Search

Shanxi-aged vinegar, a traditional Chinese grain-fermented food that is rich in polyphenols, has been shown to have therapeutic effects on a variety of diseases. However, there has been no comprehensive evaluation of the anti-inflammatory activity of polyphenols extracted from Shanxi-aged vinegar (SAVEP) to date. The anti-inflammatory activities of SAVEP, both in RAW 264.7 macrophages and mice, were extensively investigated for the potential application of SAVEP as a novel anti-inflammatory agent. In order to confirm the notion that polyphenols could improve inflammatory symptoms, SAVEP was firstly detected by gas chromatography mass spectrometry (GC-MS). In total, 19 polyphenols were detected, including 12 phenolic acids. The study further investigated the protective effect of SAVEP on lipopolysaccharide-induced inflammation in RAW264.7 macrophages and ICR mice. The results showed that compared with those of the model group, SAVEP could remarkably recover the inflammation of macrophage RAW264.7 and ICR mice. SAVEP can normalise the expression of related proteins via the suppression of MAPK/NF-κB pathway activation, inhibiting the expression of iNOS and COX-2 proteins, and consequently the production of inflammatory factors, thus alleviating inflammatory stress. These results suggest that SAVEP may have a potential function against inflammation.

The recently discovered interleukin (IL)- 32 isoform IL-32θ exerts anti-metastatic effects in the breast tumor microenvironment. However, the involvement of IL-32θ in breast cancer cell proliferation is not yet fully understood; therefore, the current study aimed to determine how IL-32θ affects cancer cell growth and evaluated the responses of IL-32θ-expressing cells to other cancer therapy. We compared the functions of IL-32θ in triple-negative breast cancer MDA-MB-231 cells that stably express IL-32θ, with MDA-MB-231 cells transfected with a mock vector. Slower growth was observed in cells expressing IL-32θ than in control cells, and changes were noted in nuclear morphology, mitotic division, and nucleolar size between the two groups of cells. Interleukin-32θ significantly reduced the colony-forming ability of MDA-MB-231 cells and induced permanent cell cycle arrest at the G1 phase. Long-term IL-32θ accumulation triggered permanent senescence and chromosomal instability in MDA-MB-231 cells. Genotoxic drug doxorubicin (DR) reduced the viability of MDA-MB-231 cells not expressing IL-32θ more than in cells expressing IL-32θ. Overall, these findings suggest that IL-32θ exerts antiproliferative effects in breast cancer cells and initiates senescence, which may cause DR resistance. Therefore, targeting IL-32θ in combination with DR treatment may not be suitable for treating metastatic breast cancer.

Due to the ever-expanding functions attributed to autophagy, there is widespread interest in understanding its contribution to human physiology; however, its specific cellular role as a stress-response mechanism is still poorly defined. To investigate autophagy's role in this regard, we repeatedly subjected cultured mouse myoblasts to two stresses with diverse impacts on autophagic flux: amino acid and serum withdrawal (Hank's balanced salt solution [HBSS]), which robustly induces autophagy, or low-level toxic stress (staurosporine, STS). We found that intermittent STS (int-STS) administration caused cell cycle arrest, development of enlarged and misshapen cells/nuclei, increased senescence-associated heterochromatic foci and senescence-associated β-galactosidase activity, and prevented myogenic differentiation. These features were not observed in cells intermittently incubated in HBSS (int-HB). While int-STS cells displayed less DNA damage (phosphorylated H2A histone family, member X content) and caspase activity when administered cisplatin, int-HB cells were protected from STS-induced cell death. Interestingly, STS-induced senescence was attenuated in autophagy related 7-deficient cells. Therefore, while repeated nutrient withdrawal did not cause senescence, autophagy was required for senescence caused by toxic stress. These results illustrate the context-dependent effects of different stressors, potentially highlighting autophagy as a distinguishing factor., (© 2020 Wiley Periodicals LLC.)

In an in vitro nanotoxicity system, cell-nanoparticle (NP) interaction leads to the surface adsorption, uptake, and changes into nuclei/cell phenotype and chemistry, as an indicator of oxidative stress, genotoxicity, and carcinogenicity. Different types of nanomaterials and their chemical composition or "corona" have been widely studied in context with nanotoxicology. However, rare reports are available, which delineate the details of the cell shape index (CSI) and nuclear area factors (NAFs) as a descriptor of the type of nanomaterials. In this paper, we propose a machine-learning-based graph modeling and correlation-establishing approach using tight junction protein ZO-1-mediated alteration in the cell/nuclei phenotype to quantify and propose it as indices of cell-NP interactions. We believe that the phenotypic variation (CSI and NAF) in the epithelial cell is governed by the physicochemical descriptors ( e.g. , shape, size, zeta potential, concentration, diffusion coefficients, polydispersity, and so on) of the different classes of nanomaterials, which critically determines the intracellular uptake or cell membrane interactions when exposed to the epithelial cells at sub-lethal concentrations. The intrinsic and extrinsic physicochemical properties of the representative nanomaterials (NMs) were measured using optical (dynamic light scattering, NP tracking analysis) methods to create a set of nanodescriptors contributing to cell-NM interactions via phenotype adjustments. We used correlation function as a machine-learning algorithm to successfully predict cell and nuclei shapes and polarity functions as phenotypic markers for five different classes of nanomaterials studied herein this report. The CSI and NAF as nanodescriptors can be used as intuitive cell phenotypic parameters to define the safety of nanomaterials extensively used in consumer products and nanomedicine.

Background: Linalool is a monoterpene compound with various potential therapeutic applications in several medical fields. Previous studies have indicated the activity of linalool against cell lines; however, its high level of toxicity restricts its use. The aim of this study was to design and manufacture compounds with a novel structure that can be used for loading linalool, to reduce its toxicity and improve its reachable ability., Methods: We synthesized and characterized a new molecule for loading linalool onto gold nanoparticles (GNPs) capped with glutathione and conjugated with a CALNN peptide. Linalool was loaded onto the GNPs via the reaction of the surface groups of both linalool and the GNPs. Moreover, the target peptide could be loaded onto the surface of the GNPs via a chemical reaction. The cytotoxic effects of linalool-GNP (LG) and linalool-GNP-CALNN peptide (LGC) conjugates against ovarian cancer cells were investigated, as were the possible mechanisms underlying the induction of apoptosis., Results: Our findings illustrated the significant antiproliferative effect of LG and LGC on SKOV-3 cells. The cytotoxicity assay demonstrated that LG and LGC were selectively toxic in cancer cells and induced apoptosis by activating caspase-8, the p53 protein, and various proteins involved in apoptosis. The present data demonstrated that LG and LGC have a high therapeutic potential and should be given particular consideration as anticancer drug-delivery systems, as LG and LGC were remarkably more cytotoxic against a cancer cell line than were linalool and GNPs alone., Conclusion: We concluded that LG and LGC are promising compounds that can be used for treating ovarian cancer (SKOV-3) cells via the induction of apoptosis through extrinsic and intrinsic pathways., Competing Interests: All authors report no conflicts of interest in this work., (© 2020 Jabir et al.)

Targeting the distinct cholesterol metabolism of tumor cells is proposed as a novel way to treat tumors. Blocking acyl-CoA cholesterol acyltransferase-1 (ACAT-1) by the inhibitor avasimible (Ava), which elevates intracellular free cholesterol levels, is shown to effectively induce apoptosis. However, Ava faces disadvantages of poor water solubility, a short half-life, and no capability for fluorescence detection, which have greatly limited its application. Herein, a fluorescent hyperstar polymer (FHSP) is developed to encapsulate Ava to improve its ability to inhibit HeLa cells and K562 cells. The results of this study show that the obtained Ava-FHSP micelles possess a high drug loading capacity of 22.7% and bright green fluorescence. Ava and Ava-FHSP are cytotoxic to both HeLa and K562 cells and cause reductions in cell size, nuclear lysis, and chromatin condensation and hindered proliferation of both cell types by causing S phase cell cycle arrest. Further mechanistic analysis indicates that Ava-FHSP reduces the protein and messenger RNA expression of ACAT-1 and significantly increases intracellular free cholesterol levels, which can increase endoplasmic reticulum stress and finally cause cell apoptosis. All these results suggest that this fluorescent hyperstar polymer represents a potential therapeutic tumor strategy by changing the cholesterol metabolism of tumor cells., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Microscopy is a powerful tool for characterizing complex cellular phenotypes, but linking these phenotypes to genotype or RNA expression at scale remains challenging. Here, we present Visual Cell Sorting, a method that physically separates hundreds of thousands of live cells based on their visual phenotype. Automated imaging and phenotypic analysis directs selective illumination of Dendra2, a photoconvertible fluorescent protein expressed in live cells; these photoactivated cells are then isolated using fluorescence-activated cell sorting. First, we use Visual Cell Sorting to assess hundreds of nuclear localization sequence variants in a pooled format, identifying variants that improve nuclear localization and enabling annotation of nuclear localization sequences in thousands of human proteins. Second, we recover cells that retain normal nuclear morphologies after paclitaxel treatment, and then derive their single-cell transcriptomes to identify pathways associated with paclitaxel resistance in cancers. Unlike alternative methods, Visual Cell Sorting depends on inexpensive reagents and commercially available hardware. As such, it can be readily deployed to uncover the relationships between visual cellular phenotypes and internal states, including genotypes and gene expression programs., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

