New insights into the cytotoxic effects of Thymus vulgaris essential oil on the human triple-negative breast cancer cell line MDA-MB-231.

Essential oils (EOs) are natural products that have gained wide interest due to their biological activities and anticancer properties through various mechanisms. The present study aimed to test the cytotoxicity of Thymus vulgaris L. (thyme) EO of Italian origin, rich in thymol (49.6%) and p-cymene (18.8%), towards the triple-negative breast cancer cell line MDA-MB-231 and to investigate the biochemical mechanisms underlying its antitumor activity. Thyme EO reduced cancer cell viability in a dose-dependent manner after 24 h treatment, with an IC50 value equal to 75.1 ± 15.2 µg/ml; simultaneously, the inhibition of cancer cell migration and colony formation capacity was evidenced. Thyme EO antiproliferative effects were related to the induction of apoptosis as demonstrated by the increased expression of the pro-apoptotic proteins Bax, cleaved caspase-3, phospho-p53, and SMAC/Diablo and by the reduction of the anti-apoptotic proteins Bcl-2, cIAP-1, cIAP-2, HIF-1α, survivin, and XIAP. Thyme EO administration led to the early formation of intracellular ROS, followed by the increment of MDA as an index of lipid peroxidation and by the decreased expression of the antioxidant enzymes catalase and PON2. The upregulation of Nrf2 mRNA expression and the strong induction of HO-1 sustained the activation of the Nrf2 pathway by thyme EO. These data showed that the EO from Thymus vulgaris L. might inhibit the malignant phenotype of MDA-MB-231, thus suggesting potential benefits against human triple-negative breast cancer.

Molecular Dynamics and Machine Learning Give Insights on the Flexibility-Activity Relationships in Tyrosine Kinome.

Tyrosine kinases are a subfamily of kinases with critical roles in cellular machinery. Dysregulation of their active or inactive forms is associated with diseases like cancer. This study aimed to holistically understand their flexibility-activity relationships, focusing on pockets and fluctuations. We studied 43 different tyrosine kinases by collecting 120 µs of molecular dynamics simulations, pocket and residue fluctuation analysis, and a complementary machine learning approach. We found that the inactive forms often have increased flexibility, particularly at the DFG motif level. Noteworthy, thanks to these long simulations combined with a decision tree, we identified a semiquantitative fluctuation threshold of the DGF+3 residue over which the kinase has a higher probability to be in the inactive form.

Erratum: Exosomal microRNAs synergistically trigger stromal fibroblasts in breast cancer.

[This corrects the article DOI: 10.1016/j.omtn.2022.02.013.].

3ß-Hydroxy-5ß-hydroxy-B-norcholestane-6ß-carboxaldehyde (SEC-B) Induces Proinflammatory Activation of Human Endothelial Cells Associated with Nitric Oxide Production and Endothelial Nitric Oxide Synthase/Caveolin-1 Dysregulation.

Oxysterols are a family of 27-carbon cholesterol oxidation derivatives found in low-density lipoproteins (LDLs) and atherosclerotic plaques where they trigger several biological responses involved in the initiation and progression of atherosclerosis. Several pieces of evidence suggest that oxysterols contribute to endothelial dysfunction (ED) due to their ability to alter membrane fluidity and cell permeability leading to inflammation, oxidative stress and apoptosis. The present study aimed to investigate the molecular events occurring in human microvascular endothelial cells (HMEC-1) in response to autoxidation-generated 3ß-hydroxy-5ß-hydroxy-B-norcholestane-6ß-carboxaldehyde (SEC-B) exposure. Our results highlight that SEC-B rapidly activates HMEC-1 by inducing oxidative stress, nitric oxide (NO) production and pro-inflammatory cytokine release. Exposure to SEC-B up to 24 h results in persistent accumulation of the vasodilator NO paralleled by an upregulation of the endothelial nitric oxide synthase (eNOS) enzyme and downregulation of Caveolin-1 (Cav-1) protein levels. Moreover, reduced expression and extracellular release of the vasoconstrictor factor endothelin-1 (ET-1) are observed. Furthermore, SEC-B stimulates the expression of the cytokines interleukin-6 (IL-6) and tumor necrosis factor-like weak inducer of apoptosis (TWEAK). This proinflammatory state leads to increased monocyte recruitment on activated HMEC-1 cells. Our findings add new knowledge on the role of SEC-B in ED and further support its potential implication in atherosclerosis.

