Apoptotic Fragmentation Research Articles

Nutraceutical phycocyanin nanoformulation for efficient drug delivery of paclitaxel in human glioblastoma U87MG cell line

To enhance the therapeutic efficacy of chemotherapy on glioblastoma U87MG cell line, paclitaxel-loaded phycocyanin nanoparticles (PTX-PcNPs) were prepared by modified desolvation process. PTX-PcNPs were characterised in terms of size, zeta potential, drug loading efficiency and drug release. Confocal laser scanning microscopy showed PTX-PcNPs could be internalised by U87MG cells. The anti-cancer activity was investigated in vitro by 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with and without photodynamic therapy. It was observed that formulation could significantly inhibit growth of U87MG cells as compared to PTX alone and also induced apoptosis, which was evidenced by presence of apoptotic bodies and nuclear fragmentation in treated cells. The present study suggests that PTX-PcNPs can act as a promising drug delivery system for cancer treatment.

In Vitro Evaluation of Photodynamic Therapy using Different Photosensitizers on Leishmania Tropica Promastigotes

Photodynamic therapy (PDT) is an alternative treatment method which uses a photosensitizer activated in the presence of light and molecular oxygen to produce reactive oxygen species that eventually target cell death by apoptosis or necrosis. The purpose of this study was to determine the efficiency of chloroaluminum phthalocyanine (AlClPc), toludine blue (TB) and phenorforbide a (PPa) mediated PDT in exerting morphological and apoptotic changes in Leishmania tropica. Parasites were incubated with increasing concentrations of TB, AlClPc and PPa for one hour and then were illuminated with a white light source for 30 min. After incubation at 26°C for 18 hours, cell viability (analyzed using a hemocytometer and XTT colorimetric assay), morphological changes (determined by giemsa staining) and apoptotic markers (analyzed by DAPI staining and DNA fragmentation assay) of Leishmania tropica parasites were evaluated as a function of TB, AlClPc and PPa concentration. Parasite viability was significantly different in between the groups treated with TB and PPa, with or without irradiation. There were no statistically significant differences in cell viability of AlClPc treated Leishmania tropica promastigotes with and without irradiation. Also, DAPI staning results indicated that apoptotic bodies and nucleus fragmentation started to be visible in AlClPc and PPa mediated PDT groups. Furthermore, DNA ladder pattern which is used to define apoptosis, was observed in irradiated AlClPc and PPa groups, but not observed in irradiated TB, non-irradiated AlClPc, TB and PPa. Although TB and AlClPc mediated PDT has been used for other Leishmania species but not on Leishmania tropica, this study is first to demonstrate the efficiency of PPa mediated PDT in a Leishmania spp. However, it is crucial to verify the photoactivity of these compounds in axenic or intracellular amastigotes and further in animal models.

Scutellarein antagonizes the tumorigenesis by modulating cytokine VEGF mediated neoangiogenesis and DFF-40 actuated nucleosomal degradation

Neoplastic cells often reside in distinctive tumor hypoxia armed with a series of adaptive responses including oxidative stress, defective apoptotic machinery and neoangiogenesis, through that further confer cell survival improvement. Plants still acts as reservoir of natural chemicals to provide newer active pharmacophores. Scutellarein is flavones which has wide range of pharmacophoral effects. In our current research, scutellarein employed for targeting oxidative stress mediated tumor angiogenesis and apoptotic nuclear fragmentation. Experimental results revealed that scutellarein has antiproliferative index against multiple cancer cell lines and diminished the oxidative stress and tumor development of murine ascitic lymphoma & inflammatory hepatocellular carcinoma. Eventual consequences lead to reduced neovessel formation by abrogating angiogeneic factors cytokine-VEGF-A, Flt-1, HIF-1α, MMP-2 and MMP-9 and reversing of evading apoptosis by activating caspase-3 activated DNA fragmentation factor (DFF-40) mediated nucleosomal degradation. In summary, our experimental evidences suggest that scutellarein has strong potentiality to attenuate the tumor development by modulating sprouting neovasculature and DFF-40 mediated apoptosis.

Phagocytosis: Hungry, Hungry Cells.

Phagocytosis is the cellular internalization and sequestration of particulate matter into a `phagosome, which then matures into a phagolysosome. The phagolysosome then offers a specialized acidic and hydrolytic milieu that ultimately degrades the engulfed particle. In multicellular organisms, phagocytosis and phagosome maturation play two key physiological roles. First, phagocytic cells have an important function in tissue remodeling and homeostasis by eliminating apoptotic bodies, senescent cells and cell fragments. Second, phagocytosis is a critical weapon of the immune system, whereby cells like macrophages and neutrophils hunt and engulf a variety of pathogens and foreign particles. Not surprisingly, pathogens have evolved mechanisms to either block or alter phagocytosis and phagosome maturation, ultimately usurping the cellular machinery for their own survival. Here, we review past and recent discoveries that highlight how phagocytes recognize target particles, key signals that emanate after phagocyte-particle engagement, and how these signals help modulate actin-dependent remodeling of the plasma membrane that culminates in the release of the phagosome. We then explore processes related to early and late stages of phagosome maturation, which requires fusion with endosomes and lysosomes. We end this review by acknowledging that little is known about phagosome fission and even less is known about how phagosomes are resolved after particle digestion.