The global incidence of breast cancer has increased. However, there are many impediments to the development of safe and effective anticancer drugs. The aim of the present study was to evaluate the effect of aviculin isolated from Lespedeza cuneata (Dum. Cours.) G. Don. (Fabaceae) on MCF-7 human breast cancer cells and determine the underlying mechanism. Using the bioassay-guided isolation by water soluble tetrazolium salt (WST-1)-based Ez-Cytox assay, nine compounds (four lignan glycosides ( 1 - 4 ), three flavonoid glycosides ( 5 - 7 ), and two phenolic compounds ( 8 and 9 )) were isolated from the ethyl acetate (EA) fraction of the L. cuneata methanolic extract. Of these, aviculin ( 2 ), a lignan glycoside, was the only compound that reduced metabolic activity on MCF-7 cells below 50% (IC 50 : 75.47 ± 2.23 μM). The underlying mechanism was analyzed using the annexin V Alexa Fluor 488 binding assay and Western blotting. Aviculin ( 2 ) was found to induce apoptotic cell death through the intrinsic apoptosis pathway, as indicated by the increased expression of initiator caspase-9, executioner caspase-7, and poly (ADP-ribose) polymerase (PARP). Aviculin ( 2 )-induced apoptotic cell death was accompanied by an increase in the Bax/Bcl-2 ratio. These findings demonstrated that aviculin ( 2 ) could induce breast cancer cell apoptosis through the intrinsic apoptosis pathway, and it can therefore be considered an excellent candidate for herbal treatment of breast cancer.

BACKGROUND Bauerane is a triterpenoid derived from the dandelion root (Taraxacum officinale). This study aimed to investigate the effects of bauerane on cell proliferation of A549 human lung cancer cells and the molecular mechanisms involved. MATERIAL AND METHODS A549 human lung adenocarcinoma cells and normal MRC-5 lung fibroblasts were grown in culture and treated with increasing doses of bauerane at 0, 2.5, 5, 10, 20, 40, 80, and 160 µM. The MTT assay was used to measure cell proliferation. Cell apoptosis was assessed by 4', 6-diamidino-2-phenylindole (DAPI), and acridine orange/ethidium bromide (AO/EB) staining. The cell cycle was evaluated by flow cytometry. Western blot measured the protein expression levels of cytochrome c, Bax, cyclin B1, Bcl-2, PI3K, p-PI3K, Akt, p-Akt, and STAT3 proteins. RESULTS Bauerane inhibited the proliferation of A549 lung cancer cells in a dose-dependent manner, with an IC₅₀ of 10 µM, with no cytotoxicity for MRC-5 cells. Bauerane treatment induced apoptosis of A549 cells, which was associated with the upregulation of Bax and down-regulation of Bcl-2. Bauerane induced S-phase arrest of A549 cells, which was dose-dependent and associated with reduced expression of cyclin B1. The findings from Western blot showed that bauerane inhibited the phosphorylation of PI3K/AKT and STAT3 signaling pathways. CONCLUSIONS Bauerane inhibited the proliferation of A549 lung cancer cells in vitro and induced cell apoptosis and cell cycle arrest in a dose-dependent manner.

Mechanosensing of the mechanical microenvironment by cells regulates cell phenotype and function. The nucleus is critical in mechanosensing, as it transmits external forces from the cellular microenvironment to the nuclear envelope housing chromatin. This study aims to elucidate how mechanical confinement affects nuclear deformation within several cell types, and to determine the role of cytoskeletal elements in controlling nuclear deformation. Human cancer cells (MDA-MB-231), human mesenchymal stem cells (MSCs), and mouse fibroblasts (L929) were seeded within polydimethylsiloxane (PDMS) microfluidic devices containing microchannels of varying cross-sectional areas, and nuclear morphology and volume were quantified via image processing of fluorescent cell nuclei. We found that the nuclear major axis length remained fairly constant with increasing confinement in MSCs and MDA-MB-231 cells, but increased with increasing confinement in L929 cells. Nuclear volume of L929 cells and MSCs decreased in the most confining channels. However, L929 nuclei were much more isotropic in unconfined channels than MSC nuclei. When microtubule polymerization or myosin II contractility was inhibited, nuclear deformation was altered only in MSCs in wide channels. This work informs our understanding of nuclear mechanics in physiologically relevant spaces, and suggests diverging roles of the cytoskeleton in regulating nuclear deformation in different cell types., Competing Interests: The authors declare no conflict of interest.

Ligustilide (LIG) is the main lipophilic component of the Umbelliferae family of pharmaceutical plants, including Radix angelicae sinensis and Ligusticum chuanxiong. LIG shows various pharmacological properties associated with anti-inflammation and anti-apoptosis in several kinds of cell lines. However, the therapeutic effects of LIG on chondrocyte apoptosis remain unknown. In this study, we investigated whether LIG had an anti-apoptotic activity in sodium nitroprusside (SNP)-stimulated chondrocyte apoptosis and could delay cartilage degeneration in a surgically induced rat OA model, and elucidated the potential mechanisms. In vitro studies revealed that LIG significantly suppressed chondrocyte apoptosis and cytoskeletal remodelling, which maintained the nuclear morphology and increased the mitochondrial membrane potential. In terms of SNP, LIG treatment considerably reduced the expression levels of cleaved caspase-3, Bax and inducible nitric oxide synthase and increased the expression level of Bcl-2 in a dose-dependent manner. The LIG-treated groups presented a significantly suppressed expression of activating transcription factor 2 and phosphorylation of Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). The inhibitory effect of LIG was enhanced by the p38 MAPK inhibitor SB203580 or the JNK inhibitor SP600125 and offset by the agonist anisomycin. In vivo studies demonstrated that LIG attenuated osteoarthritic cartilage destruction by inhibiting the cartilage chondrocyte apoptosis and suppressing the phosphorylation levels of activating transcription factor 2, JNK and p38 MAPK. This result was confirmed by histological analyses, micro-CT, TUNEL assay and immunohistochemical analyses. Collectively, our studies indicated that LIG protected chondrocytes against SNP-induced apoptosis and delayed articular cartilage degeneration by suppressing JNK and p38 MAPK pathways., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Skin is exposed to various physico-chemical cues. Keratinocytes, a major component of the skin epidermis, directly interact with the surrounding extracellular matrix, and thus, biochemical and biophysical stimulations from the matrix regulate the function of keratinocytes. Although it was reported that inflammatory responses of skin were altered by an applied mechanical force, understanding how the keratinocytes sense the mechanical stimuli and regulate a cytokine secretion remains unclear. Here, we designed a device that is able to apply chemo-mechanical cues to keratinocytes and assess their proinflammatory cytokine IL-6 production. We showed that when chemical stimuli were applied with mechanical stimuli simultaneously, the IL-6 production markedly increased compared to that observed with a single stimulus. Quantitative structural analysis of cellular components revealed that the applied mechanical stretch transformed the cell morphology into an elongated shape, increased the cell size, and dictated the distribution of focal adhesion complex. Our results suggest that the mechanical cue-mediated modulation of focal adhesion proteins and actin cytoskeleton translates into intracellular signaling associated with the IL-6 production particularly in skin sensitization. Our study can be applied to understand proinflammatory responses of skin under altered biophysical environments of the skin.

