Changes In Nuclear Morphology Research Articles

Odorous Compounds from Poultry Manure Induce DNA Damage, Nuclear Changes, and Decrease Cell Membrane Integrity in Chicken Liver Hepatocellular Carcinoma Cells.

Animal breeding and management of organic wastes pose a serious problem to the health of livestock and workers, as well as the nearby residents. The aim of the present study was to determine the mechanisms of toxicity of selected common odorous compounds from poultry manure, including ammonia, dimethylamine (DMA), trimethylamine (TMA), butyric acid, phenol, and indole. We measured their genotoxic and cytotoxic activity in the model chicken cell line (LMH), in vitro, by comet assay and lactate dehydrogenase assay, respectively. We also made microscopic observations of any morphological changes in these cells by DAPI staining. Four compounds, namely ammonia, DMA, TMA, and butyric acid increased DNA damage in a dose-dependent manner (p < 0.05), reaching genotoxicity as high as 73.2 ± 1.9%. Phenol and indole induced extensive DNA damage independent of the concentration used. Ammonia, DMA, and TMA caused a dose-dependent release of lactate dehydrogenase (p < 0.05). The IC50 values were 0.02%, 0.05%, and 0.1% for DMA, ammonia and TMA, respectively. These compounds also induced nuclear morphological changes, such as chromatin condensation, shrinkage, nuclear fragmentation (apoptotic bodies), and chromatin lysis. Our study exhibited the damaging effects of odorous compounds in chick LMH cell line.

Anticancer effects of an extract from the scallop Patinopecten yessoensis on MCF-7 human breast carcinoma cells.

Patinopecten yessoensis, is a species of scallop and a marine bivalve mollusk. In traditional East Asian medicine, scallop meat is used as a drug for the treatment of diabetes, pollakisuria, and indigestion. The present study was conducted in order to examine the potential anticancer effects of scallop flesh extract (SE) on MCF-7 human breast cancer cells. An MTT assay was used to evaluate cell viability and flow cytometry was used for the assessment of cell cycle distribution and apoptosis. The alteration in protein expression level was determined by western blot analysis, and the amounts of docosahexaenoic acid and eicosapentaenoic acid in the SE were measured by gas chromatography. SE inhibited the growth of MCF-7 human breast cancer cells in a dose-dependent manner by inducing G0/G1 phase arrest. The cell cycle arrest was associated with the upregulation of p53 and p21, and downregulation of G1 phase-associated cyclin D1/cyclin-dependent kinase (Cdk) 4 and cyclin E1/Cdk 2. In addition, SE-mediated cell cycle arrest was associated with the promotion of apoptosis, as indicated by the expression of apoptosis-associated proteins and changes in nuclear morphology. SE appeared to induce the mitochondrial apoptotic cascade, as indicated by a decreased expression of Bcl-2, activation of Bcl-2 associated X protein, release of cytochrome c, decrease in procaspase-3, and an increase in cleaved-poly (ADP-ribose) polymerase (PARP). Furthermore, the expression levels of Fas-associated via death domain and cleaved caspase-8 were increased in a SE dose-dependent manner. Taken together, these results suggest that the intrinsic and extrinsic pathways of apoptosis are associated with the anticancer effects of SE on MCF-7 cells. Thus, SE may be a suitable candidate for the treatment and prevention of human breast cancer.

Whole Exome Sequencing Identifies Truncating Variants in Nuclear Envelope Genes in Patients With Cardiovascular Disease.