Exosomal microRNAs synergistically trigger stromal fibroblasts in breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. TNBC progression is sustained by recruitment of a strong tumor microenvironment (TME) mainly composed of cancer-associated fibroblasts (CAFs) able to endorse tumor hallmarks. Increasing evidences demonstrate that exosomes mediate the crosstalk between cancer cells and the TME. We examined TNBC-derived exosomes and their microRNA (miRNA) cargo in activation of normal fibroblasts (NFs) toward CAFs. We demonstrated that TNBC cell-derived exosomes increased NF collagen contraction and migration alongside CAF molecular markers. Exosome-activated fibroblasts promoted the invasion potential of normal breast epithelial cells, as assessed by an organotypic co-culture assay that resembled the in vivo context. We also investigated TNBC cell-derived exosome cargo in activating NFs to CAFs by performing small RNA sequencing. We found that the synergistic action of miR-185-5p, miR-652-5p, and miR-1246 boosted fibroblast migration and contraction, promoting specific CAF subspecialization toward a pro-migratory functional state. These data highlight the role of breast cancer cells in re-education of the TME and their contribution to tumor evolution.

Probing Interplays between Human XBP1u Translational Arrest Peptide and 80S Ribosome.

The ribosome stalling mechanism is a crucial biological process, yet its atomistic underpinning is still elusive. In this framework, the human XBP1u translational arrest peptide (AP) plays a central role in regulating the unfolded protein response (UPR) in eukaryotic cells. Here, we report multimicrosecond all-atom molecular dynamics simulations designed to probe the interactions between the XBP1u AP and the mammalian ribosome exit tunnel, both for the wild type AP and for four mutant variants of different arrest potencies. Enhanced sampling simulations allow investigating the AP release process of the different variants, shedding light on this complex mechanism. The present outcomes are in qualitative/quantitative agreement with available experimental data. In conclusion, we provide an unprecedented atomistic picture of this biological process and clear-cut insights into the key AP-ribosome interactions.

The effects of Acyclovir administration to NCI-H1975 non-small cell lung cancer cells.

The biochemical mechanisms by which the antiviral drug Acyclovir (ACV) may induce anticancer effects even without detecting human herpesviruses (HHVs) are still poorly understood. Herein, we investigated for the first time how NCI-H1975 non-small cell lung cancer cells responded in vitro to ACV administration by exploring mitochondrial damage and apoptosis induction. We confirmed ACV ability to cause the inhibition of cancer cell growth even without detecting intracellular HHVs; the drug also significantly inhibited the colony formation capacity of NCI-H1975 cells. Cell cycle analysis revealed an increase of the sub-G1 hypodiploid peak after ACV treatment; the activation of caspase-3 and the presence of DNA laddering sustained the capacity of the drug to induce apoptotic cell death. Regarding mitochondrial toxicity, a reduction of mitochondrial membrane potential, altered mitochondrial size and shape, and mtDNA damage were found after ACV administration. Furthermore, an increment of intracellular reactive oxygen species levels as well as the upregulation of NudT3 involved in DNA repair mechanisms were observed. Altogether, these findings suggest that mitochondria may be possible initial targets and/or sites of ACV cytotoxicity within cancer cells in the absence of intracellular HHVs.

miR-34c-3p targets CDK1 a synthetic lethality partner of KRAS in non-small cell lung cancer.