Background: Treatment for melanoma is a challenging clinical problem, and some new strategies are worth exploring., Purpose: The objective of this study was to investigate the in vitro and in vivo anti-melanoma effects of hydroxyapatite nanoparticles (HANPs) and discuss the involved material factors., Materials and Methods: Five types of HANPs, ie, HA-A, HA-B, HA-C, HA-D, and HA-E, were prepared by wet chemical method combining with polymer template and appropriate post-treatments. The in vitro effects of the as-prepared five HANPs on inhibiting the viability of A375 melanoma cells and inducing the apoptosis of the cells were evaluated by Cell Counting Kit-8 analysis, cell nucleus morphology observation, flow cytometer, and PCR analysis. The in vivo anti-melanoma effects of HANPs were studied in the tumor model of nude mice., Results: The five HANPs had different physicochemical properties, including morphology, size, specific surface area (SSA), crystallinity, and so on. By the in vitro cell study, it was found that the material factors played important roles in the anti-melanoma effect of HANPs. Among the as-prepared five HANPs, HA-A with granular shape, smaller size, higher SSA, and lower crystallinity exhibited best effect on inhibiting the viability of A375 cells. At the concentration of 200 μg/mL, HA-A resulted in the lowest cell viability (34.90%) at day 3. All the HANPs could induce the apoptosis of A375 cells, and the relatively higher apoptosis rates of the cells were found in HA-A (20.10%) and HA-B (19.41%) at day 3. However, all the HANPs showed no inhibitory effect on the viability of the normal human epidermal fibroblasts. The preliminary in vivo evaluation showed that both HA-A and HA-C could delay the formation and growth speed of melanoma tissue significantly. Likely, HA-A exhibited better effect on inhibiting the growth of melanoma tissue than HA-C. The inhibition rate of HA-A for tumor tissue growth reached 49.1% at day 23., Conclusion: The current study confirmed the anti-melanoma effect of HANPs and provided a new idea for the clinical treatment of melanoma., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

The anti-lung cancer activity of volatile oil from Alpinia officinarum (VOAO) and the underlying mechanism has not been studied. Herein, VOAO was extracted by steam distillation and its components were identified by GC-MS analysis. Cells viability was measured by an MTT assay and the cell survival capacity was tested via a colony formation assay. Apoptosis cells were detected using the Annexin V-FITC/PI method. Hoechst 33342 cell unclear staining was employed to evaluate the nuclear morphology change. The mitochondrial membrane potential was detected by a JC-1 staining assay. Bcl-2, Mcl-1 and cleaved caspase-3 proteins were quantified by immune blotting. Furthermore, VOAO anti-cancer activity was evaluated on an A549 cell xenograft nude mice model. Our results have revealed that VOAO could inhibit the cell viability of lung carcinoma cells and the colony formation capacity. VOAO downregulates Bcl-2 and Mcl-1 and triggers dysfunction of the mitochondrial membrane potential. VOAO further activates caspase-3 cleavage and induces lung cancer cells apoptosis. In addition, VOAO administration significantly suppresses lung cancer growth in xenograft mice without obvious hepatotoxicity. We conclude that VOAO could be an effective, low cytotoxicity natural component for treatment of lung carcinoma.

Triple negative breast cancer (TNBC) cells express increased levels of the pro-inflammatory and pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8), which promotes their proliferation and migration. Because TNBC patients are unresponsive to current targeted therapies, new therapeutic strategies are urgently needed. While proteasome inhibition by bortezomib (BZ) or carfilzomib (CZ) has been effective in treating hematological malignancies, it has been less effective in solid tumors, including TNBC, but the mechanisms are incompletely understood. Here we report that proteasome inhibition significantly increases expression of IL-8, and its receptors CXCR1 and CXCR2, in TNBC cells. Suppression or neutralization of the BZ-induced IL-8 potentiates the BZ cytotoxic and anti-proliferative effect in TNBC cells. The IL-8 expression induced by proteasome inhibition in TNBC cells is mediated by IκB kinase (IKK), increased nuclear accumulation of p65 NFκB, and by IKK-dependent p65 recruitment to IL-8 promoter. Importantly, inhibition of IKK activity significantly decreases proliferation, migration, and invasion of BZ-treated TNBC cells. These data provide the first evidence demonstrating that proteasome inhibition increases the IL-8 signaling in TNBC cells, and suggesting that IKK inhibitors may increase effectiveness of proteasome inhibitors in treating TNBC., Competing Interests: The authors have declared that no competing interests exist.

Purpose: To evaluate acetylation of histone H3, chromatin remodeling, nuclear size and shape, DNA ploidy, and distribution of nucleolus organizing regions (NORs) in corneal epithelial and stromal cells of diabetic and nondiabetic rats., Methods: Diabetes was induced by a single intraperitoneal injection of alloxan. All diabetic rats (n = 20) included in the study had 4 weeks of moderate-to-severe hyperglycemia (plasma glucose levels >400 mg/dL). Acetylated histone H3 levels were quantified in corneal tissue using a colorimetric assay. Chromatin remodeling, nuclear sizes (area/perimeter) and shapes (circularity), and DNA ploidies were evaluated from Feulgen-stained tissue sections using video image analysis. Distributions of NORs were studied in tissue sections impregnated with silver ions. Ophthalmic clinical parameters, including corneal sensitivity, were investigated. Twenty nondiabetic rats were used as controls., Results: Acetylation of histone H3 was reduced in the corneas of the diabetic rats. Nuclei in corneal epithelial cells of diabetic rats compacted chromatin, increased in size, modified their shapes, and elevated DNA ploidy. The only nuclear change observed in the corneal stromal cells of diabetic rats was chromatin decompaction. The size of the silver-stained NOR did not differ between the study samples. The corneal sensitivity in diabetic rats was 51.8% lower than that in nondiabetic rats., Conclusions: The results of this study show that alloxan-induced diabetes altered the histone H3 acetylation pattern and compromised the chromatin supraorganization in corneal tissue/cells. Continued research is needed to understand the clinical and morphofunctional significance of changes in corneal cell nuclei of diabetic individuals.

The leucine aminopeptidase inhibitor, benzyloxycarbonyl-leucine-chloromethylketone (z-L-CMK), was found to be toxic and readily induce cell death in Jurkat T cells. Dose-response studies show that lower concentration of z-L-CMK induced apoptosis in Jurkat T cells whereas higher concentration causes necrosis. In z-L-CMK-induced apoptosis, both the initiator caspases (-8 and -9) and effector caspases (-3 and -6) were processed to their respective subunits. However, the caspases remained intact in z-L-CMK-induced necrosis. The caspase inhibitor, z-VAD-FMK inhibited z-L-CMK-mediated apoptosis and caspase processing but has no effect on z-L-CMK-induced necrosis in Jurkat T cells. The high mobility group protein B1 (HMGB1) protein was found to be released into the culture medium by the necrotic cells and not the apoptotic cells. These results indicate that the necrotic cell death mediated by z-L-CMK at high concentrations is via classical necrosis rather than secondary necrosis. We also demonstrated that cell death mediated by z-L-CMK was associated with oxidative stress via the depletion of intracellular glutathione (GSH) and increase in reactive oxygen species (ROS), which was blocked by N-acetyl cysteine. Taken together, the results demonstrated that z-L-CMK is toxic to Jurkat T cells and induces apoptosis at low concentrations, while at higher concentrations the cells die of necrosis. The toxic side effects in Jurkat T cells mediated by z-L-CMK are associated with oxidative stress via the depletion of GSH and accumulation of ROS.

Despite the low case fatality, Zika virus (ZIKV) infection has been associated with microcephaly in infants and Guillain-Barré syndrome. Antiviral and vaccine developments against ZIKV are still ongoing; therefore, in the meantime, preventing the disease transmission is critical. Primarily transmitted by Aedes species mosquitoes, ZIKV also can be sexually transmitted. We used AG129 mice lacking interferon-α/β and -γ receptors to study the testicular pathogenesis and sexual transmission of ZIKV. Infection of ZIKV progressively damaged mouse testes, increased testicular oxidative stress as indicated by the levels of reactive oxygen species, nitric oxide, glutathione peroxidase 4, spermatogenesis-associated-18 homolog in sperm and pro-inflammatory cytokines including IL-1β, IL-6, and G-CSF. We then evaluated the potential role of the antioxidant ebselen (EBS) in alleviating the testicular pathology with ZIKV infection. EBS treatment significantly reduced ZIKV-induced testicular oxidative stress, leucocyte infiltration and production of pro-inflammatory response. Furthermore, it improved testicular pathology and prevented the sexual transmission of ZIKV in a male-to-female mouse sperm transfer model. EBS is currently in clinical trials for various diseases. ZIKV infection could be on the list for potential use of EBS, for alleviating the testicular pathogenesis with ZIKV infection and preventing its sexual transmission.