The genetic variation underlying many heritable forms of cardiovascular disease is incompletely understood, even in patients with strong family history or early age at onset. We used whole exome sequencing to detect pathogenic variants in 55 patients with suspected monogenic forms of cardiovascular disease. Diagnostic analysis of established disease genes identified pathogenic variants in 21.8% of cases and variants of uncertain significance in 34.5% of cases. Three patients harbored heterozygous nonsense or splice-site variants in the nucleoporin genes NUP37, NUP43, and NUP188, which have not been implicated previously in cardiac disease. We also identified a heterozygous splice site variant in the nuclear envelope gene SYNE1 in a child with severe dilated cardiomyopathy that underwent transplant, as well as in his affected father. To confirm a cardiovascular role for these candidate genes in vivo, we used morpholinos to reduce SYNE1, NUP37, and NUP43 gene expression in zebrafish. Morphant embryos displayed cardiac abnormalities, including pericardial edema and heart failure. Furthermore, lymphoblasts from the patient carrying a SYNE1 splice-site variant displayed changes in nuclear morphology and protein localization that are consistent with disruption of the nuclear envelope. These data expand the repertoire of pathogenic variants associated with cardiovascular disease and validate the diagnostic and research use of whole exome sequencing. We identify NUP37, NUP43, and NUP188 as novel candidate genes for cardiovascular disease, and suggest that dysfunction of the nuclear envelope may be an under-recognized component of inherited cardiac disease in some cases.

Lamin A and microtubules collaborate to maintain nuclear morphology

ABSTRACT Lamin A (LA) is a critical structural component of the nuclear lamina. Mutations within the LA gene (LMNA) lead to several human disorders, most striking of which is Hutchinson-Gilford Progeria Syndrome (HGPS), a premature aging disorder. HGPS cells are best characterized by an abnormal nuclear morphology known as nuclear blebbing, which arises due to the accumulation of progerin, a dominant mutant form of LA. The microtubule (MT) network is known to mediate changes in nuclear morphology in the context of specific events such as mitosis, cell polarization, nucleus positioning and cellular migration. What is less understood is the role of the microtubule network in determining nuclear morphology during interphase. In this study, we elucidate the role of the cytoskeleton in regulation and misregulation of nuclear morphology through perturbations of both the lamina and the microtubule network. We found that LA knockout cells exhibit a crescent shape morphology associated with the microtubule-organizing center. Furthermore, this crescent shape ameliorates upon treatment with MT drugs, Nocodazole or Taxol. Expression of progerin, in LA knockout cells also rescues the crescent shape, although the response to Nocodazole or Taxol treatment is altered in comparison to cells expressing LA. Together these results describe a collaborative effort between LA and the MT network to maintain nuclear morphology.

Francisella novicida inhibits spontaneous apoptosis and extends human neutrophil lifespan.

Francisella novicida is a Gram-negative bacterium that is closely related to the highly virulent facultative intracellular pathogen, Francisella tularensis Data published by us and others demonstrate that F. tularensis virulence correlates directly with its ability to impair constitutive apoptosis and extend human neutrophil lifespan. In contrast, F. novicida is attenuated in humans, and the mechanisms that account for this are incompletely defined. Our published data demonstrate that F. novicida binds natural IgG that is present in normal human serum, which in turn, elicits NADPH oxidase activation that does not occur in response to F. tularensis As it is established that phagocytosis and oxidant production markedly accelerate neutrophil death, we predicted that F. novicida may influence the neutrophil lifespan in an opsonin-dependent manner. To test this hypothesis, we quantified bacterial uptake, phosphatidylserine (PS) externalization, and changes in nuclear morphology, as well as the kinetics of procaspase-3, -8, and -9 processing and activation. To our surprise, we discovered that F. novicida not only failed to accelerate neutrophil death but also diminished and delayed apoptosis in a dose-dependent, but opsonin-independent, manner. In keeping with this, studies of conditioned media (CM) showed that neutrophil longevity could be uncoupled from phagocytosis and that F. novicida stimulated neutrophil secretion of CXCL8. Taken together, the results of this study reveal shared and unique aspects of the mechanisms used by Francisella species to manipulate neutrophil lifespan and as such, advance understanding of cell death regulation during infection.

Detection and Quantification of Nuclear Morphology Changes in Apoptotic Cells by Fluorescence Microscopy and Subsequent Analysis of Visualized Fluorescent Signals.

