomas (LLC). However, no studies have addressed its anti-metastatic actions. In this study, we showed that treatment of LLC cells with mastic oil within a range of non-toxic concentrations (0.01-0.04% v/v): (a) abrogated their Matrigel invasion and migration capabilities in transwell assays; (b) reduced the levels of secreted MMP-2; (c) restricted phorbol ester-induced actin remodeling and (d) limited the length of neo-vessel networks in tumor microenvironment in the model of chick embryo chorioallantoic membrane. Moreover, exposure of LLC and endothelial cells to mastic oil impaired their adhesive interactions in a co-culture assay and reduced the expression of key adhesion molecules by endothelial cells upon their stimulation with tumor necrosis factor-alpha. Overall, this study provides novel evidence supporting a multipotent role for mastic oil in prevention of crucial processes related to cancer metastasis.

The soluble guanylyl cyclase inhibitor NS-2028 reduces vascular endothelial growth factor-induced angiogenesis and permeability.

Nitric oxide (NO) is known to promote vascular endothelial growth factor (VEGF)-stimulated permeability and angiogenesis. However, effector molecules that operate downstream of NO in this pathway remain poorly characterized. Herein, we determined the effect of soluble guanylyl cyclase (sGC) inhibition on VEGF responses in vitro and in vivo. Treatment of endothelial cells (EC) with VEGF stimulated eNOS phosphorylation and cGMP accumulation; pretreatment with the sGC inhibitor 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one (NS-2028) blunted cGMP levels without affecting VEGF-receptor phosphorylation. Incubation of cells with NS-2028 blocked the mitogenic effects of VEGF. In addition, cells in which sGC was inhibited exhibited no migration and sprouting in response to VEGF. To study the mechanisms through which NS-2028 inhibits EC migration, we determined the effects of alterations in cGMP levels on p38 MAPK. Initially, we observed that inhibition of sGC attenuated VEGF-stimulated activation of p38. In contrast, the addition of 8-Br-cGMP to EC stimulated p38 phosphorylation. The addition of cGMP elevating agents (BAY 41-2272, DETA NO and YC-1) enhanced EC migration. To test whether sGC also mediated the angiogenic effects of VEGF in vivo, we used the rabbit cornea assay. Animals receiving NS-2028 orally displayed a reduced angiogenic response to VEGF. As increased vascular permeability occurs prior to new blood vessel formation, we determined the effect of NS-2028 in vascular leakage. Using a modified Miles assay, we observed that NS-2028 attenuated VEGF-induced permeability. Overall, we provide evidence that sGC mediates the angiogenic and permeability-promoting activities of VEGF, indicating the significance of sGC as a downstream effector of VEGF-triggered responses.

A transcriptomic computational analysis of mastic oil-treated Lewis lung carcinomas reveals molecular mechanisms targeting tumor cell growth and survival.

BACKGROUND: Mastic oil from Pistacia lentiscus variation chia, a blend of bioactive terpenes with recognized medicinal properties, has been recently shown to exert anti-tumor growth activity through inhibition of cancer cell proliferation, survival, angiogenesis and inflammatory response. However, no studies have addressed its mechanisms of action at genome-wide gene expression level. METHODS: To investigate molecular mechanisms triggered by mastic oil, Lewis Lung Carcinoma cells were treated with mastic oil or DMSO and RNA was collected at five distinct time points (3-48 h). Microarray expression profiling was performed using Illumina mouse-6 v1 beadchips, followed by computational analysis. For a number of selected genes, RT-PCR validation was performed in LLC cells as well as in three human cancer cell lines of different origin (A549, HCT116, K562). PTEN specific inhibition by a bisperovanadium compound was applied to validate its contribution to mastic oil-mediated anti-tumor growth effects. RESULTS: In this work we demonstrated that exposure of Lewis lung carcinomas to mastic oil caused a time-dependent alteration in the expression of 925 genes. GO analysis associated expression profiles with several biological processes and functions. Among them, modifications on cell cycle/proliferation, survival and NF-kappaB cascade in conjunction with concomitant regulation of genes encoding for PTEN, E2F7, HMOX1 (up-regulation) and NOD1 (down-regulation) indicated some important mechanistic links underlying the anti-proliferative, pro-apoptotic and anti-inflammatory effects of mastic oil. The expression profiles of Hmox1, Pten and E2f7 genes were similarly altered by mastic oil in the majority of test cancer cell lines. Inhibition of PTEN partially reversed mastic oil effects on tumor cell growth, indicating a multi-target mechanism of action. Finally, k-means clustering, organized the significant gene list in eight clusters demonstrating a similar expression profile. Promoter analysis in a representative cluster revealed shared putative cis-elements suggesting a common regulatory transcription mechanism. CONCLUSIONS: Present results provide novel evidence on the molecular basis of tumor growth inhibition mediated by mastic oil and set a rational basis for application of genomics and bioinformatic methodologies in the screening of natural compounds with potential cancer chemopreventive activities.