Lung cancer is still the leading cause of death by cancer worldwide despite advances both in its detection and therapy. Multiple oncogenic driver alterations have been discovered, opening the prospective for new potential therapeutic targets. Among them, KRAS mutations represent the most frequent oncogene aberrations in non-small cell lung cancer (NSCLC) patients with a negative prognostic impact, but effective therapies targeting KRAS are not well characterized yet. Here, we demonstrate that the microRNA miR-34c-3p is a positive prognostic factor in KRAS-mutated NSCLC patients. Firstly, looking at the TGCA dataset, we found that high miR-34c-3p expression correlated with longer survival of KRAS-mutated NSCLC patients. In vitro assays on immortalized and patient-derived primary NSCLC cells revealed that miR-34c-3p overexpression increased apoptosis and lowered proliferation rate in KRASmut cells. Computational analysis and in vitro assays identified CDK1, one of the most promising lethal targets for KRAS-mutant cancer, as a target of miR-34c-3p. Moreover, the combination of CDK1 inhibition (mediated by RO3306) and miR-34c-3p overexpression resulted in an additive effect on the viability of KRASmut-expressing cells. Altogether, our findings demonstrate that miR-34c-3p is a novel biomarker that may allow tailored treatment for KRAS-mutated NSCLC patients.

Serum changes in sTWEAK and its scavenger receptor sCD163 in ultramarathon athletes running the 24-h race.

In the present investigation, the serum changes of sTWEAK levels, a multifunctional cytokine involved in tissue response to acute injury and inflammation, and of its scavenger receptor sCD163, were monitored for the first time in ultramarathon athletes running the 24-h competition, an extremely demanding race in terms of muscular and physiological exertion. To this aim, venous blood samples were collected from each participant (n = 22, M = 12, F = 10) both before and immediately after the 24-h running. Other than sTWEAK and sCD163, the common serum biomarkers of inflammation (namely CRP and IL-6) and tissue injury (such as CPK, LDH, CPK-MB, troponin-I, and NT-proBNP) were evaluated. All parameters were within the reference ranges at baseline, indicating no alterations of the normal physiological processes before the competition; on the contrary, most biomarkers of tissue damage and inflammation strongly increased after the ultramarathon race. Interestingly, a significant decrement of sTWEAK levels associated with an increment of its scavenger receptor sCD163 was observed at post-race. Positive relationships were evidenced between IL-6 and sCD163 levels and the markers of cardiac damage troponin-I and NT-proBNP. On the contrary, sTWEAK showed an inverse correlation with IL-6 and NT-proBNP. This study opens the way to further investigations aimed at clarifying the role of TWEAK pathway during the prolonged ultraendurance activity, paying particular attention to the link of IL-6, CD163 and TWEAK with the cardiac function.

Modulating the Crosstalk between the Tumor and the Microenvironment Using SiRNA: A Flexible Strategy for Breast Cancer Treatment.

Tumorigenesis is a complex and multistep process in which sequential mutations in oncogenes and tumor-suppressor genes result in enhanced proliferation and apoptosis escape. Over the past decades, several studies have provided evidence that tumors are more than merely a mass of malignant cancer cells, with the tumor microenvironment (TME) also contributing to cancer progression. For this reason, the focus of cancer research in recent years has shifted from the malignant cancer cell itself to the TME and its interactions. Since the TME actively participates in tumor progression, therapeutic strategies targeting it have created great interest. In this context, much attention has been paid to the potential application of small interfering RNA (siRNA), a class of non-coding RNA that has the ability to downregulate the expression of target genes in a sequence-specific way. This is paving the way for a novel therapeutic approach for the treatment of several diseases, including cancer. In this review, we describe recent efforts in developing siRNA therapeutics for the treatment of breast cancer, with particular emphasis on TME regulation. We focus on studies that adapt siRNA design to reprogram/re-educate the TME and eradicate the interplay between cancer cells and TME.

Synthesis of new dihydroberberine and tetrahydroberberine analogues and evaluation of their antiproliferative activity on NCI-H1975 cells.