Apoptosis results in specific and stage-dependent morphological alterations of the cell nucleus, including pyknosis and cell shrinking. The experimental investigation of apoptotic processes is still challenging and routinely based on the assessment of molecular events like chromatin fragmentation and caspase enzyme activity. Alternatively, the establishment of a fluorescence microscopy nuclear morphology assay would provide a simple and robust low-cost method for detection and quantification of apoptotic cascades. Model cell lines LNCaP and MDA-MB-231 were incubated in the presence of the apoptosis-inducer cycloheximide (CHX). After evaluation of apoptotic cascades by terminal deoxynucleotidyl transferase-dUTP nick-end labeling (TUNEL) assay, stained cell nuclei were analyzed regarding area, perimeter, major and minor axis, as well as brightness of nuclear fluorescence signal. When compared to vehicle-treated control cells, administration of CHX led to significantly reduced cell growth and elevated rates of chromatin fragmentation of both cell lines as shown by cell counting and TUNEL assay, respectively. These apoptotic effects were accompanied by apoptosis-specific modulations of the nuclei demonstrated by diminished nuclear morphology parameters, such as area, perimeter, major and minor axis, as well as elevated levels of nuclear staining intensity. We present a computerized method for apoptosis detection and quantification using images of fluorescent dye-stained cell nuclei. The advantages of this nuclear morphology assay include the (i) ability to routinely assess apoptosis by a fast, highly reproducible low-cost technique, (ii) applicability of an experimental approach analyzing high numbers of single nuclei and (iii) detection of apoptosis in early, as well as late, stages of the apoptotic cascade.

Novel purification of 1’S-1’-Acetoxychavicol acetate from Alpinia galanga and its cytotoxic plus antiproliferative activity in colorectal adenocarcinoma cell line SW480

Alpinia galanga (L.) Willd. is a valuable medicinal crop found in specific tropical regions of southeast Asia. Its crude extracts are well known for their wide medicinal properties and many compounds identified from these extracts are of great interest currently. 1’S-1’-Acetoxychavicol acetate (ACA) obtained from rhizomes of A.galanga is one such well-illustrated compound. This study strives to progress and simplifies the purification protocol for ACA from A.galanga rhizomes. It also studies the cytotoxicity and antiproliferative activity of ACA against Dukes’ type B, colorectal adenocarcinoma (SW480). HPLC standardisation was carried out for purification of ACA from rhizomes of Alpinia galanga. MTT assay was executed to estimate the IC50 value of ACA against SW480 cell line. This value was used to study the apoptosis, nuclear morphological changes and mitochondrial membrane permeability using Acridine orange/ethidium bromide, DAPI, and JC-1 staining. The DNA fragmentation assay was used to substantiate the nuclear fragmentation of DNA observed in the DAPI staining. Further, cell cycle analysis was performed using flow cytometry to study the exact stage of the cell cycle where SW480 cells are arrested due to ACA, western blot analysis of relevant genes were done to further understand at molecular level. A comprehensive 1.89g of 1’S-1’-Acetoxychavicol acetate (ACA) was recovered from 500g of A.galanga rhizomes. ACA significantly suppressed the proliferation of SW480 cells at an IC50 of 80μM (48h). The mode of SW480 cell death due to ACA was initially identified as apoptosis and cell cycle halted at G0/G1 checkpoint with considerable DNA damage and mitochondrial depolarization. The expression of p21 was increased and concomitantly Cyclin D was downregulated in ACA treated in comparison to control. This study suggests that 1’S-1’-Acetoxychavicol acetate has potent anti-colorectal adenocarcinoma activity.

A novel real-time imaging technique to quantify mechanisms of cell death in neutrophils