Mensacarcin is a highly oxygenated polyketide that was first isolated from soil-dwelling Streptomyces bacteria. It exhibits potent cytostatic properties (mean of 50% growth inhibition = 0.2 μm) in almost all cell lines of the National Cancer Institute (NCI)-60 cell line screen and relatively selective cytotoxicity against melanoma cells. Moreover, its low COMPARE correlations with known standard antitumor agents indicate a unique mechanism of action. Effective therapies for managing melanoma are limited, so we sought to investigate mensacarcin's unique cytostatic and cytotoxic effects and its mode of action. By assessing morphological and biochemical features, we demonstrated that mensacarcin activates caspase-3/7-dependent apoptotic pathways and induces cell death in melanoma cells. Upon mensacarcin exposure, SK-Mel-28 and SK-Mel-5 melanoma cells, which have the BRAF V600E mutation associated with drug resistance, showed characteristic chromatin condensation as well as distinct poly(ADP-ribose)polymerase-1 cleavage. Flow cytometry identified a large population of apoptotic melanoma cells, and single-cell electrophoresis indicated that mensacarcin causes genetic instability, a hallmark of early apoptosis. To visualize mensacarcin's subcellular localization, we synthesized a fluorescent mensacarcin probe that retained activity. The natural product probe was localized to mitochondria within 20 min of treatment. Live-cell bioenergetic flux analysis confirmed that mensacarcin disturbs energy production and mitochondrial function rapidly. The subcellular localization of the fluorescently labeled mensacarcin together with its unusual metabolic effects in melanoma cells provide evidence that mensacarcin targets mitochondria. Mensacarcin's unique mode of action suggests that it may be a useful probe for examining energy metabolism, particularly in BRAF-mutant melanoma, and represent a promising lead for the development of new anticancer drugs., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Context: Clinacanthus nutans Lindau (Acanthaceae) is a medicinal plant that has been reported to have anti-inflammatory, antiviral, antimicrobial and antivenom activities. In Malaysia, it has been widely claimed to be effective in various cancer treatments but scientific evidence is lacking., Objective: This study investigates the chemical constituents, anti-proliferative, and apoptotic properties of C. nutans root extracts., Materials and Methods: The roots were subjected to solvent extraction using methanol and ethyl acetate. The anti-proliferative effects of root extracts were tested at the concentrations of 10 to 50 μg/mL on MCF-7 and HeLa by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay for 72 h. Morphological changes were observed under light microscope. Pro-apoptotic effects of root extracts were examined using flow cytometric analysis and RT-PCR. The chemical compositions of root extracts were detected using GC-MS., Results: The proliferation of MCF-7 cells was inhibited with the IC 50 values of 35 and 30 μg/mL, respectively, for methanol and ethyl acetate root extracts. The average inhibition of HeLa cells was ∼25%. Induction of apoptosis in MCF-7 was supported by chromatin condensation, down-regulation of BCL2 and unaltered expression of BAX. However, only ethyl acetate extract caused the loss of mitochondrial membrane potential. GC-MS analysis revealed the roots extracts were rich with terpenoids and phytosterols., Discussion and Conclusions: The results demonstrated that root extracts promote apoptosis by suppressing BCL2 via mitochondria-dependent or independent manner. The identified compounds might work solely or cooperatively in regulating apoptosis. However, further studies are required to address this.

Hospital effluents contain myriad of mutagens and genotoxins capable of increasing DNA damage in aquatic biota. African mudfish, Clarias gariepinus, are exposed to genotoxins when cultured in swamps and derelict water bodies often contaminated by effluents. Moreover, its DNA is susceptible to xenobiotic-induced lesions since it lacks L-gulonolactone oxidase and hence cannot synthesize L-ascorbic acid. This study investigated 96-h acute toxicity and protective effects of dietary ascorbic acid (AA) against micronucleus (MN) and abnormal nuclear (NAs) formation in C. gariepinus exposed to sub-lethal concentrations of hospital effluent. Six concentrations (0.5-3.0%) of the effluent were selected to determine the 96-h acute toxicity of the effluent in C. gariepinus, after range finding test. Fish were exposed to sub-lethal concentrations (0.08-1.30%) of the 96 h LC 50 . Two other groups were exposed to the 96 h LC 50 (1.30%) of the effluent +50 and +100 mg/kg of dietary ascorbic for 7 days, and MN and NAs assessed in peripheral erythrocytes. The 96 h LC 50 (1.30%) was 1.18 times more toxic than the 24 h LC 50 (1.54%), indicating that the toxicity of the effluent increased with exposure duration. MN, nuclear bud, enucleated, fragmented nucleus (apoptosis), and necrotic erythrocytes significantly increase in effluent treated fish. Dietary AA reduced MN from 6.35-fold (1.30% treated group) to 3.72-fold (1.30% + 50 mg AA) and 3.54-fold (1.30% + 100 mg AA). Also, AA reduced total NAs from 2.26-fold (1.30%) to 1.40-fold (1.30% + 50 mg AA) and 1.06-fold (1.30% + 100 mg AA) compared to the control. Heavy metals and physicochemical parameters analyzed in the tested effluent possibly induced the mortality and cytogenotoxicity in C. gariepinus, and this was ameliorated by dietary AA.

Metal complexes are still broadly used as the first line of the treatment for different types of tumors nowadays. Carboplatin and oxaliplatin were authorized for clinical use, even though there is little information on the mutagenic profile associated to their usage. This study evaluated the cytostatic effects and the induction of complex genomic alterations after 24-h treatment of CHO-K1 cells to concentrations of 12.5-800 μM of carboplatin and oxaliplatin in the cytokinesis-block micronucleus assay (CBMN-Cyt). The results demonstrated that carboplatin and oxaliplatin significantly increased the frequency of micronuclei (MN), nucleoplasmatic bridges (NPBs), and nuclear buds (NBUDs). On one hand, oxaliplatin induces significantly more chromosomal abnormalities than carboplatin at concentrations of 12.5 and 25 μM. On the other hand, carboplatin, in cells exposed to concentrations of 50 and 100 μM, is more efficient than oxaliplatin in the induction of chromosomal instability events. Both drugs cause significant reduction in the cytokinesis-block proliferation index, demonstrating their cytostatic effects at concentrations 50-800 μM. The results of this study shed more light on the characterization of biological effects associated with the exposure to carboplatin and oxaliplatin.

Acute promyelocytic leukemia (APL) is a special subtype of acute myeloid leukemia that responds to treatment with all‑trans retinoic acid and arsenic trioxide. However, severe side effects and drug resistance limit the effectiveness of these treatments. Hence, new drugs for APL are required urgently. Shikonin, an active naphthoquinone derived from the Chinese medical herb Zi Cao exerts antitumor activity in several cancers. In the present study, the effects of shikonin on proliferation and apoptosis in NB4 cells, as well as related mechanisms were assessed. Treatment of NB4 cells with shikonin inhibited proliferation in a concentration‑ and time‑dependent manner. The cell cycle was arrested in the G1 phase. NB4 cells treated with shikonin exhibited more apoptosis and higher levels of cleaved caspase‑3 and poly ADP‑ribose polymerase than control cells. Western blotting results demonstrated that the expression of p‑p38 mitogen‑activated protein kinase (p‑p38MAPK) and p‑c‑Jun N‑terminal kinase (p‑JNK) was increased significantly by shikonin treatment, while the expression of p‑ERK and c‑Myc was decreased. In summary, these findings indicated that shikonin inhibited cell proliferation and induced apoptosis partly through modulation of the MAPKs and downregulation of c‑Myc.

The present study aimed to investigate the neuroprotective effect of a novel synthetic compound (5zou) on differentiated PC12 cells against 6‑hydroxydopamine (6‑OHDA) and L‑glutamic acid (L‑Glu) neurotoxin‑induced cell injury and the potential mechanisms involved. 5zou is a 2, 2‑disubstituted 1,2‑dihydropyridine. PC12 cells were treated with 6‑OHDA and L‑Glu to establish neurotoxic cell models. MTT assay, DCFH‑DA staining, Fluo‑4‑AM staining, JC‑1 staining and western blotting were used to determine the changes in cell viability, intracellular reactive oxygen species concentration, Ca2+ influx, mitochondrial membrane potential and the protein expressions of B‑cell lymphoma‑2 (Bcl‑2) and B‑cell lymphoma‑extra large (Bcl‑xL). Morphological analysis demonstrated the effect of 5zous on neuritogenesis and differentiation in PC12 cells. The results suggested that 5zou rescued the cell viability, intracellular ROS level, Ca2+ influx, mitochondrial membrane potential, and expression of Bcl‑2 and Bcl‑xL, which were altered by 6‑OHDA and L‑Glu. The study confirmed that 5zou has neuroprotective effects on neurotoxin‑induced differentiated PC12 cells injury, potentially via the mitochondrial apoptosis pathway.