Deoxyrhapontigenin, a Natural Stilbene Derivative Isolated From Rheum undulatum L. Induces Endoplasmic Reticulum Stress-Mediated Apoptosis in Human Breast Cancer Cells.

Although current chemotherapeutic agents are active at the beginning of therapy, the most common risk is the development of resistance during later stages in almost all cancer types including breast cancer. Hence, investigation of novel drugs is still a priority goal for cancer treatment. The objective of the present study is to investigate the anticancer effect of a derivative of stilbene, deoxyrhapontigenin (DR) isolated from Rheum undulatum L. root extracts against the chemoresistant MCF-7/adr and its parental MCF-7 human breast cancer cells. The morphological images indicate that DR induces an extensive cytoplasmic vacuolation in breast cancer cells. Mechanistic investigations revealed that DR treatment causes endoplasmic reticulum (ER) dilation and upregulated the expression of ER stress markers GRP78, IRE1α, eIF2α, CHOP, JNK, and p38. Subsequently, we also identified that DR increases the levels of apoptotic fragment of PARP (89 kDa) in breast cancer cells. Blocking the expression of one of the components of the ER stress–mediated apoptosis pathway, CHOP using siRNA significantly decreased DR-induced apoptotic cleavage of PARP. In summary, the present study suggests that the induction of ER stress–mediated apoptosis by DR may account for its cytotoxic effects in human breast cancer cells.

Hemolin triggers cell survival on fibroblasts in response to serum deprivation by inhibition of apoptosis.

Fibroblasts are the main cellular component of connective tissues and play important roles in health and disease through the production of collagen, fibronectin and growth factors. Under certain conditions, such as wound healing, fibroblasts intensify their metabolic demand, while the restriction of nutrients affect matrix composition, cell metabolism and behavior. In lepidopterans, wound healing is regulated by ecdysteroid hormones, which upregulate multifunctional proteins such as hemolin. However, the role of hemolin in cell proliferation and wound healing is not clear. rLosac is a recombinant hemolin from the caterpillar Lonomia obliqua whose proliferative and cytoprotective effects on endothelial cells have been described. Here, we show that rLosac induces a marked cell survival effect on fibroblast submitted to serum deprivation, which is observable as early as 24h, as demonstrated through the MTT assay, as well as an increase in migration of human dermal fibroblasts (HDF). No effects on cell proliferation or cell cycle distribution of fibroblasts in normal conditions were observed, suggesting that rLosac induces an effect in stressful conditions such serum deprivation but not when nutrient are sufficient. By flow cytometry, rLosac caused an apparent dose-dependent increase in cells in the S phase of the cell cycle and a significant reduction of cells with fragmented DNA. Furthermore, treatment with rLosac results in a significant decrease in the production of reactive oxygen species and in the loss of mitochondrial membrane potential, indicating that a reduction in oxidative stress is involved in rLosac-mediated cytoprotection. Our results also show an up-regulation of Bcl-2 and a down-regulation of Bax protein levels, inhibition of cytochrome c release and a reduction in caspase-3 levels, all considered critical factors for apoptosis. Moreover, rLosac treatment reduces the morphological changes induced by prolonged serum deprivation including the emergence of apoptotic bodies, nucleus fragmentation, cytoplasmic vacuolization and loss of extracellular matrix organization. The wound scratch test assay revealed that rLosac could enhance wound healing in vitro. Altogether, these findings suggest that rLosac strongly induces cellular protection in conditions of stress by serum deprivation preventing damage and loss of mitochondrial function by inhibiting apoptosis. This finding opens a new perspective to further understand the role of hemolin proteins during cellular processes such as wound healing and development.

Ammonium ions improve the survival of glutamine-starved hybridoma cells.