Abstract Neutrophils have distinct mechanisms of cell death, including the formation of neutrophil extracellular traps (NETs- a process called NETosis). NETosis is characterized by reactive oxygen species generation, chromatin decondensation, membrane rupture and extrusion of a meshwork of chromatin bound to granule peptides. Objective, efficient techniques to quantify NETosis are required. We developed a new method to automatically quantify the percentage of neutrophils undergoing NETosis using the IncuCyte ZOOM™ (Essen BioScience, Inc.) platform to image neutrophils at 20× in a 96-well plate and a dual-dye system dependent on membrane integrity to stain DNA, collecting both phase and fluorescence images. DNA in healthy control neutrophils were stained with a cell membrane permeable NUCLEAR-ID Red DNA dye. Neutrophils were then incubated with Sytox, a membrane-impermeable DNA dye, used to assess cell death. Conditions were run in triplicate and 3 images per well were captured at 10-minute intervals. A processing definition was set and optimized to count all neutrophils (NUCLEAR-ID stained) and neutrophils undergoing cell death (Sytox stained) using fluorescence intensity and stained area size to identify cells undergoing NETosis. Changes in nuclear morphology, quantified by the processing definition, distinguished PMA, Ionomycin and SLE sera induced NETosis from staurosporine induced apoptosis. Findings were confirmed and correlated with the gold-standard of immunofluorescence microscopy. This novel real time assay distinguishes types of neutrophil cell death and quantifies NETosis in a rapid, accurate and reproducible way, significantly improving the study of neutrophils.

Interaction of silver and gold nanoparticles in mammalian cancer: as real topical bullet for wound healing— A comparative study

The present study evaluates in vitro cytotoxic effects and the mode of interaction of biologically synthesized Ag and Au nanoparticles (NPs) using Brassica oleracea L. var. capitata f. rubra (BOL) against HT-1080 cancer cells and bacterial cells as well as their wound healing efficacy using a mouse model. UV-visible spectroscopy, scanning electron microscopy, high-resolution transmission electron microscopy, and energy-dispersive X-ray analysis have ascertained the formation of nano-sized Ag and Au particles. Fourier transform infrared analysis has confirmed that polyphenol and amide groups in BOL act as capping as well as reducing agents. The free radical scavenging activity under in vitro conditions is found to be higher for the Ag NPs when compared to the Au NPs. Acridine orange-ethidium bromide dual staining and comet assay have indicated that the cytotoxic effects are mediated through nuclear morphological changes and DNA damage. The intracellular localization of Ag and Au NPs in HT-1080 cells and their subsequent effect on apoptosis and necrosis were analyzed by flow cytometry while the mode of interaction was established by scanning electron microscopy under field emission mode and by bio-transmission electron microscopy. These methods of analysis clearly revealed that the Ag and Au NPs have easily entered and accumulated into the cytosol and nucleus, resulting in activation of inflammatory and apoptosis pathways, which in turn cause damage in DNA. Further, mRNA and protein expression of caspase-3 and caspase-7, TNF-α, and NF-κB have provided sufficient clues for induction of intrinsic and extrinsic apoptosis and inflammatory pathways in Ag NP- and Au NP-treated cells. Evaluation of wound healing properties of Ag and Au NPs using a mouse model indicates rapid healing of wounds. In addition, no clear toxic effects and no nuclear abnormalities in peripheral blood cells are observed. Ag NPs appear to be a better anticancer therapeutic agent than Au NPs. Nonetheless, both Ag NPs and Au NPs show potential for promoting topical wound healing without any toxic effects. Graphical abstract Schematic representation of biological synthesis of Ag and Au NPs and its application on cancer and wound healing.

Mdivi‑1 attenuates sodium azide‑induced apoptosis in H9c2 cardiac muscle cells.

The aim of the current study was to investigate the effect of mitochondrial division inhibitor 1 (Mdivi-1) in sodium azide-induced cell death in H9c2 cardiac muscle cells. Mdivi-1 is a key inhibitor of the mitochondrial division protein dynamin-related protein 1 (Drp1). Mdivi-1 was added to H9c2 cells for 3 h, after which, the cells were treated with sodium azide for 24 h. Cell viability was measured by Cell Counting kit-8 assay. DAPI staining was used to observe nuclear morphology changes by microscopy. To further investigate the role of mitochondria in sodium azide-induced cell death, mitochondrial membrane potential (ΔΨm) and the cellular ATP content were determined by JC-1 staining and ATP-dependent bioluminescence assay, respectively. Reactive oxygen species (ROS) production was also assessed by use of the specific probe 2′,7′-dichlorodihydrofluorescein diacetate. In addition, the expression of Drp1 and of the apoptosis-related proteins BCL2 apoptosis regulator (Bcl-2), and BCL2 associated X (Bax) was determined by western blotting. The present findings demonstrated that pretreatment with Mdivi-1 attenuated sodium azide-induced H9c2 cell death. Mdivi-1 pretreatment also inhibited the sodium azide-induced downregulation of Bcl-2 expression and upregulation of Bax and Drp1 expression. In addition, the mitochondrion was revealed to be the target organelle of sodium azide-induced toxicity in H9c2 cells. Mdivi-1 pretreatment moderated the dissipation of ΔΨm, preserved the cellular ATP contents and suppressed the production of ROS. The results suggested that the mechanism of sodium azide-induced cell death in H9c2 cells may involve the mitochondria-dependent apoptotic pathway. The present results indicated that Mdivi-1 may have a cardioprotective effect against sodium azide-induced apoptosis in H9c2 cardiac muscle cells.