Cadmium (Cd) is a well-known heavy metal that causes environmental pollution and human health problems. Several studies attempted to assess Cd toxicity in vitro and in vivo. However, the systemic profile of cadmium toxicity has not been studied well. In the present study, we assessed the toxicity of Cd on human peripheral blood lymphocytes and monocytes and gene expression, using a system toxicological approach. Cd effect on cell viability and morphology were analyzed by MTT assay and AO/EB staining respectively. Mitochondrial membrane potential depletion and reactive oxygen singlet generation were assessed by flow cytometry. Effects of Cd treatment on gene expression were also studied. Significant reduction in cell viability and disintegration of nuclear morphology were observed in Cd-treated cells. Cd exposure enhanced the loss of mitochondrial membrane potential through oxidative stress. Dose-dependent upregulation of GSTM3 and downregulation of GSR gene expression were observed. TNF gene expression decreased as the level of Cd exposure increased. We analyzed the toxicological effects of Cd on more than 45 proteins for biological target identification. These system toxicological studies suggested that Cd induced toxicity through various biological processes such as oxidative stress, oxidation-reduction, and ROS and hydrogen peroxide generation. Additionally, Cd affects the cellular component network and modulates the expression level of oxidative stress-related genes., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Chemotherapy-induced peripheral neuropathy (CIPN) is a major, dose-limiting adverse effect experienced by cancer patients. Advancements in mechanism-based risk mitigation and effective treatments for CIPN can be aided by suitable in vitro assays. To this end, we developed a multiparametric morphology-centered rat dorsal root ganglion (DRG) assay. Morphologic alterations in subcellular structures of neurons and non-neurons were analyzed with an automated microscopy system. Stains for NeuN (a neuron-specific nuclear protein) and Tuj-1 (β-III tubulin) were used to identify neuronal cell nuclei and neuronal cell bodies/neurites, respectively. Vimentin staining (a component of Schwann cell intermediate filaments) was used to label non-neuronal supporting cells. Nuclei that stained with DAPI, but lacked NeuN represented non-neuronal cells. Images were analyzed following 24 h of continuous exposure to CIPN-inducing agents and 72 h after drug removal to provide a dynamic measure of recovery from initial drug effects. Treatment with bortezomib, cisplatin, eribulin, paclitaxel or vincristine induced a dose-dependent loss of neurite/process areas, mimicking the 'dying back' degeneration of axons, a histopathological hallmark of clinical CIPN in vivo. The IC50 for neurite loss was within 3-fold of the maximal clinical exposure (Cmax) for all five CIPN-inducing drugs, but was >4- or ≥ 28-fold of the Cmax for 2 non-CIPN-inducing agents. Compound-specific effects, eg, neurite fragmentation by cisplatin or bortezomib and enlarged neuronal cell bodies by paclitaxel, were also observed. Collectively, these results support the use of a quantitative, morphologic evaluation and a DRG cell culture model to inform risk and examine mechanisms of CIPN., (Published by Oxford University Press on behalf of the Society of Toxicology 2017. This work is written by US Government employees and is in the public domain in the US.)

Ataxia telangiectasia is a rare genetic disease and no therapy is currently available. Glucocorticoid analogues have been shown to improve the neurological symptoms of treated patients. In the present study ataxia telangiectasia and wild type cells were used as a cellular model and treated with dexamethasone. The cells were subsequently investigated for membrane and whole cell mechanical properties by atomic force microscopy. In addition, cytoskeleton protein dynamics and nuclear shapes were assayed by fluorescence microscopy, while western blots were used to assess actin and tubulin content. At the macro level, dexamethasone directly modified the cell shape, Young's modulus and cytoskeleton protein dynamics. At the nano level, the roughness of the cell surface and the local nano-mechanical proprieties were found to be affected by Dexa. Our results show that ataxia telangiectasia and wild type cells are affected by Dexa, although there are dissimilarities in some macro-level and nano-level features between the tested cell lines. The Young's modulus of the cells appears to depend mainly on nuclear shape, with a slight contribution from the tested cytoskeleton proteins. The current study proposes that dexamethasone influences ataxia telangiectasia cell membranes contents, cell components and cell shape.

Vacuole fusion is a necessary process for the establishment of a large central vacuole, which is the central location of various hydrolytic enzymes and other factors involved in death at the beginning of plant programmed cell death (PCD). In our report, the fusion of vacuoles has been presented in two ways: i) small vacuoles coalesce to form larger vacuoles through membrane fusion, and ii) larger vacuoles combine with small vacuoles when small vacuoles embed into larger vacuoles. Regardless of how fusion occurs, a large central vacuole is formed in rice (Oryza sativa) aleurone cells. Along with the development of vacuolation, the rupture of the large central vacuole leads to the loss of the intact plasma membrane and the degradation of the nucleus, resulting in cell death. Stabilizing or disrupting the structure of actin filaments (AFs) inhibits or promotes the fusion of vacuoles, which delays or induces PCD. In addition, the inhibitors of the vacuolar processing enzyme (VPE) and cathepsin B (CathB) block the occurrence of the large central vacuole and delay the progression of PCD in rice aleurone layers. Overall, our findings provide further evidence for the rupture of the large central vacuole triggering the PCD in aleruone layers.

Cell death triggered by photodynamic therapy can occur through different mechanisms: apoptosis, necrosis or autophagy. However, recent studies have demonstrated the existence of other mechanisms with characteristics of both necrosis and apoptosis. These new cell death pathways, collectively termed regulated necrosis, include a variety of processes triggered by different stimuli. In this study, we evaluated the cell death mechanism induced by photodynamic treatments with two photosensitizers, meso-tetrakis (4-carboxyphenyl) porphyrin sodium salt (Na-H 2 TCPP) and its zinc derivative Na-ZnTCPP, in two human breast epithelial cell lines, a non-tumoral (MCF-10A) and a tumoral one (SKBR-3). Viability assays showed that photodynamic treatments with both photosensitizers induced a reduction in cell viability in a concentration-dependent manner and no dark toxicity was observed. The cell death mechanisms triggered were evaluated by several assays and cell line-dependent results were found. Most SKBR-3 cells died by either necrosis or apoptosis. By contrast, in MCF-10A cells, necrotic cells and another cell population with characteristics of both necrosis and apoptosis were predominant. In this latter population, cell death was PARP-dependent and translocation of AIF to the nucleus was observed in some cells. These characteristics are related with parthanatos, being the first evidence of this type of regulated necrosis in the field of photodynamic therapy.

How spatial organization of the genome depends on nuclear shape is unknown, mostly because accurate nuclear size and shape measurement is technically challenging. In large cell populations of the yeast Saccharomyces cerevisiae, we assessed the geometry (size and shape) of nuclei in three dimensions with a resolution of 30 nm. We improved an automated fluorescence localization method by implementing a post-acquisition correction of the spherical microscopic aberration along the z-axis, to detect the three dimensional (3D) positions of nuclear pore complexes (NPCs) in the nuclear envelope. Here, we used a method called NucQuant to accurately estimate the geometry of nuclei in 3D throughout the cell cycle. To increase the robustness of the statistics, we aggregated thousands of detected NPCs from a cell population in a single representation using the nucleolus or the spindle pole body (SPB) as references to align nuclei along the same axis. We could detect asymmetric changes of the nucleus associated with modification of nucleolar size. Stereotypical modification of the nucleus toward the nucleolus further confirmed the asymmetric properties of the nuclear envelope., Competing Interests: The authors declare no competing or financial interests., (© 2016. Published by The Company of Biologists Ltd.)