Dihydroberberine (DHBER), the partially reduced form of the alkaloid berberine (BER), is known to exhibit important biological activities. Despite this fact, there have been only few studies that concern the biological properties of functionalized DHBER. Attracted by the potentiality of this latter compound, we have realized the preparation of new arylhydrazono-functionalized DHBERs, starting from BER and some α-bromohydrazones. On the other hand, also the fully reduced form of BER, namely tetrahydroberberine (THBER), and its derivatives have proven to present different biological activities. Therefore, the obtained arylhydrazono-functionalized DHBERs were reduced to the corresponding arylhydrazono-THBERs. The antiproliferative activity of both arylhydrazono-DHBERs and -THBERs has been evaluated on NCI-H1975 lung cancer cells.

miR-216a Acts as a Negative Regulator of Breast Cancer by Modulating Stemness Properties and Tumor Microenvironment.

Breast cancer is the most frequent malignancy in females in terms of both incidence and mortality. Underlying the high mortality rate is the presence of cancer stem cells, which divide indefinitely and are resistant to conventional chemotherapies, so causing tumor relapse. In the present study, we identify miR-216a-5p as a downregulated microRNA in breast cancer stem cells vs. the differentiated counterpart. We demonstrate that overexpression of miR-216a-5p impairs stemness markers, mammosphere formation, ALDH activity, and the level of Toll-like receptor 4 (TLR4), which plays a significant role in breast cancer progression and metastasis by leading to the release of pro-inflammatory molecules, such as interleukin 6 (IL-6). Indeed, miR-216a regulates the crosstalk between cancer cells and the cells of the microenvironment, in particular cancer-associated fibroblasts (CAFs), through regulation of the TLR4/IL6 pathway. Thus, miR-216a has an important role in the regulation of stem phenotype, decreasing stem-like properties and affecting the cross-talk between cancer cells and the tumor microenvironment.

Phytochemical composition, antioxidant and antiproliferative activities and effects on nuclear DNA of ethanolic extract from an Italian mycelial isolate of Ganoderma lucidum.

ETHNOPHARMACOLOGICAL RELEVANCE: Ganoderma lucidum (Curtis) P. Karst. (also known as Linghzhi and Reishi) is the most appreciated and revered medicinal mushroom across many Asian countries, but its properties have also attracted interest in Western countries. Indeed, in the West, it is now commercially available as a dietary supplement in preparations mainly made from spores, fruiting bodies and mycelia. It is employed in both nutraceutical and pharmacological formulations either for its immuno-modulating anti-inflammatory properties or as an effective adjuvant therapy in the treatment of several chronic diseases as well as in cancer treatment. AIM OF THE STUDY: The aim of this investigation was to show the phytochemical composition and antioxidant and antiproliferative activities of an ethanolic extract from an Italian mycelial isolate of Ganoderma lucidum and to assess its effects on nuclear DNA. MATERIALS AND METHODS: LC/ESI-MS and tandem mass spectrometry MSMS were used to obtain structural identification of ethanolic G. lucidum extract constituents. Antioxidant activities were determined by the DPPH method, chelating effect on Fe2+ and lipoxygenase inhibition while cytotoxic activities using the MTT assay. Effects on nuclear DNA were evaluated using the DNA nicking assay in a cell-free system and the fast halo assay performed on oxidatively injured human U937 cells; apoptosis induction was investigated using the non-denaturing fast halo assay and DNA laddering detection. RESULTS: This extract was rich in several bioactive compounds, mainly phenolic and triterpenic acids. It showed antioxidant activity and protective effects in oxidatively injured DNA in cell-free analyses and antiproliferative, genotoxic, and proapoptotic effects in the cell model. CONCLUSIONS: Italian G. lucidum mycelium isolate appears to be a source of various natural compounds that may have applications as chemopreventive agents or functional foods.

Acyclovir induces cell cycle perturbation and apoptosis in Jurkat leukemia cells, and enhances chemotherapeutic drug cytotoxicity.