BackgroundAs a consequence of a reprogrammed metabolism, cancer cells are dependent on the amino acid l-glutamine for their survival, a phenomenon that currently forms the basis for the generation of new, cancer-specific therapies. In this paper, we report on the role which ammonium ions, a product of glutaminolysis, play on the survival of l-glutamine-deprived Sp2/0-Ag14 mouse hybridoma cells.ResultsThe supplementation of l-glutamine-starved Sp2/0-Ag14 cell cultures with either ammonium acetate or ammonium chloride resulted in a significant increase in viability. This effect did not depend on the ability of cells to synthesize l-glutamine, and was not affected by the co-supplementation with α-ketoglutarate. When we examined the effect of ammonium acetate and ammonium chloride on the induction of apoptosis by glutamine deprivation, we found that ammonium salts did not prevent caspase-3 activation or cytochrome c leakage, indicating that they did not act by modulating core apoptotic processes. However, both ammonium acetate and ammonium chloride caused a significant reduction in the number of l-glutamine-starved cells exhibiting apoptotic nuclear fragmentation and/or condensation.ConclusionAll together, our results show that ammonium ions promote the survival of l-glutamine-deprived Sp2/0-Ag14 cells and modulate late-apoptotic events. These findings highlight the complexity of the modulation of cell survival by l-glutamine, and suggest that targeting survival-signaling pathways modulated by ammonium ions should be examined as a potential anti-cancer strategy.

Biomarkers of Neutrophil‐Mediated Liver Injury in Patients with Alcoholic Liver Disease

Alcoholic liver disease (ALD) is a multi‐stage liver disorder associated with chronic ethanol abuse. The most serious syndrome associated with ALD is alcoholic hepatitis (AH), a type of acute‐on‐chronic liver failure that commonly occurs on a backdrop of alcoholic cirrhosis (AC). Neutrophil (PMN) accumulation in the liver is noted during AH, and is hypothesized to be a major mediator of the injury. This study was initiated to better define mechanisms of PMN mediated liver injury in human patients, using both mechanistic plasma biomarkers, and liver biopsies from AC and AH patients. We hypothesized PMN mediated ROS production exacerbated liver injury in patients with AH. Healthy volunteers, patients with clinically diagnosed AH, or abstinent AC patients were admitted to the IRB approved study, and whole blood was acquired for analysis of PMN activity and an array of mechanistic plasma biomarkers previously shown to provide insight into clinical liver injury, including cytokeratin‐18 levels, caspase‐3 activity, glutamate dehydrogenase (GDH) activity, and cytokine levels. In addition, hepatic cell death and PMN activation were measured in liver biopsies from patients with AH or AC via immunohistochemistry. Flow cytometry was used to ascertain the activation status of circulating PMNs. CD11b, a verified marker of neutrophil activation, was significantly elevated in neutrophils from AH patients, and was elevated in AC patients, but not significantly so. This was consistent with a substantial elevation in plasma IL‐8 levels, a known neutrophil chemoattractant, in the AH population. Baseline values of ROS production and phagocytic capacity were elevated in AC and AH patients, indicating low‐level basal stimulation of neutrophils. Surprisingly though, total hepatic neutrophils levels were similar in liver biopsies of AC and AH patients. Moreover, both groups stained positive for hypochlorous acid modified proteins, a gold standard histological marker for neutrophil activation, suggesting active neutrophils in both populations. Interestingly, plasma M30 levels, an apoptotic fragment of cytokeratin‐18 released during epithelial cell death, and plasma M65 levels, a necrotic fragment of cytokeratin‐18, were substantially elevated above control values, despite the fact that traditional markers of liver injury such as plasma ALT and plasma GDH were not elevated above controls, nor was there any detectable plasma caspase‐3 activity. Of note, AH patients had significantly higher plasma M65 values than AC patients, indicating increased total cell death. There was a small increase in TUNEL positive cells in AH patients (2–4 fold versus control), but this was not consistent with the dramatic elevation in M30/M65 levels (20–100 fold versus control). M30 staining of biopsies indicated only minor levels of M30 arose from the liver. In conclusion, neutrophil mediated hepatocyte cell death is likely a limited player in acute AH, although this does not preclude the possibility that neutrophil mediated ROS production contributes to the fibrotic or cirrhotic phenotype in these patients, as neutrophils are constitutively activated in the populations.Support or Funding InformationThis work was supported by a clinical pilot grant from Kansas University Medical Center. Additional support came from the “Training Program in Environmental Toxicology” T32 ES007079‐26A2 (to B.L.W.) from the National Institute of Environmental Health Sciences

Primula auriculata Extracts Exert Cytotoxic and Apoptotic Effects against HT-29 Human Colon Adenocarcinoma Cells.

Primula auriculata (Tootia) is one of the most important local medicinal plants in Hamedan district, Iran. To investigate cytotoxicity and apoptosis induction of crude methanolic extract and different fraction of it, we compared several methods on HT-29 human colon Adenocarcinoma cells. Cancer cell proliferation was measured by 3-(4, 5‑dimethylthiazolyl)2, 5‑diphenyl‑tetrazolium bromide (MTT) assay and apoptosis induction was analyzed by fluorescence microscopy (acridin orange/ethidium bromide, annexin V/propidium iodide staining, TUNEL assay and Caspase-3 activity assay). Crude methanolic extract (CM) inhibited the growth of malignant cells in a dose-dependent manner. Among solvent fractions, the dichloromethane fraction (CF) was found to be the most toxic compared to other fractions. With double staining methods, high percentage of 40 µg/mL of (CM) and (CF) treated cells exhibited typical characteristics of apoptotic cells. Apoptosis induction was also revealed by apoptotic fragmentation of nuclear DNA and activation of caspas-3 in treated cells. These findings indicate that crude methanolic extract and dichloromethan fraction of P.auriculata induced apoptosis and inhibited proliferation in colon cancer cells and could be used as a source for new lead structures in drug design to combat colon cancer.