In routine course of life, nonsteroidal anti-inflammatory drugs (NSAIDs) are widely prescribed antipyretic, analgesic, and anti-inflammatory drugs. It is a well-proposed notion that treatment of NSAIDs may induce anti-proliferative effects in numerous cancer cells. Ibuprofen from isobutylphenylpropanoic acid is NSAID and used to relieve fever, pain, and inflammation. It is also used for juvenile idiopathic arthritis, rheumatoid arthritis, patent ductus arteriosus, and for pericarditis. Despite few emerging studies have expanded the fundamental concept that the treatment of NSAIDs influences apoptosis in cancer cells, however the NSAID-mediated precise mechanisms that determine apoptosis induction without producing adverse consequences in variety of cancer cells are largely unknown. In our present study, we have observed that ibuprofen reduces proteasome activity, enhances the aggregation of ubiquitylated abnormal proteins, and also elevates the accumulation of crucial proteasome substrates. Ibuprofen treatment causes mitochondrial abnormalities and releases cytochrome c into cytosol. Perhaps, the more detailed study is needed in the future to elucidate the molecular mechanisms of NSAIDs that can induce apoptosis without adverse effects and produce effective anti-tumor effects and consequently help in neurodegeneration and ageing.

Hepatocellular carcinoma is the most prevalent primary liver tumor and is among the top ten cancer that affect the world population. Its development is related, in most cases, to the existence of chronic liver injury, such as in cirrhosis. The knowledge about the correlation between chronic inflammation and cancer has driven new researches with anti-inflammatory agents that have potential for the development of antitumor drugs. Gallic acid is a phenolic acid found in many natural products and have shown anti-inflammatory, anti-tumor, anti-mutagenic and antioxidant actions. The purpose of this study was to investigate the effect of gallic acid on acute and chronic cell proliferation and inflammatory parameters of hepatocellular carcinoma cells (HepG2), as well as to investigate the mechanisms involved. Results showed that the gallic acid decreased the proliferation of HepG2 cells in a dose-dependent manner (Trypan blue exclusion assay), without causing necrosis (LDH assay). We observed a significant increase in the percentage of small and regular nuclei (Nuclear Morphometric Analysis assay), a significant induction of apoptosis by Annexin V-FITC and PI assay and no interference with the cell cycle using the FITC BrdU Flow Kit. We observed a significant reduction in the levels of IL-8 and increased levels of IL-10 and IL-12 (Cytometric Bead Array Human Inflammation Assay). Furthermore, gallic acid caused no cancer cells regrowth at a long term (Cumulative Population Doubling assay). According to these results, gallic acid showed a strong potential as an anti-tumor agent in hepatocellular carcinoma cells., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Zinc oxide nanoparticles (ZnO NPs) are widely used in a variety of products used in daily life. However, their impact on human health has not been completely elucidated. This study was designed to investigate the cytotoxicity associated with ZnO NPs, the role of dissolution in the toxicity of ZnO NPs, the molecular mechanisms and mode of cell death induced by ZnO NPs in human aortic endothelial cells (HAECs), and the protective effects of the antioxidant alpha-lipoic acid (LA). ZnO NPs significantly reduced cell viability in a dose- and time-dependent manner, resulted in intracellular oxidative stress and cell membrane leakage when treated with doses of 8-50 μg/mL for 12 and 24 h in HAECs. The toxicity was produced by undissolved ZnO NPs but not dissolved Zn(2+) and metal impurities. Exposure to ZnO NPs was found to induce apoptosis at 12 h and necrosis after 24 h. Apoptosis was confirmed using reactive oxygen species that triggered a decrease in mitochondria membrane potential, increase in Cyt-C release, activation of caspases 3 and caspases9 and increase in the ratio of Bax/Bcl-2. Futhermore, ZnO NPs could activate the Fas death receptor pathway. In addition, the antioxidant LA was able to protect HAECs from apoptosis induced by ZnO NPs., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Central events in the mitochondrial-dependent cell death pathway include the disruption of mitochondrial membrane potential, which causes the release of apoptogenic molecules leading to cell death. Based on the cytotoxic mechanism of deltamethrin (DLM), we examined the neuroprotective mechanisms of rosiglitazone (RGZ), which is against DLM-induced neuronal cell death. In this study, we found that DLM induces apoptosis in SH-SY5Y cells as demonstrated by the activation of caspase-3 and nuclear condensation. In addition, neuronal cell death in response to DLM was due to mitochondrial dependent-apoptosis pathways since DLM increased cytochrome c release into the cytosol and activated caspase-9. DLM exposure reduced PINK1 expression, and pretreatment with RGZ significantly reduced cytochrome c release and caspase-9 activation. RGZ also attenuated the reduction of complex I activity, mitochondrial membrane potential, and ATP levels. Pretreatment with RGZ significantly enhanced PINK1 expression in DLM-exposed cells. In addition, RGZ increased cytosolic PINK1 by inhibiting mitochondrial translocation of PINK1. Interestingly, RGZ fails to rescue DLM-induced mitochondrial dysfunction both in PINK1 knockdown and PPAR-γ antagonist treated cells. Results from this study suggest that RGZ exerts anti-apoptotic effects against DLM-induced cytotoxicity by attenuation of mitochondrial dysfunction through cytosolic PINK1-dependent signaling pathways., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Cistanoside A (C. A) was one of phenylethanol glycosides isolated from Cistanche deserticola, a tonic in traditional Chinese medicine. In our previous research, we demonstrated that Cistanoside A (C. A) possess the protective activities on CCl 4 induced hepatotoxicity in mice, such as increasing free radicals clearing activities, alleviating lipid-overoxidation damage, and improving respiratory chain function in mitochondria. Meanwhile, our previous research also demonstrated C.A possess protective activities on alcohol induced hepatotoxicity in mice, shown in ameliorate the hepatic function indices, lightening steatosis and inflammatory infiltration, increasing free radicals clearing activities, alleviating lipid-overoxidation damage, and alleviating energy metabolism in mitochondria. The aim of this research was to evaluate the effects of Cistanoside A (C. A) on ethanol-induced damage in primary cultured mouse hepatocytes, and probe into the mechanism related. Using fluorescent staining, flow cytometer, immunohistochemistry analysis, and Western blotting, we demonstrated that C.A could enhance the survival rate of the primary cultured hepatocytes, alleviate apoptosis and necrosis, the mechanism was involved with enhance the expression of apoptosis inhibition factor bcl-2, and inhibition the expression of immediate early genes c-fos., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

The deleterious effects of ethanol (EtOH) on the brain have been widely described, but its effects on the neuronal cytoskeleton during differentiation have not yet been firmly established. In this context, our aim was to investigate the direct effect of EtOH on cortical neurons during the period of differentiation. Primary cultures of cortical neurons obtained from 1-day-old rats were exposed to EtOH after 7days of culture, and viability and morphology were analyzed at structural and ultrastructural levels after 24-h EtOH exposure. EtOH caused a significant reduction of 73±7% in the viability of cultured cortical neurons, by preferentially inducing apoptotic cellular death. This effect was accompanied by an increase in caspase 3 and 9 expression. Furthermore, EtOH induced a reduction in total dendrite length and in the number of dendrites per cell. Ultrastructural studies showed that EtOH increased the number of lipidic vacuoles, lysosomes and multilamellar vesicles and induced a dilated endoplasmatic reticulum lumen and a disorganized Golgi apparatus with a ring-shape appearance. Microtubules showed a disorganized distribution. Apposition between pre- and postsynaptic membranes without a defined synaptic cleft and a delay in presynaptic vesicle organization were also observed. Synaptophysin and PSD95 expression, proteins pre- and postsynaptically located, were reduced in EtOH-exposed cultures. Overall, our study shows that EtOH induces neuronal apoptosis and changes in the cytoskeleton and membrane proteins related with the establishment of mature synapses. These direct effects of EtOH on neurons may partially explain its effects on brain development., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Olive leaf extract is characterized by a high content of polyphenols (oleuropein, hydroxytyrosol and their derivatives), which is associated with its therapeutic properties. The objective of the present research was to evaluate the antifungal activity of olive leaf extract against Candida albicans ATCC 10231 and C. dubliniensis CBS 7987 strains. Minimum inhibitory concentrations (MIC) of the extract were determined by several in vitro assays. The extract showed a concentration depended effect on the viability of C. albicans with MIC value of 46.875 mg mL-1 and C. dubliniensis with MIC value 62.5 mg mL-1. Most sensitive methods for testing the antifungal effect of the extracts were the trypan blue exclusion method and fluorescent dye exclusion method while MIC could not be determined by the method according to the EUCAST recommendation suggesting that herbal preparations contain compounds that may interfere with this susceptibility testing. The fluorescent dye exclusion method was also used for the assessment of morphological changes in the nuclei of treated cells. According to the obtained results, olive leaf extract is less effective against the tested strains than hydroxytyrosol, an olive plant constituent tested in our previous study.