Aloe-emodin Induces Apoptosis in Human Liver HL-7702 Cells through Fas Death Pathway and the Mitochondrial Pathway by Generating Reactive Oxygen Species.

Aloe-emodin (1,8-dihydroxy-3-hydroxymethyl-anthraquinone) is one of the primary active compounds in total rhubarb anthraquinones isolated from some traditional medicinal plants such as Rheum palmatum L. and Cassia occidentalis, which induce hepatotoxicity in rats. Thus, the aim of this study was to determine the potential cytotoxic effects and the underlying mechanism of aloe-emodin on human normal liver HL-7702 cells. The CCK-8 assays demonstrated that aloe-emodin decreased the viability of HL-7702 cells in a dose-dependent and time-dependent manner. Aloe-emodin induced S and G2/M phase cell cycle arrest in HL-7702 cells. This apoptosis was further investigated by flow cytometry and nuclear morphological changes by DAPI staining, respectively. Moreover, aloe-emodin provoked the production of intracellular reactive oxygen species and the depolarization of mitochondrial membrane potential (MMP). Further studies by western blot indicated that aloe-emodin dose-dependently up-regulated the levels of Fas, p53, p21, Bax/Bcl-2 ratio, and cleaved caspase-3, -8, -9, and subsequent cleavage of poly(ADP-ribose)polymerase (PARP). Taken together, these results suggest that aloe-emodin inhibits cell proliferation of HL-7702 cells and induces cell cycle arrest and caspase-dependent apoptosis via both Fas death pathway and the mitochondrial pathway by generating reactive oxygen species, indicating that aloe-emodin should be taken into account in the risk assessment for human exposure. Copyright © 2017 John Wiley & Sons, Ltd.

Artemisinin protects PC12 cells against β-amyloid-induced apoptosis through activation of the ERK1/2 signaling pathway

Accumulating evidence displays that an abnormal deposition of amyloid beta-peptide (Aβ) is the primary cause of the pathogenesis of Alzheimer's disease (AD). And therefore the elimination of Aβ is regarded as an important strategy for AD treatment. The discovery of drug candidates using culture neuronal cells against Aβ peptide toxicity is believed to be an effective approach to develop drug for the treatment of AD patients. We have previously showed that artemisinin, a FDA-approved anti-malaria drug, has neuroprotective effects recently. In the present study, we aimed to investigate the effects and potential mechanism of artemisinin in protecting neuronal PC12 cells from toxicity of β amyloid peptide. Our studies revealed that artemisinin, in clinical relevant concentration, protected and rescued PC12 cells from Aβ25–35-induced cell death. Further study showed that artemisinin significantly ameliorated cell death due to Aβ25–35 insult by restoring abnormal changes in nuclear morphology, lactate dehydrogenase, intracellular ROS, mitochondrial membrane potential and activity of apoptotic caspase. Western blotting analysis demonstrated that artemisinin activated extracellular regulated kinase ERK1/2 but not Akt survival signaling. Consistent with the role of ERK1/2, preincubation of cells with ERK1/2 pathway inhibitor PD98059 blocked the effect of artemisinin while PI3K inhibitor LY294002 has no effect. Moreover, Aβ1-42 also caused cells death of PC12 cells while artemisinin suppressed Aβ1-42 cytotoxicity in PC12 cells. Taken together, these results, at the first time, suggest that artemisinin is a potential protectant against β amyloid insult through activation of the ERK1/2 pathway. Our finding provides a potential application of artemisinin in prevention and treatment of AD.