Cytokeratin (CK)-18 and CK18 Fragments As Sensitive and Specific Biomarkers of Acute Hepato-Intestinal Gvhd: Results of a Prospective Observational Study

Introduction:Preliminary data have suggested that CK18, a highly abundant protein in liver and gut epithelial cells, and its apoptotic degradation product CK18 fragments (CK18F) could indicate apoptotic end organ damage induced by acute hepato-intestinal GVHD and, thus, may be used as an acute GVHD biomarker (Luft et al, Blood 2011; 118:1685). Here, we present the first results of a clinical study aiming at prospective validation of the capacity of CK18/ CK18F to predict acute hepato-intestinal GvHD activity and its response to treatment.Patients and methods:Main endpoints of the study were (1) prediction of imminent acute hepato-intestinal GvHD by comparing CK18/CK18F serum levels measured 7 to 14 days prior to symptom onset to with baseline levels (pre-conditioning, at transplant and one week after alloSCT) of the same subject; and (2) prediction of response to GVHD treatment by measuring CK18/CK18F kinetics 3, 7 and 14 days after start of immunosuppressive therapy for hepato-intestinal GvHD. Eligible were all adult patients undergoing allogeneic stem cell transplantation (alloSCT) at our institution from December 2008 onwards. Target sample size was 100. Blood samples were collected in the study population prior to the initiation of the conditioning regimen and from the day of transplantation on a weekly basis for one year. The M30-Apoptosense ELISA kit was used for measuring serum levels of CK18 fragments, CK18 was analyzed with the M65 (EpiDeath) ELISA Kit (both provided by PEVIVA, Nacka, Sweden). Serum levels measured at the pre-specified landmarks were compared by Wilcoxon's signed rank test. The study was registered at ClinicalTrials.gov (Identifier: NCT00935324).Results:Between December 2008 to February 2011, 109 patients were enrolled. The median age was 53 years (range 19 - 69), 72 were male. The underlying disease was acute myeloid leukemia in 39 patients, myeloproliferative / myelodysplastic disorders in 18 patients, acute lymphoblastic leukemia in 10 patients, and lymphoproliferative malignancies in 42 patients. Donors were matched related (36), matched unrelated (52), or mismatched unrelated (21). Stem cell source was peripheral blood in 100 patients and bone marrow in 9 patients. Reduced intensity conditioning protocols (n = 89) prevailed over myeloablative (n = 7) and aplasia conditioning protocols (n = 13). Immunosuppression was based on a calcineurin inhibitor (CNI) backbone with mycophenolate or short-course methotrexate, plus ATG in mismatched or unrelated transplants. Patients received pravastatin (20 mg daily dose) for routine prophylaxis of CNI-induced vascular complications as per institutional policy as of 2010.Acute hepato-intestinal GVHD grade II-IV developed in 38 patients (34,9 %) at a median of 53 days post transplant and was steroid-refractory in 12 patients. In patients with biopsy-proven acute hepato-intestinal GVHD, CK18 significantly raised 7 to 14 days before clinical GVHD onset and can therefore serve as an early predictor of impending acute hepato-intestinal GVHD (median at baseline vs preGVHD, 149 ng/ml vs 527 ng/ml; p=0.012), and peaked at symptom onset (median 1281 ng/ml; p=0.003, compared to baseline). CK18F kinetics paralleled that of CK18 but was less pronounced. In contrast, patients with isolated acute skin GVHD, patients without acute GVHD (cut-off day+53), and patients with GVHD-typical symptoms but a negative biopsy did not show any increase CK18 or CK18F in serum (Figure 1). However, CK18/CK18F levels were not correlated with acute GVHD severity. Neither marker predicted refractory GVHD or non-relapse mortality after acute GVHD at any of the time points measured.Conclusions:This prospective study validates CK18 and CK18F as specific biomarkers suitable for prediction and diagnosis of suspected imminent and clinically manifest acute hepato-intestinal GVHD. In contrast, neither CK18 nor CK18F can be used to predict the prognosis of an ongoing GVHD episode. Interventional trials employing CK18 as trigger for pre-emptive GVHD therapy seem to be warranted. [Display omitted] DisclosuresHegenbart:Janssen: Honoraria, Other: travel support. Ho:Genzyme/Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Luft:Immundiagnostik AG: Research Funding.