In the present study, we investigate the function of ROS-AKT-mTOR axis on the apoptosis, proliferation and autophagy of MC3T3-E1 cells, and the proliferation of MC3T3-E1 cells after autophagy inhibition under high glucose conditions. MC3T3-E1 cells cultured in vitro were divided into the following groups: normal control group, N-acetylcysteine (NAC) group, 11.0 mM high glucose group, 11.0 mM high glucose + NAC group, 22.0 mM high glucose group, 22.0 mM high glucose + NAC group, CQ group, 22.0 mM high glucose + CQ group, 3-MA group and 3-MA + 22.0 mM high glucose group. ROS production was measured by DCFH-DA fluorescent probe. Cell proliferation was measured by MTT assay. Cells in different groups were stained with Annexin V-FITC/PI, and then apoptosis rate was detected by flow cytometry. Nucleus morphology was observed under fluorescence microscope after being incubated with Honchest33258. Protein expression was measured using Western blotting and immunofluorescence. Cell apoptosis and proliferation in high glucose group were increased and decreased, respectively, in a dose-dependent manner. Autophagy was significantly induced in high glucose group, even though different concentration of glucose induced autophagy in different stages of autophagy. ROS production in MC3T3-E1 cells was remarkably increased in high glucose group, but not in a dose-dependent manner. NAC, as an antioxidant, reduced ROS production and ameliorated cell apoptosis, proliferation abnormity and autophagy caused by high glucose. Expression of p-AKT and p-mTOR proteins were dramatically decreased in high glucose group, and NAC reversed their expression. In addition, 3-MA, an inhibitor of autophagy, significantly decreased the proliferation of MC3T3-E1 cells. When cocultured with 22.0 mM glucose that induced autophagy, proliferation of MC3T3-E1 cells was not affected compared to 22.0 mM high glucose group. Our present findings reveal that high glucose affects apoptosis, proliferation and autophagy of MC3T3-E1 cells through ROS-AKT-mTOR axis. In addition, autophagy inhibition does not affect the proliferation of MC3T3-E1 cells under high glucose conditions., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

L-asparaginase is an enzyme used as a chemotherapeutic agent, mainly for treating acute lymphoblastic leukemia. In this study, the gene of L-asparaginase from Zymomonas mobilis was cloned in pET vectors, fused to a histidine tag, and had its codons optimized. The L-asparaginase was expressed extracellularly and intracellularly (cytoplasmically) in Escherichia coli in far larger quantities than obtained from the microorganism of origin, and sufficient for initial cytotoxicity tests on leukemic cells. The in silico analysis of the protein from Z. mobilis indicated the presence of a signal peptide in the sequence, as well as high identity to other sequences of L-asparaginases with antileukemic activity. The protein was expressed in a bioreactor with a complex culture medium, yielding 0.13 IU/mL extracellular L-asparaginase and 3.6 IU/mL intracellular L-asparaginase after 4 h of induction with IPTG. The cytotoxicity results suggest that recombinant L-asparaginase from Z. mobilis expressed extracellularly in E.coli has a cytotoxic and cytostatic effect on leukemic cells.

The number of people afflicted with oral carcinoma in Japan has increased in recent years. Although preoperative neoadjuvant therapy with cisplatin and 5-fluorouracil are performed, chemotherapeutic response varies widely among the patients. With the aim of establishing novel indices to predict the therapeutic response to chemotherapy, we investigated the relationship between morphological features of pre-treatment oral carcinoma nuclei and the chemotherapeutic response using quantifying morphology of cell nuclei in pathological specimen images. We measured 4 morphological features of the nucleus of oral squamous cell carcinoma cases classified by the response to chemotherapy: No Change (NC) group, Partial Response (PR) group and Complete Response (CR) group. Furthermore, we performed immunohistochemical staining for p53 and Ki67 and calculated their positive rates in cancer tissues. Compactness and symmetry of the nucleus were significantly higher and nuclear edge response was significantly lower in cancer cells with lower chemotherapeutic responses compared high chemotherapeutic responders. As for positive rates of p53 and Ki67, there were no significant differences between any of the response groups. Morphological features of cancer cell nuclei in pathological specimens are sensitive predictive factors for the chemotherapeutic response to oral squamous cell carcinoma.

Though special AT-rich sequence-binding protein 2 (SATB2) is reported as a transcriptional regulator of skeletal development and osteogenic differentiation, the underlying mechanism of SATB2 is not fully understood. Herein, we report that SATB2 is localized to the mitotic microtubules, the centrosome, and midbodies in mitotic cells with alpha-tubulin. Moreover, siRNA-mediated disruption of SATB2 in H460 cells caused the defect of nuclear morphology and multinucleate cells. SATB2 siRNA knockdown reduced the viability and downregulated the CDK2 expression in SKOV3 cells. Consistently, cell cycle analysis demonstrated that the silencing of SATB2 induced cell-cycle G1 arrest. Furthermore, proteosomal inhibitor MG132 treatment rescued the downregulation of CDK2 in SATB2-silenced SKOV3 cells. Taken together, our findings suggest that SATB2 regulates the mitosis of cell cycle and affects G1 cell cycle via interaction with CDK2.

The nucleus has a smooth, regular appearance in normal cells, and its shape is greatly altered in human pathologies. Yet, how the cell establishes nuclear shape is not well understood. We imaged the dynamics of nuclear shaping in NIH3T3 fibroblasts. Nuclei translated toward the substratum and began flattening during the early stages of cell spreading. Initially, nuclear height and width correlated with the degree of cell spreading, but over time, reached steady-state values even as the cell continued to spread. Actomyosin activity, actomyosin bundles, microtubules, and intermediate filaments, as well as the LINC complex, were all dispensable for nuclear flattening as long as the cell could spread. Inhibition of actin polymerization as well as myosin light chain kinase with the drug ML7 limited both the initial spreading of cells and flattening of nuclei, and for well-spread cells, inhibition of myosin-II ATPase with the drug blebbistatin decreased cell spreading with associated nuclear rounding. Together, these results show that cell spreading is necessary and sufficient to drive nuclear flattening under a wide range of conditions, including in the presence or absence of myosin activity. To explain this observation, we propose a computational model for nuclear and cell mechanics that shows how frictional transmission of stress from the moving cell boundaries to the nuclear surface shapes the nucleus during early cell spreading. Our results point to a surprisingly simple mechanical system in cells for establishing nuclear shapes., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Doxorubicin (DOX) is a well-known drug used to treat a wide range of solid tumor and hematological malignancies, but the use of this drug is now restricted owing to its severe side effects, including normal cellular toxicity. This study was conducted to evaluate the potency of self-assembled betulinic acid (SA-BA) against DOX induced chemotherapeutic toxicity in human peripheral blood lymphocytes (PBLs). The isolated betulinic acid from the bark of Ziziphus jujuba tree was purified by column chromatography and characterized by FT-IR, XRD, (1)H NMR and self-assembly property was investigated by SEM imaging. DOX treatment produced significant reduction of viability of PBLs mainly by lowering cellular anti-oxidant pool and elevating the reactive oxygen species level. Pre-treatment with SA-BA followed by DOX exposure for 24h protected the PBLs from DOX induced oxidative stress. Potent anti-apoptotic role of SA-BA was also confirmed by FACS analysis and western blot assay. Severe inflammation is one of the major concerns in DOX treatment. We found that pre-treatment with SA-BA on PBLs significantly protected the PBLs from DOX induced inflammation. Thus, our finding confirms that SA-BA can be used to ameliorate the cytotoxic effects of DOX, which can be a helpful strategy during DOX mediated chemotherapy in cancer patients., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

The development of vascular scaffolds with controlled mechanical properties and stimulatory effects on biological activities of endothelial cells still remains a significant challenge to vascular tissue engineering. In this work, we reported an innovative approach to prepare a new type of vascular scaffolds with anisotropically and heterogeneously aligned patterns using electrospinning technique with unique wire spring templates, and further investigated the structural effects of the patterned electrospun scaffolds on mechanical properties and angiogenic differentiation of human umbilical vein endothelial cells (HUVECs). Results showed that anisotropically aligned patterned nanofibrous structure was obtained by depositing nanofibers on template in a structurally different manner, one part of nanofibers densely deposited on the embossments of wire spring and formed cylindrical-like structures in the transverse direction, while others loosely suspended and aligned along the longitudinal direction, forming a three-dimensional porous microstructure. We further found that such structures could efficiently control the mechanical properties of electrospun vascular scaffolds in both longitudinal and transverse directions by altering the interval distances between the embossments of patterned scaffolds. When HUVECs were cultured on scaffolds with different microstructures, the patterned scaffolds distinctively promoted adhesion of HUVECs at early stage and proliferation during the culture period. Most importantly, cells experienced a large shape change associated with cell cytoskeleton and nuclei remodeling, leading to a stimulatory effect on angiogenesis differentiation of HUVECs by the patterned microstructures of electrospun scaffolds, and the scaffolds with larger distances of intervals showed a higher stimulatory effect. These results suggest that electrospun scaffolds with the anisotropically and heterogeneously aligned patterns, which could efficiently control the mechanical properties and bioactivities of the scaffolds, might have great potential in vascular tissue engineering application.