Protective effects of mastic oil from Pistacia lentiscus variation chia against experimental growth of lewis lung carcinoma.

Mastic oil from Pistacia lentiscus variation chia, a traditionally used dietary flavoring agent with medicinal properties, has been shown to exert in vitro antitumor activities, but no study has addressed in vivo efficacy and mechanisms of action. Presently, we demonstrated that treatment of immunocompetent mice with mastic oil (45 mg/kg body weight, intraperitoneally, 3 times a wk for approximately 3 wk) significantly inhibited tumor growth (56.4% +/- 5.7 maximum reduction in tumor volumes) without toxicity. Analysis of tumors by immunohistochemistry and ELISA indicated that this effect is associated with increased apoptosis, reduced neovascularization, and inhibition of chemokine expression. Likewise mastic oil reduced vascular endothelial growth factor and chemokine release by Lewis lung carcinoma (LLC) cells. Furthermore, mastic oil administration decreased small guanosine triphosphatases (GTPases) Ras, RhoA and nuclear factor-kappa-B-dependent reporter gene expression in vivo and in vitro, indicating a mechanistic link between mastic oil activities and blocking of relevant signaling and transcription pathways. A dose-response comparison with perillyl alcohol and alpha-pinene, two of its components, revealed a higher efficacy of mastic oil, pointing to a beneficial collective interaction among its ingredients. Conclusively, our results provide novel in vivo evidence of mastic oil inhibitory effects on tumor growth and set a rational basis for its future application in cancer prevention.

Zoledronic acid is effective against experimental malignant pleural effusion.

RATIONALE: Aminobiphosphonates, such as zoledronic acid (ZA), exert potent indirect antitumor effects and are currently being tested against human solid tumors. The antitumor actions of aminobiphosphonates, including angiostasis, are relevant to the pathogenesis of malignant pleural effusion (MPE), but no study has addressed the efficacy of these compounds against malignant pleural disease. OBJECTIVES: Here we hypothesized that treatment of immunocompetent mice with ZA would halt tumor progression in a mouse model of adenocarcinoma-induced MPE. METHODS: To induce MPE in mice, Lewis lung carcinoma cells were delivered directly into the pleural space. Subsequently, animals were treated with ZA in both a prevention and a regression protocol. MEASUREMENTS AND MAIN RESULTS: ZA treatment resulted in significant reductions in pleural fluid accumulation and tumor dissemination, while it significantly prolonged survival. These effects of ZA were linked to enhanced apoptosis of pleural tumor cells, decreased formation of new vessels in pleural tumors, and reduced pleural vascular permeability. In addition, ZA was able to inhibit the recruitment of mononuclear cells to pleural tumors, with concomitant reductions in matrix metalloproteinase-9 release into the pleural space. Finally, ZA limited the expression of proinflammatory and angiogenic mediators, as well as the activity of small GTP proteins Ras and RhoA, in tumor cells in vivo and in vitro. CONCLUSIONS: ZA is effective against experimental MPE, suggesting that this intervention should be considered for testing in clinical trials.

Inhaled activated protein C attenuates lung injury induced by aerosolized endotoxin in mice.

The serine protease activated protein C (APC) possesses prominent anticoagulant and anti-inflammatory actions. In this study, we investigated the effect of inhaled recombinant human (rh) APC in a murine lung injury model. Animals inhaled 10 mg of Pseudomonas lipopolysaccharide (LPS) in 3 mL normal saline (NS); 30 min prior to LPS, mice were pretreated with inhaled rhAPC (4 mg/3 mL NS; APC+LPS group) or NS (LPS group). A control animal group inhaled vehicle (NS) twice. 24 h later, total cells and cell-types, protein content, and the cytokines tumor necrosis factor-alpha, interleukin (IL)-6, macrophage inflammatory protein-1alpha, and mouse keratinocyte-derived chemokine (a homolog of human IL-8) were estimated in bronchoalveolar lavage fluid (BALF). Lung pathology given as total histology score (THS), wet/dry lung weight ratios, and lung vascular cell adhesion molecule (VCAM)-1 expression were additionally assessed. rhAPC inhalation attenuated the aerosolized LPS-induced increases of: total cells, neutrophils and macrophages in BALF, lung tissue VCAM-1 protein levels, and THS. Total protein levels and cytokines in BALF, and wet/dry weight ratios were increased in the LPS group, but rhAPC pretreatment did not significantly alter the LPS-induced responses. In conclusion, in this murine septic model of lung injury, inhaled rhAPC appears to attenuate lung inflammation, without reversing the observed increases in lung permeability and BALF cytokines. This effect may be associated with leukocyte trafficking modifications, related, at least in part, to VCAM-1 reduction.