Saturated Free Fatty Acids Induce Trophoblast Lipoapoptosis

Obesity during pregnancy increases the risk for maternal complications like gestational diabetes, preeclampsia, and maternal inflammation. Maternal obesity also increases the risk of childhood obesity, fetalintrauterine growth restriction (IUGR) and diabetes to the children. Earlier studies demonstrated placental trophoblast apoptosis occurs in patients with preeclampsia and IUGR due to hypoxia. Increased circulating free fatty acids (FFAs) in obesity due to adipose lipolysis induces lipoapoptosis tohepatocytes, cholangiocytes, and pancreatic‐β‐cells. During the third trimester of pregnancy, there is an increase in maternal lipolysis and release of FFAs into the circulation. It is currently unknown if increased FFAs during maternal obesity cause placental cell lipoapoptosis. Palmitate, the predominant circulating FFA, has been shown to induce blastocyst lipoapoptosis and exposure of blastocysts before implantation to palmitate results in a smaller sized fetus. Here, we hypothesis the involvement of trophoblastlipoapoptosis in placental dysfunction during maternal obesity.MethodsApoptosis was assessed by characteristic nuclear morphology staining with DAPI, and caspase 3/7 activity assay. Cleaved PARP and cleaved caspase 3 were examined by Immunoblot.ResultsTreatment of trophoblastcell lines, JEG‐3 and JAR cells with palmitate (PA) or stearate (SA)‐induced trophoblastlipoapoptosis as evidenced by a significant increase in apoptotic nuclearmorphology changes and caspase 3/7 activity. We observed that saturated FFAs caused a concentration‐dependent increase in placental cell lipoapoptosis. We also observed that palmitoleate, a mono‐unsaturated omega‐7 fatty acid mitigated placental cell lipoapoptosis caused due to PA exposure.In conclusion, we show that saturated FFAs induce trophoblast lipoapoptosis. The signaling mechanism of FFA‐induced trophoblast lipoapoptosis are now under investigation.Support or Funding InformationNIH:P20GM104320

DT-13, a saponin monomer 13 of the Dwarf lilyturf tuber, synergized with vinorelbine to induce mitotic arrest via activation of ERK signaling pathway in NCI-H1299 cells.

Vinorelbine (NVB) is a semi-synthetic vinca alkaloid that is approved for the clinical therapy of lung cancer. However, the clinical application of NVB was limited because of the acquisition of resistance and inacceptable toxicity. Therefore, it is of great interest to develop low-cytotoxic drugs that can synergize with NVB. DT-13, a saponin monomer 13 of the Dwarf lilyturf tuber, showed inhibitory effects on tumor metastasis and angiogenesis in the previous studies. Here, we found that DT-13 combined with NVB exhibited synergistic effect to inhibit the cell proliferation in human lung cancer NCI-H1299 cells rather than human embryonic lung fibroblasts WI-38. The combination of DT-13 and NVB significantly inhibited the colony formation, induced cellular and nuclear morphological changes, and triggered cell cycle arrest at mitotic phase. Furthermore, MAPK signaling pathway was activated by the combination treatment, and the activation of ERK was required for the induction of mitotic arrest. Taken together, DT-13 combined with NVB exhibited synergistic anticancer effect in NCI-H1299 cells, and DT-13 may be a candidate agent for adjuvant chemotherapy of NVB in lung cancer.