AIMS AND METHODS: Many antiviral agents have been reported to present direct cytotoxic activity in cancer, showing antiproliferative and proapoptotic effects through different mechanisms. In the present study, we took into account the cytotoxic action of the antiviral drug acyclovir (ACV) on leukemia cells, by investigating cell cycle perturbations and apoptosis induction upon drug administration to three still unexplored cell lines, namely Jurkat, U937, and K562. At the same time, the cytotoxicity of cisplatin (CDDP) and 5­fluorouracil (5­FU) in combination with ACV was assessed, thus to evaluate if the antiviral agent could enhance cancer cell sensitivity to these chemotherapeutic drugs. FINDINGS AND SIGNIFICANCE: Our results showed that ACV cytotoxic action was maximum in Jurkat cells (acute T cell leukemia), which showed a dose- and time-dependent reduction of cell viability after drug exposure. The flow cytometric analysis of cell cycle revealed a delay/block in S phase and an increase of the sub-G1 peak upon ACV administration, thereby indicating apoptotic cell death. The activation of caspase-3 and the presence of nuclear DNA fragmentation confirmed the induction of apoptosis in ACV-treated cells. Interestingly, the pre-treatment of Jurkat cells with ACV for 72 h or 7 days increased CDDP and 5-FU cytotoxicity, suggesting enhanced leukemia cell sensitivity to these anticancer drugs.

Aptamer-miR-34c Conjugate Affects Cell Proliferation of Non-Small-Cell Lung Cancer Cells.

MicroRNAs (miRNAs) are key regulators of different human processes that represent a new promising class of cancer therapeutics or therapeutic targets. Indeed, in several tumor types, including non-small-cell lung carcinoma (NSCLC), the deregulated expression of specific miRNAs has been implicated in cell malignancy. As expression levels of the oncosuppressor miR-34c-3p are decreased in NSCLC compared to normal lung, we show that reintroduction of miR-34c-3p reduces NSCLC cell survival in vitro. Further, in order to deliver the miR-34c-based therapeutic selectively to tumor cells, we took advantage of a reported nucleic acid aptamer (GL21.T) that binds and inhibits the AXL transmembrane receptor and is rapidly internalized in the target cells. By applying methods successfully used in our laboratory, we conjugated miR-34c to the GL21.T aptamer as targeting moiety for the selective delivery to AXL-expressing NSCLC cells. We demonstrate that miR-34c-3p and the GL21.T/miR-34c chimera affect NSCLC cell proliferation and are able to overcome acquired RTK-inhibitor resistance by targeting AXL receptor. Thus, the GL21.T/miR-34c chimera exerts dual inhibition of AXL at functional and transcriptional levels and represents a novel therapeutic tool for the treatment of NSCLC.

Insights into the homo-oligomerization properties of N-terminal coiled-coil domain of Ebola virus VP35 protein.

The multifunctional Ebola virus (EBOV) VP35 protein is a key determinant of virulence. VP35 is essential for EBOV replication, is a component of the viral RNA polymerase and participates in nucleocapsid formation. Furthermore, VP35 contributes to EBOV evasion of the host innate immune response by suppressing RNA silencing and blocking RIG-I like receptors' pathways that lead to type I interferon (IFN) production. VP35 homo-oligomerization has been reported to be critical for its replicative function and to increase its IFN-antagonism properties. Moreover, homo-oligomerization is mediated by a predicted coiled-coil (CC) domain located within its N-terminal region. Here we report the homo-oligomerization profile of full-length recombinant EBOV VP35 (rVP35) assessed by size-exclusion chromatography and native polyacrylamide gel electrophoresis. Based on our biochemical results and in agreement with previous experimental observations, we have built an in silico 3D model of the so-far structurally unsolved EBOV VP35 CC domain and performed self-assembly homo-oligomerization simulations by means of molecular dynamics. Our model advances the understanding of how VP35 may associate in different homo-oligomeric species, a crucial process for EBOV replication and pathogenicity.

Antiproliferative activity of vitexin-2-O-xyloside and avenanthramides on CaCo-2 and HepG2 cancer cells occurs through apoptosis induction and reduction of pro-survival mechanisms.