Porcine deltacoronavirus induces apoptosis in swine testicular and LLC porcine kidney cell lines in vitro but not in infected intestinal enterocytes in vivo

We compared the mechanisms of porcine delatacoronavirus (PDCoV) induced death of infected enterocytes in vivo and infected LLC porcine kidney (LLC-PK) and swine testicular (ST) cells in vitro. We conducted histologic analysis and immunofluorescence (IF) staining for the detection of PDCoV antigens, and TUNEL assay in singly or serially cut tissue sections from the small and large intestines of four, 11- to 14-day-old gnotobiotic pigs, inoculated orally with 8.8–11.0log10 genomic equivalents (GE) of US PDCoV strains OH-FD22 or OH-FD100 (n=3), or mock (n=1). Similar comparative assays were done on LLC-PK and ST cells inoculated with the cell-adapted PDCoV strain OH-FD22-P44 (passage 44) in cell culture medium with 2.5–10μg/ml of trypsin and 1% pancreatin, respectively. At post-inoculation days 3–4, infected pigs showed severe watery diarrhea and/or vomiting and mainly, diffuse, severe atrophic enteritis, with mild to moderate cytoplasmic vacuolation of the enteroctyes lining the atrophied villous epithelium. By IF, PDCoV antigens were evident in villous or crypt epithelial cells. No PDCoV antigen-positive, small and large intestinal villous or crypt epithelial cells, of which cytoplasm was also either vacuolated or morphologically normal, showed positive TUNEL staining. In contrast, by double IF and TUNEL staining, most of the TUNEL-positive signals (apoptotic nuclear fragmentation) were found in PDCoV antigen-positive LLC-PK and ST cells that also showed cytopathic effects, such as cell rounding, detachment and clumping in clusters. Secondary annexin V/propidium iodide (PI) staining revealed increased numbers of annexin V- or PI-positive LLC-PK and ST cells at 21h after inoculation, compared to the negative controls. Thus, PDCoV does not induce apoptosis in the infected intestinal enterocytes in vivo, but in two infected cell lines of swine origin, LLC-PK and ST cells.

Cytokeratin 18-Aspartate396 apoptotic fragment for fibrosis detection in patients with non-alcoholic fatty liver disease and chronic viral hepatitis

BackgroundThe combination of non-invasive markers for the detection of fibrosis in patients with chronic liver diseases is still a matter of debate. AimsTo test the performance of cytokeratin18-Aspartate396 alone or in combination with transient elastography as a marker of fibrosis, compared to liver biopsy as gold standard. MethodsIn 259 prospectively enrolled patients with chronic liver diseases, clinical, biochemical, and histological features were assessed. Serum cytokeratin18-Aspartate396 and Fibroscan were performed within 6 months prior to liver biopsy. ResultsCytokeratin18-Aspartate396 levels predicted both significant and advanced fibrosis in non-alcoholic fatty liver disease group, correctly identifying 83.7% and 80.8% of cases, respectively. Liver stiffness performed best in predicting severe fibrosis in patients with chronic viral infection, correctly identifying 78.7% of chronic hepatitis B and 88.6% of chronic hepatitis C subjects. The combination of cytokeratin18-Aspartate396 and liver stiffness improved their diagnostic performance for the detection of significant and advanced fibrosis in non-alcoholic fatty liver disease group, only (sensitivity=78.3%, specificity=90.7%; sensitivity=91.7%, specificity=71.6%, respectively). ConclusionCytokeratin18-Aspartate396 and liver stiffness can improve the non-invasive prediction of significant and advanced fibrosis in patients with non-alcoholic fatty liver disease, while in hepatitis B and C virus infected patients their combined use had no advantage over the diagnostic accuracy of transient elastography alone.

Biological and Health-promoting Activity of Vinification Byproducts Produced in Spanish Vineyards

Several by-products are produced in the Spanish agricultural system. Among them, fresh and vinified grape skins represent an abundant source of phenols with a potential nutraceutical value. Fresh grape skin extracts (FGSE) and vinification of grape skin extracts (VGSE) obtained by a microwave-assisted method have been chemically and biologically characterised. Their role in the maintenance of genetic stability was stated by in vivo genotoxic and antigenotoxic evaluations (Drosophilla melanogaster wing spot test), as well as by their potential chemopreventive effect (in an HL60 in vitro model). Total phenolic, anthocyanin and resveratrol contents were chemically characterised in the two extracts, showing some qualitative differences. Both extracts and resveratrol were not mutagenic in the Drosophila somatic mutation and recombination tests, and exerted antigenotoxic activities against hydrogen peroxide. They also showed cytotoxic activity to HL60 leukaemia cells, with an IC 50 of 4.5µL/mL, 4.6µL/mL and 98µM respectively and induced apoptotic internucleosomic fragmentation in the HL60 cell line.