DNA analysis of cattle parasitic protozoan Tritrichomonas foetus after photodynamic therapy

Photodynamic therapy (PDT) is a modality of therapy that involves the activation of photosensitive substances and the generation of cytotoxic oxygen species and free radicals to promote the selective destruction of target tissues. This study analyzed the application of PDT to Tritrichomonas foetus, a scourged and etiological agent of bovine trichomoniasis, a sexually transmitted infectious disease. As it is an amitochondrial and aerotolerant protozoan, it produces energy under low O2 tension via hydrogenosome. T. foetus from an axenic culture was incubated with photosensitizer tetrasulfonated aluminium phthalocyanine and then irradiated with a laser source (InGaAIP) at a density of 4.5Jcm−2. The DNA integrity of the control and treated group parasites was analyzed by conventional gel electrophoresis and comet assay techniques. In previous results, morphological changes characterized by apoptotic cell death were observed after T. foetus was submitted to PDT treatment. In the treated groups, T. foetus DNA showed a higher concentration of small fragments, about 200pb, in gel electrophoresis after PDT. In the comet assay, the DNA tail percentage was significantly higher in the treated groups. These results demonstrate that PDT leads to DNA fragmentation with changes in nuclear morphology and apoptotic features.

Toxicological evaluation of the topoisomerase inhibitor, etoposide, in the model animal Caenorhabditis elegans and 3T3-L1 normal murine cells.

Etoposide, a topoisomerase II inhibitor, has been widely used as a clinical anticancer drug to treat diverse cancer patients. Since not only rapidly dividing cancer cells but also the cells of normal human tissues and every living organism in environmental ecosystems have topoisomerases, it is crucial to study the toxicity of etoposide in other organisms in addition to cancer cells. In this study, we evaluated the toxicity of etoposide in both a soil nematode, Caenorhabditis elegans, and 3T3-L1 normal murine cells. Etoposide significantly retarded the growth, egg laying, and hatching in C. elegans. Etoposide also affected the reproductive gonad tissue, decreased the number of germ cells and induced abnormally enlarged nuclei in C. elegans. In addition, etoposide inhibited 3T3-L1 cell proliferation, with IC50 values of 37.8 ± 7.3 and 9.8 ± 1.8 μM after 24 and 48 hours of treatment, respectively, via the induction of cell cycle arrest at the G2/M phase and apoptotic cell death. Etoposide also induced nuclear enlargement in 3T3-L1 normal murine cells. The reproductive toxicity and abnormal nuclear morphological changes seemed to correlate with the adverse effects of etoposide. We suggest that these experimental platforms, i.e., the toxicological evaluation of both nematodes and 3T3-L1 cells, may be useful to study the mechanisms underlying the side effects of chemicals, including topoisomerase inhibitors.

Matrine Suppresses the ER-positive MCF Cells by Regulating Energy Metabolism and Endoplasmic Reticulum Stress Signaling Pathway.

Matrine (C15 H24 N2 O), an alkaloid that is one of the main active components from Sophora flavescens. Matrine has been demonstrated to have therapeutic effects on various solid tumors, including breast cancer, but the mechanism still needs further study. Endoplasmic reticulum (ER)-positive Michigan Cancer Foundation cells were cultured, and matrine was added in various amounts to measure the dose-dependent and time-dependent cytotoxicity. Hoechst 33258 staining was used to observed nuclear morphological changes. Apoptosis was measured by AnnexinV/PI double staining assay kit. Intracellular adenosine triphosphate and glycometabolism were detected by assay kit. The protein levels GRP78, p-eIF2α, CHOP, cytochrome c, and HexokinaseII were analyzed. Mechanistic investigations revealed that matrine treatment causes ER dilation and up-regulated the expression of ER stress markers GRP78, eIF2α, and CHOP, increases the levels of apoptotic in Michigan Cancer Foundation cells, subsequently, blocking the ER stress-mediated apoptosis pathway, significantly decreased matrine-induced apoptotic but still has significant difference between control group. In addition, matrine not only promoted the occurrence of ER stress but also inhibited the expression of hexokinase II, down-regulated energy metabolism. In summary, the present study suggests that the induction of ER stress-mediated apoptosis by matrine and down-regulated energy metabolism may account for its cytotoxic effects in human breast cancer cells. Copyright © 2017 John Wiley & Sons, Ltd.

Sea Buckthorn Leaf Extract Inhibits Glioma Cell Growth by Reducing Reactive Oxygen Species and Promoting Apoptosis.