Mastic oil from Pistacia lentiscus var. chia inhibits growth and survival of human K562 leukemia cells and attenuates angiogenesis.

Mastic oil from Pistacia lentiscus var. chia, a natural plant extract traditionally used as a food additive, has been extensively studied for its antimicrobial activity attributed to the combination of its bioactive components. One of them, perillyl alcohol (POH), displays tumor chemopreventive, chemotherapeutic, and antiangiogenic properties. We investigated whether mastic oil would also suppress tumor cell growth and angiogenesis. We observed that mastic oil concentration and time dependently exerted an antiproliferative and proapoptotic effect on K562 human leukemia cells and inhibited the release of vascular endothelial growth factor (VEGF) from K562 and B16 mouse melanoma cells. Moreover, mastic oil caused a concentration-dependent inhibition of endothelial cell (EC) proliferation without affecting cell survival and a significant decrease of microvessel formation both in vitro and in vivo. Investigation of underlying mechanism(s) demonstrated that mastic oil reduced 1) in K562 cells the activation of extracellular signal-regulated kinases 1/2 (Erk1/2) known to control leukemia cell proliferation, survival, and VEGF secretion and 2) in EC the activation of RhoA, an essential regulator of neovessel organization. Overall, our results underscore that mastic oil, through its multiple effects on malignant cells and ECs, may be a useful natural dietary supplement for cancer prevention.

Perillyl alcohol is an angiogenesis inhibitor.

Aberrant angiogenesis is essential for the progression of solid tumors and hematological malignancies. Thus, antiangiogenic therapy is one of the most promising approaches to control cancer. In the present work, we examined the ability of perillyl alcohol (POH), a dietary monoterpene with well-established tumor chemopreventive and chemotherapeutic activity, to interfere with the process of angiogenesis. POH remarkably prevented new blood vessel growth in the in vivo chicken embryo chorioallantoic membrane assay and proved to be effective in inhibiting the morphogenic differentiation of cultured endothelial cells into capillary-like networks both in collagen gel and Matrigel models. In addition, POH reduced the cell number in a proliferation assay and induced apoptosis of endothelial cells as indicated by the POH-mediated increase of caspase-3 activity and DNA fragmentation. Consistent with the observed antisurvival effect, POH treatment resulted in a significant inhibition of Akt phosphorylation in endothelial cells. Finally, POH was able to differentially modulate the release of two important angiogenic regulators: vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2). POH decreased the release of VEGF from cancer cells but stimulated the expression of Ang2 by endothelial cells, indicating that it might suppress neovascularization and induce vessel regression. Overall, these data underscore the antiangiogenic potential of POH and suggest that POH, in addition to its anticancer activity, may be an effective agent in the treatment of angiogenesis-dependent diseases.

Studies on the catalytic behaviour of a cholinesterase-like abzyme in an AOT microemulsion system.

The hydrolytic activity of a monoclonal catalytic antibody (9A8) (abzyme) with acetylcholinesterase-like activity was investigated in water-in-oil (w/o) microemulsions (reverse micelles) based on sodium bis-2-(ethylhexyl)sulfosuccinate (AOT) in isooctane, using p- and o-nitrophenylacetate (p-and o-NPA) as substrates. The dependence of the abzyme hydrolytic activity on the molar ratio of water to surfactant (w(o)) showed a bell-shaped curve, presenting a maximum at w(o)=11.1. An increase of the AOT concentration at constant w(o), resulted in a decrease of the catalytic activity suggesting a possible inhibition effect of the surfactant. The incorporation of the abzyme into the reverse micelle system caused a blue shift of the fluorescence emission maximum by a magnitude of 7-10 nm depending on the w(o) value. This result indicates that the antibody molecule, or a large part of it, is located in the aqueous microphase of the system. Kinetic studies showed that the hydrolysis of p-and o-NPA in microemulsion system as well as in aqueous solution follows Michaelis-Menten kinetics. The catalytic efficiency (k(cat)/K(m)) in w/o microemulsion was significant lower than in aqueous solution.