Ultrastructural Changes of the Smooth Muscle in Esophageal Atresia

Esophageal atresia (EA) with or without tracheo-esophageal fistula (TEF) is a relatively rare congenital anomaly. Despite the advances in the management techniques and neonatal intensive care, esophageal dysmotility remains a very common problem following EA/TEF repair. Our current study aimed to describe the most significant ultrastructural changes of the smooth muscle cells (SMCs) trying to highlight some of the underlying mechanisms of esophageal dysmotility following EA/TEF repair. Twenty-three biopsies were obtained from the tip of the lower esophageal pouch (LEP) of 23 patients during primary repair of EA/TEF. Light microscopic examination was performed with hematoxylin and eosin (HE), and Van Gieson’s stains. Ultrastructural examination was done using transmission electron microscopy (TEM). Histopathological examination showed distortion of smooth muscle layer and deposition of an abundant amount of fibrous tissue in-between smooth muscles. Using TEM, SMCs exhibited loss of the cell-to-cell adhesion, mitochondrial vacuolation, formation of myelin figures, and apoptotic fragmentation. There were also plasmalemmal projections and formation of ghost bodies. Interestingly, SMCs were found extending pseudopodia-like projections around adjacent collagen fibers. Engulfed collagen fibers by SMCs underwent degradation within autophagic vacuoles. Degeneration of SMCs and deposition of abundant extracellular collagen fibers are prominent pathological changes in LEP of EA/TEF. These changes might contribute to the pathogenesis of esophageal dysmotility in patients who have survived EA/TEF.

Abstract 5232: Changes in plasma circulating DNA composition during focal ablative therapy for liver cancer

Abstract Circulating cell-free DNA (cfDNA) may potentially serve as a diagnostic or therapeutic biomarker. We tested our established methodologies for cfDNA profiling on blood samples obtained during focal ablative treatments for liver cancer, given that such interventions may cause abrupt spillage of circulating tumor DNA (ctDNA) into the bloodstream. Following written informed consent, serial blood samples from 13 patients with hepatocellular carcinoma (HCC) were drawn into cfDNA-optimized collection tubes before treatment, 2-3 hours post-treatment, and a few weeks after. Nine patients underwent radiofrequency ablation and 4 patients received transarterial chemoembolization. The cfDNA concentration was assessed by quantitative PCR of Alu-amplicons (Alu-101 and Alu-226) on 13 pre-treatment samples, 12 immediate post-treatment samples and 8 follow-up samples. Aberrant cfDNA methylation at genes GSTP1 and RASSF1 was assessed by pyrosequencing to investigate tumor-specific cfDNA. Mean cfDNA levels of patients with HCC increased by an average of 3.6-fold (up to 9.3-fold) from baseline immediately after treatment (p = 0.0019). Levels measured 1-15 weeks after treatment returned to on average 87% of the baseline. Each specimen contained both high molecular weight DNA (consistent with abrupt release of DNA from disrupted cells) and smaller apoptotic cfDNA fragments that ranged in size of 160-180 bp. DNA integrity (Alu-226/Alu-101) was similar before treatment, immediately after, and in the follow-up samples. Compared to the paired leukocyte DNA methylation levels, higher methylation levels at RASSF1 and/or GSTP1 were observed in all 13 pre-treatment cfDNA samples, supporting at least partial tumor origin of the isolated cfDNA. RASSF1 methylation moderately correlated with GSTP1 methylation (r = 0.55, p<0.001). Excluding one patient with exceptionally high RASSF1 and GSTP1 methylation levels, the averaged amount of RASSF1/GSTP1-methylated ctDNA correlated with the tumor size prior to the procedure (r = 0.72, p = 0.008), while no association was found between the AFP levels and tumor burden. The characteristics of cfDNA in cancer patients may change significantly following therapeutic interventions. We have established an improved workflow for cfDNA quantification and characterization in plasma that can be applied to the peri-treatment setting and are in the process of evaluating ctDNA analysis protocols for EDTA blood as well. These preliminary observations in a limited number of specimens support the potential of quantifying ctDNA as a marker of tumor cell destruction. Preliminary observation of a correlation between aberrantly-methylated cfDNA levels and HCC tumor size suggests that pre-treatment cfDNA profiling may inform on tumor burden. Further research will be needed to validate the clinical significance of the quantitative and epigenetic changes in cfDNA samples from patients with HCC. Citation Format: Maarit I. Tiirikainen, Karolina Peplowska, Min-Ae Song, Kyle Miyazaki, Linda L. Wong, Sandi A. Kwee. Changes in plasma circulating DNA composition during focal ablative therapy for liver cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5232. doi:10.1158/1538-7445.AM2015-5232

Otago Glaucoma Surgery Outcome Study: Further Histology and Immunohistochemistry of Molteno Implant Blebs.