Background: The poor response to chemotherapy and the brief response to vemurafenib in metastatic melanoma patients, make the identification of new therapeutic approaches an urgent need. Interestingly the increased expression and activity of the Aurora kinase B during melanoma progression suggests it as a promising therapeutic target., Methods: The efficacy of the Aurora B kinase inhibitor barasertib-HQPA was evaluated in BRAF mutated cells, sensitive and made resistant to vemurafenib after chronic exposure to the drug, and in BRAF wild type cells. The drug effectiveness has been evaluated as cell growth inhibition, cell cycle progression and cell migration. In addition, cellular effectors of drug resistance and response were investigated., Results: The characterization of the effectors responsible for the resistance to vemurafenib evidenced the increased expression of MITF or the activation of Erk1/2 and p-38 kinases in the newly established cell lines with a phenotype resistant to vemurafenib. The sensitivity of cells to barasertib-HQPA was irrespective of BRAF mutational status. Barasertib-HQPA induced the mitotic catastrophe, ultimately causing apoptosis and necrosis of cells, inhibited cell migration and strongly affected the glycolytic metabolism of cells inducing the release of lactate. In association i) with vemurafenib the gain in effectiveness was found only in BRAF(V600K) cells while ii) with nab-paclitaxel, the combination was more effective than each drug alone in all cells., Conclusions: These findings suggest barasertib as a new therapeutic agent and as enhancer of chemotherapy in metastatic melanoma treatment.

Croton membranaceus aqueous root extract (CMARE) is among the widely used phytotherapeutics in Ghana for the management of benign prostatic hyperplasia (BPH) and prostate cancer. However, the mechanism of action of CMARE remains to be elucidated. This study aimed to establish whether apoptosis is involved in the antiproliferative effect of CMARE on human BPH-1 cells. We determined the effect of treatment with 0, 1, 3, and 5 mg/mL CMARE for 24, 48, and 72 h on the viability and morphology of BPH-1 cells using the MMT assay and phase-contrast microscopy, respectively. We examined the apoptosis-inducing effects of CMARE after 48 h at the cellular level using Hoescht 33258 and JC-1 dye staining and flow cytometry analysis. We performed reverse transcription polymerase chain reaction and Western blotting to confirm the apoptotic effects of CMARE at the molecular level. CMARE induced a significant dose-dependent inhibition in the proliferation of BPH-1 cells (P < 0.05) and an alteration in their morphology and a reduction their density. Furthermore, CMARE induced dose-dependent staining of the nuclear chromatin, significant DNA fragmentation with G₀/G₁ sub-diploid cells (P < 0.01), and loss of the mitochondrial membrane potential in the treated cells compared to the controls after 48 h (P < 0.01). Additionally, while CMARE induced a significant upregulation of the mRNA and protein levels of Bax, those of Bcl2 did not change significantly. Therefore, induction of mitochondria-dependent apoptosis of BPH-1 cells may be a possible mechanism of action of CMARE.

Photodynamic therapy (PDT) involves the administration and activation of photosensitizing reagents in cancer tissues to induce cytotoxicity. Here we examined the effects of 5-5- (4-N, N-diacetoxylphenyl)-10,15,20- tetraphenylporphyrin (DTPP) -mediated PDT on cell morphology, viability, cell cycle, and cytotoxicity in a murine lung adenocarcinoma cell line. LA795 murine lung adenocarcinoma cell line was used in the study, with cellular uptake of DTPP being quantified by a UV-visible spectrophotometer. The subcellular localization of DTPP was detected by confocal laser scanning microscopy, alteration of cell morphology after PDT was observed by an inverted light microscope, and late-stage apoptosis was examined by terminal dUTP nick end labeling (TUNEL) . The effects of influencing factors on cytotoxicity of PDT in LA795 cells was investigated with varying concentrations of DTPP, energy densities, power densities, and antioxidants by 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Effects of PDT on cell cycle and plasma membrane integrity were studied by flow cytometry analysis. The uptake of DTPP by LA795 cells reached maximum after incubation for 24 h. Confocal laser scanning microscopy showed that DTPP was mainly in the mitochondrion, and slight localization was detected in the lysosomes. Cellular inhibitory effects increased with increased irradiation dose and DTPP concentration, while unactivated DTPP had low toxicity. Flow cytometry analysis revealed that DTPP-PDT-treated cells showed S phase arrest. Cell membrane damage initiation, repair, and irreversible damage were observed at 2, 4, and 5 h after DTPP-PDT , respectively. Together, our results demonstrated cell apoptosis, compromised viability, and cell cycle S phase arrest of LA795 in response to DTPP-PDT , while no effect on the lung cancer cells was observed with irradiation or photosensitizer treatment alone.

Type 1 diabetes is chronic disease with numerous complications and currently no cure. Tissue engineering strategies have shown promise in providing a therapeutic solution, but maintenance of islet function and survival within these therapies represents a formidable challenge. The islet microenvironment may hold the key for proper islet maintenance. To elucidate the microenvironmental conditions necessary for improved islet function and survival, three-dimensional (3D) polyacrylamide cell scaffolds were fabricated with stiffnesses of 0.1 and 10 kPa to regulate the spatial and mechanical control of biosignals. Specifically, we show a significant increase in insulin mRNA expression of 3D primary mouse islet-derived and Min6-derived β-cell clusters grown on compliant 0.1 kPa scaffolds. Moreover, these compliant 0.1 kPa scaffolds also increase glucose sensitivity in Min6-derived β-cell clusters as demonstrated by the increased glucose stimulation index. Our data suggest that stiffness-specific insulin processing is regulated through the myosin light chain kinase (MLCK) and Rho-associated protein kinase (ROCK) mechanosensing pathways. Additionally, β-catenin is required for regulation of stiffness-dependent insulin expression. Through activation or inhibition of β-catenin signaling, reversible control of insulin expression is achieved on the compliant 0.1 kPa and overly stiff 10 kPa substrates. Understanding the role of the microenvironment on islet function can enhance the therapeutic approaches necessary to treat diabetes for improving insulin sensitivity and response.

We sought to use splice-switching antisense oligonucleotides to produce a model of accelerated ageing by enhancing expression of progerin, translated from a mis-spliced lamin A gene (LMNA) transcript in human myogenic cells. The progerin transcript (LMNA Δ150) lacks the last 150 bases of exon 11, and is translated into a truncated protein associated with the severe premature ageing disease, Hutchinson-Gilford progeria syndrome (HGPS). HGPS arises from de novo mutations that activate a cryptic splice site in exon 11 of LMNA and result in progerin accumulation in tissues of mesodermal origin. Progerin has also been proposed to play a role in the 'natural' ageing process in tissues. We sought to test this hypothesis by producing a model of accelerated muscle ageing in human myogenic cells. A panel of splice-switching antisense oligonucleotides were designed to anneal across exon 11 of the LMNA pre-mRNA, and these compounds were transfected into primary human myogenic cells. RT-PCR showed that the majority of oligonucleotides were able to modify LMNA transcript processing. Oligonucleotides that annealed within the 150 base region of exon 11 that is missing in the progerin transcript, as well as those that targeted the normal exon 11 donor site induced the LMNA Δ150 transcript, but most oligonucleotides also generated variable levels of LMNA transcript missing the entire exon 11. Upon evaluation of different oligomer chemistries, the morpholino phosphorodiamidate oligonucleotides were found to be more efficient than the equivalent sequences prepared as oligonucleotides with 2'-O-methyl modified bases on a phosphorothioate backbone. The morpholino oligonucleotides induced nuclear localised progerin, demonstrated by immunostaining, and morphological nuclear changes typical of HGPS cells. We show that it is possible to induce progerin expression in myogenic cells using splice-switching oligonucleotides to redirect splicing of LMNA. This may offer a model to investigate the role of progerin in premature muscle ageing.