PURPOSE: CaCo-2 colon cancer cells and HepG2 liver cancer cells represent two malignant cell lines, which show a high resistance to apoptosis induced by the conventional anticancer drugs. Vitexin-2-O-xyloside (XVX) and avenanthramides (AVNs) are naturally occurring dietary agents from Beta vulgaris var. cicla L. and Avena sativa L., respectively. The aim of this work was to evaluate the antiproliferative effects and the reduction of the pro-survival mechanisms exerted by XVX and AVNs, used individually and in combination, in CaCo-2 and HepG2 cancer cells. METHODS: XVX and AVNs were isolated by liquid chromatography and characterized by HPLC-PDA-MS. The XVX and AVN antiproliferative effects were evaluated through sulforhodamine B method, while their pro-apoptotic effects through caspase activity assays. RTqPCR was used to investigate the modulation of the pro-survival factors baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5), hypoxia inducible factor 1 A (HIF1A), and vascular endothelial growth factor A (VEGFA). Cellular antioxidant activity (CAA) was investigated by means of DCFH-DA assay, whereas chemical antioxidant capacity was evaluated by the ORAC method. RESULTS: XVX and AVNs, both individually and in combination, inhibited the proliferation of CaCo-2 and HepG2 cancer cells, through activation of caspases 9, 8, and 3. XVX and AVNs downregulated the pro-survival genes BIRC5, HIF1A, and VEGFA. The CAA assay showed that AVNs exhibited strong antioxidant activity inside both CaCo-2 and HepG2 cells. CONCLUSIONS: The antiproliferative activity of the XVX + AVNs mixture represents an innovative treatment, which is effective against two types of cancer cells characterized by high resistance to the conventional anticancer drugs.

Dynamics behind affinity maturation of an anti-HCMV antibody family influencing antigen binding.

The investigation of antibody affinity maturation and its effects on antigen binding is important with respect to understanding the regulation of the immune response. To shed light on this crucial process, we analyzed two Igs neutralizing the human cytomegalovirus: the primary germline antibody M2J1 and its related mature antibody 8F9. Both antibodies target the AD-2S1 epitope of the gB envelope protein and are considered to establish similar interactions with the cognate antigen. We used molecular dynamics simulations to understand the effect of mutations on the antibody-antigen interactions. The results provide a qualitative explanation for the increased 8F9 peptide affinity compared with that of M2J1. The emerging atomistic-detailed description of these complexes reveals the molecular effects of the somatic hypermutations occurring during affinity maturation.

Reduced cell viability and apoptosis induction in human thyroid carcinoma and mesothelioma cells exposed to cidofovir.

Besides its well-recognized antiviral activity, Cidofovir (CDV) has been shown to exert anticancer properties both within in vitro and in vivo models. The aim of this study was to evaluate the effects of CDV on still unexplored cultured cancer cells from human mesothelioma as well as breast, colon, liver, lung, prostate, and thyroid carcinomas. Overall, a dose- and time-dependent inhibition of cell viability was observed after CDV exposure. To clarify the mechanisms underlying CDV action, apoptotic cell death was investigated in two infected cell lines [Ist-Mes1 and Ist-Mes2 mesothelioma cells (SV40+)] and in two uninfected cell lines (NCI-H2425 mesothelioma cells and FTC-133 thyroid cancer cells), which resulted the most sensitive to CDV treatment. Reduced expression of procaspase-3 and increased expression of PARP p85 fragment were observed in both infected and uninfected mesothelioma cells, indicating apoptosis induction by CDV in a virus-independent manner. Similarly, the increase of the pro-apoptotic proteins p53, cytochrome c and caspase-3, the decrease of the survival protein Bcl-x, and the increment of Bax/Bcl-2 ratio revealed the occurrence of apoptosis in CDV-treated FTC-133. The presence of nuclear DNA fragmentation confirmed apoptotic cell death by CDV. Overall, our findings warrant further investigations to explore the therapeutic potential of CDV for human mesothelioma and follicular thyroid carcinoma.