To describe findings of light microscopic examination of Molteno implant bleb capsules. Histological and immunohistochemical features of bleb capsules including distribution of apoptotic cells and cell fragments were examined in 11 eyes 0.2 to 30.4 years after Molteno implants. In the superficial layer of capsules, high proportions of cells showed cytological features of apoptosis, with a higher proportion of cells showing specific immunohistochemical features of apoptosis with a mean of 22% (range, 3%-40%) of cells staining positively for Fas ligand. This demonstrated that cells migrating into the superficial capsule were replaced over approximately 1 to 6 days. In the deeper layer a mean of 8% (range, 3%-38%) of apoptosing cells stained positively for Fas ligand. These lower proportions of positively staining cells and cell fragments in the deeper layers, and the presence of occasional positively staining cells on the inner surface of capsules, demonstrated continuous migration of cells into the deeper layers associated with breakdown of connective tissue matrix and the release of numerous membrane-bound vesicles. These findings demonstrated unexpectedly rapid turnover of cells in the superficial layer of the bleb capsule, where most cells were efficiently phagocytosed by nearby monocytic cells, macrophages, and histiocytes; the remaining cells migrating into the deeper layers completed apoptosis and disintegrated with release of collagenolytic enzymes and Fas ligand positive presumed "death messengers" that were carried toward the superficial layers by the aqueous.

Evidence for a subventricular zone neural stem cell phagocytic activity stimulated by the vitamin K-dependent factor protein S.

Neural stem cells, whose major reservoir in the adult mammalian brain is the subventricular zone (SVZ), ensure neuropoiesis, a process during which many generated cells die. Removal of dead cells and debris by phagocytes is necessary for tissue homeostasis. Using confocal and electron microscopy, we demonstrate that cultured SVZ cells phagocytose both 1 and 2 µm latex beads and apoptotic cell-derived fragments. We determine by flow cytometry that phagocytic cells represent more than 10% of SVZ cultured cells. Phenotyping of SVZ cells using nestin, GFAP, Sox2, or LeX/SSEA and quantification of aldehyde dehydrogenase (ALDH) activity, reveals that cells with neural stem-cell features phagocytose and represent more than 30% of SVZ phagocytic cells. In vivo, nestin-, Sox2-, and ALDH-expressing neural stem-like cells engulfed latex beads or apoptotic cell-derived fragments that were injected into mice lateral brain ventricles. We show also that SVZ cell phagocytic activity is an active process, which depends both on cytoskeleton dynamic and on recognition of phosphatidylserine eat-me signal, and is stimulated by the vitamin K-dependent factor protein S (ProS). ProS neutralizing antibodies inhibit SVZ cell phagocytic activity and exposure of SVZ cells to apoptotic cell-derived fragments induces a transient Mer tyrosine kinase receptor (MerTK) phosphorylation. Conversely, MerTK blocking antibodies impair both basal and ProS-stimulated SVZ cell phagocytic activity. By revealing that neural stem-like cells act within the SVZ neurogenic niche as phagocytes and that the ProS/MerTK path represents an endogenous regulatory mechanism for SVZ cell phagocytic activity, the present report opens-up new perspectives for both stem cell biology and brain physiopathology.

Porcine parvovirus infection induces apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated pathway

Porcine parvovirus (PPV) infection has been reported to induce the cytopathic effects (CPE) in some special host cells and contribute the occurrence of porcine parvovirus disease, but the molecular mechanisms underlying PPV-induced CPE are not clear. In this study, we investigated the morphological and molecular changes of porcine kidney cell line (PK-15 cells) infected with PPV. The results showed that PPV infection inhibited the viability of PK-15 cells in a time and concentration dependent manner. PPV infection induced typical apoptotic features including chromatin condensation, apoptotic body formation, nuclear fragmentation, and Annexin V-binding activity. Further studies showed that Bax was increased and translocated to mitochondria, whereas Bcl-2 was decreased in PPV-infected cells, which caused mitochondrial outer-membrane permeabilization, resulting in the release of mitochondrial cytochrome c, followed by caspase-9 and caspase-3 activation. However, the expression of Fas and Fas ligand (FasL) did not appear significant changes in the process of PPV-induced apoptosis. Moreover, PPV infection activated p53 signaling, which was involved in the activation of apoptotic signaling induced by PPV infection via regulation of Bax and Bcl-2. Taken together, our results demonstrated that PPV infection induced apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated apoptosis pathway. This study may contribute to shed light on the molecular pathogenesis of PPV infection.

Catalase inhibition in diabetic rats potentiates DNA damage and apoptotic cell death setting the stage for cardiomyopathy.