Hippophae rhamnoides L., also known as sea buckthorn (SBT), possesses a wide range of biological and pharmacological activities. However, the underlying mechanism is largely unknown. The present study examined whether SBT leaf extract could inhibit proliferation and promote apoptosis of rat glioma C6 cells. The results revealed that the treatment with SBT leaf extract inhibited proliferation of rat C6 glioma cells in a dose-dependent manner. SBT-induced reduction of C6 glioma cell proliferation and viability was accompanied by a decrease in production of reactive oxygen species (ROS), which are critical for the proliferation of tumor cells. SBT treatment not only significantly upregulated the expression of the pro-apoptotic protein Bcl-2-associated X (Bax) but also promoted its localization in the nucleus. Although increased expression and nuclear translocation of Bax were observed in SBT-treated C6 glioma cells, the induced nuclear morphological change was distinct from that of typical apoptotic cells in that most of SBT-treated cells were characterized by convoluted nuclei with cavitations and clumps of chromatin. All of these results suggest that SBT leaf extract could inhibit the rapid proliferation of rat C6 glioma cells, possibly by inducing the early events of apoptosis. Thus, SBT may serve as a potential therapeutic candidate for the treatment of glioma.

Centchroman induces redox-dependent apoptosis and cell-cycle arrest in human endometrial cancer cells.

Centchroman (CC) or Ormeloxifene has been shown to induce apoptosis and cell cycle arrest in various types of cancer cells. This has, however, not been addressed for endometrial cancer cells where its (CC) mechanism of action remains unclear. This study focuses on the basis of antineoplasticity of CC by blocking the targets involved in the cell cycle, survival and apoptosis in endometrial cancer cells. Ishikawa Human Endometrial Cancer Cells were cultured under estrogen deprived medium, exposed to CC and analyzed for proliferation and apoptosis. Additionally, we also analyzed oxidative stress induced by CC. Cell viability studies confirmed the IC50 of CC in Ishikawa cells to be 20µM after 48h treatment. CC arrests the cells in G0/G1 phase through cyclin D1 and cyclin E mediated pathways. Phosphatidylserine externalization, nuclear morphology changes, DNA fragmentation, PARP cleavage, and alteration of Bcl-2 family protein expression clearly suggest ongoing apoptosis in the CC treated cells. Activation of caspase 3 & 9, up-regulation of AIF and inhibition of apoptosis by z-VAD-fmk clearly explains the participation of the intrinsic pathway of programmed cell death. Further, the increase of ROS, loss of MMP, inhibition of antioxidant (MnSOD, Cu/Zn-SOD and GST) and inhibition of apoptosis with L-NAC suggests CC induced oxidative stress leading to apoptosis via mitochondria mediated pathway. Therefore, CC could be a potential therapeutic agent for the treatment of Endometrial Cancer adjunct to its utility as a contraceptive and an anti-breast cancer agent.

Essential oil from Siegesbeckia pubescens induces apoptosis through the mitochondrial pathway in human HepG2 cells

Siegesbeckia pubescens (SP) has been used as a traditional medicine for the treatment of and inflammatory diseases. However, the activities of SP against hepatocellular carcinoma and the related mechanisms remain unclear. The present study aimed to examine the effects of the essential oil of SP (SPEO) on the proliferation of hepatocellular carcinoma cells and the possible mechanisms. The growth inhibition of HepG2 cells was analyzed by MTT assay. Hoechst 33258 and fluorescence microscopy were utilized to observe the nuclear morphological changes of apoptotic cells. Flow cytometry was used to detect cell apoptosis and cell cycle. The expressions of the target proteins were detected by Western blotting. The results showed that SPEO obviously inhibited the proliferation of HepG2 cells in a dose-dependent manner. SPEO activated a series of apoptotic proteins in HepG2 cells, increasing expression levels of Bax, caspase-3 and caspase-9, and decreasing the bcl-2 expression level. SPEO displayed promising anti-hepatocellular carcinoma activities in vitro, partly by inducing apoptosis in HepG2 cells through activating the mitochondrial pathway.