Diabetes is a risk factor for cardiovascular disease that has a multifactorial etiology, with oxidative stress as an important component. Our previous observation of a significant diabetes-related increase in rat cardiac catalase (CAT) activity suggested that CAT could play a major role in delaying the development of diabetic cardiomyopathy. Thus, in the present work, we examined the effects of the daily administration of the CAT inhibitor, 3-amino-1,2,4-triazole (1 mg/g), on the hearts of streptozotocin (STZ)-induced diabetic rats. Administration of CAT inhibitor was started from the 15th day after the last STZ treatment (40 mg/kg/5 days), and maintained until the end of the 4th or 6th weeks of diabetes. Compared to untreated diabetic rats, at the end of the observation period, CAT inhibition lowered the induced level of cardiac CAT activity to the basal level and decreased CAT protein expression, mediated through a decline in the nuclear factor erythroid-derived 2-like 2 /nuclear factor-kappa B p65 (Nrf2/NF-κB p65) subunit ratio. The perturbed antioxidant defenses resulting from CAT inhibition promoted increased H₂O₂production (P < 0.05) and lipid peroxidation (P < 0.05). Generated cytotoxic stimuli increased DNA damage (P < 0.05) and activated pro-apoptotic events, observed as a decrease (P < 0.05) in the ratio of the apoptosis regulator proteins Bcl-2/Bax, increased (P < 0.05) presence of the poly(ADP-ribose) polymerase-1 (PARP-1) 85 kDa apoptotic fragment and cytoplasmic levels of cytochrome C. These findings confirm an important function of CAT in the suppression of events leading to diabetes-promoted cardiac dysfunction and cardiomyopathy.

Abstract 2595: Deubiquitinase USP9X inhibition induces apoptosis in metastatic breast cancer cells

Abstract Highly metastatic Triple Negative (ER/PR and HER2 negative) breast cancer (TNBC) comprises 20% of all breast cancers but accounts for a disproportionate share of mortality due to its aggressive nature and limited therapeutic options. Thus novel metastatic breast cancer targets and inhibitors need to be defined. Usp9x is a deubiquitinase that has been shown to regulate the stability and function of many signaling, cell cycle regulatory and survival proteins and has emerged as a negative prognosticator and potential therapeutic target in several cancers. Usp9x functions in polarization and survival of tumor cells derived from various sites of origin. Additional studies have shown that Usp9x expression is required to maintain growth of glioblastoma and medulloblastomas and it suppresses the apoptotic effects of targeted-, chemo- and radio-therapy in a broad spectrum of tumors. Previous studies also demonstrated that Usp9x and Mcl-1 protein levels are highly elevated in ductal breast carcinoma when compared to normal tissue. Thus, we hypothesized that Usp9x may play a role in tumor and metastasis promoting functions in breast cancer. We first assessed Usp9x activity and total protein expression in a panel of benign (HME, MCF10A), tumorigenic (MCF7 and T47D) and highly metastatic (SUM149 and MDAMB231) human breast cell lines and found up-regulation of Usp9x enzymatic activity and total protein in tumorigenic and metastatic breast cancers as compared to benign breast cells. Increased Usp9x expression also correlated with increased levels of Mcl-1. Knockdown (KD) of Usp9x in four TNBC cell lines [MDAMB231, MDAMB436, BT549 and SUM159] triggered phenotypic apoptotic events like membrane blebbing and nuclear fragmentation, which were further confirmed by the presence of activated caspases, cleaved PARP and reduced Mcl-1 protein expression. Importantly Usp9x KD specifically induced apoptosis in metastatic breast cancer cells with a mesenchymal stem cell-like phenotype (EpCAM+/CD24-/CD44+ population of cells) and fully suppressed TNBC colony growth and acinar structures when grown on Matrigel. Usp9x KD in tumorigenic MCF7 cells resulted in growth inhibition with G2 arrest, without evidence of apoptosis, whereas Usp9x KD had no effect on the growth or survival of benign breast cells. To examine the therapeutic potential of Usp9x inhibitors in breast cancer we employed EOAI3402143 (G9), a small molecule Usp9x inhibitor developed in our laboratory with efficacy in myeloma tumor regression in mice. G9 inhibited Usp9x activity, reduced Mcl-1 levels and induced apoptosis in TNBC cells within 4h of inhibitor treatment. G9 also phenocopied the effect of Usp9x KD in 3D culture with nM activity. Collectively, our data shows that Usp9x plays a role in breast tumor growth and survival and its inhibition in tumorigenic and highly metastatic breast cancers may provide a novel therapy for the treatment of metastatic breast cancer. Citation Format: Anupama Pal, Ming Luo, Max Wicha, Moshe Talpaz, Nicholas Donato. Deubiquitinase USP9X inhibition induces apoptosis in metastatic breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2595. doi:10.1158/1538-7445.AM2014-2595