Changes In Nuclear Morphology Research Articles

Cold Atmospheric Plasma Treatment Induces Anti-Proliferative Effects in Prostate Cancer Cells by Redox and Apoptotic Signaling Pathways.

One of the promising possibilities of the clinical application of cold plasma, so-called cold atmospheric plasma (CAP), is its application on malignant cells and cancer tissue using its anti-neoplastic effects, primarily through the delivery of reactive oxygen and nitrogen species (ROS, RNS). In this study, we investigated the impact of CAP on cellular proliferation and consecutive molecular response mechanisms in established prostate cancer (PC) cell lines. PC cells showed a significantly reduced cell growth following CAP treatment as a result of both an immediate increase of intracellular peroxide levels and through the induction of apoptosis indicated by annexin V assay, TUNEL assay, and the evaluation of changes in nuclear morphology. Notably, co-administration of N-acetylcysteine (NAC) completely neutralized CAP effects by NAC uptake and rapid conversion to glutathione (GSH). Vitamin C could not counteract the CAP induced effects on cell growth. In summary, relatively short treatments with CAP of 10 seconds were sufficient to induce a significant inhibition of cancer proliferation, as observed for the first time in urogenital cancer. Therefore, it is important to understand the mode of CAP related cell death and clarify and optimize CAP as cancer therapy. Increased levels of peroxides can alter redox-regulated signaling pathways and can lead to growth arrest and apoptosis. We assume that the general intracellular redox homeostasis, especially the levels of cellular GSH and peroxidases such as peroxiredoxins affect the outcome of the CAP treatment.

Induction of Mitochondrial Dependent Apoptosis in Human Leukemia K562 Cells by Meconopsis integrifolia: A Species from Traditional Tibetan Medicine.

Objectives: Meconopsis integrifolia (M. integrifolia) is one of the most popular members in Traditional Tibetan Medicine. This study aimed to investigate the anticancer effect of M. integrifolia and to detect the underlying mechanisms of these effects. Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and trypan blue assay were used to evaluate the cytotoxicity of M. integrifolia. Changes in cell nuclear morphology and reactive oxygen species (ROS) level were observed by fluorescent microscopy. Apoptosis ratio, DNA damage and mitochondrial membrane potential (MMP) loss were analyzed by flow cytometry. Western blotting assay was adopted to detect the proteins related to apoptosis. Immunofluorescence was used to observe the release of cytochrome C. Results: The obtained data revealed that M. integrifolia could significantly inhibit K562 cell viability, mainly by targeting apoptosis induction and cell cycle arrest in G2/M phase. Collapse in cell morphology, chromatin condensation, DNA damage and ROS accumulation were observed. Further mechanism detection revealed that mitochondrion might be a key factor in M. integrifolia-induced apoptosis. Conclusions: M. integrifolia could induce mitochondria mediated apoptosis and cell cycle arrest in G2/M phase with little damage to normal cells, suggesting that M. integrifolia might be a potential and efficient anticancer agent that deserves further investigation.

Features of cell death, mitochondrial activation and caspase dependence of rabbit anti-T-lymphocyte globulin signaling in lymphoblastic Jurkat cells are distinct from classical apoptosis signaling of CD95

Rabbit anti-T-lymphocyte-globulin (ATG) is used for immunosuppression in organ and stem cell transplantation. The aim of this study was to investigate ATG-induced cell death compared to CD95-signaling of apoptosis. We measured features of cell death at the cell membrane, mitochondria, nuclei and caspase-3 cleavage. We used the following inhibitors: the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (O-Me)-fluoromethyl ketone (zVAD-fmk), the serine protease inhibitors 3,4 dichloroisocoumarin (DCI) and N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK) and the reducing agent N-acetycysteine (NAC). ATG-induced cellular changes were rapid, included mitochondrial membrane permeability (MMP) induction and annexin V/propidium iodide (PI) positivity but little caspase-3 activation and nuclear morphology changes. MMP was not sensitive to caspase inhibition, serine protease inhibition with DCI moderately reduced MMP. These findings were in contrast to CD95-signaling. Interestingly, TLCK massively augmented CD95-induced MMP which could be abrogated by NAC. In conclusion, ATG-signaling differs in features and kinetics from CD95-induced apoptosis with caspase-independent mechanisms involved in MMP.

Resistance of lung cancer cells grown as multicellular tumour spheroids to zinc sulfophthalocyanine photosensitization.

Photodynamic therapy (PDT) is phototherapeutic modality used in the treatment of neoplastic and non-neoplastic diseases. The photochemical interaction of light, photosensitizer (PS) and molecular oxygen produces singlet oxygen which induces cell death. Zinc sulfophthalocyanine (ZnPcSmix) has been shown to be effective in A549 monolayers, multicellular tumor spheroids (MCTSs) (250 µm) and not on MCTSs with a size of 500 µm. A549 cells used in this study were grown as MCTSs to a size of 500 µm in order to determine their susceptibility to PDT. ZnPcSmix distribution in MCTSs and nuclear morphology was determined using a fluorescent microscope. Changes in cellular responses were evaluated using cell morphology, viability, proliferation, cytotoxicity, cell death analysis and mitochondrial membrane potential. Untreated MCTSs, showed no changes in cellular morphology, proliferation, cytotoxicity and nuclear morphology. Photoactivated ZnPcSmix also showed no changes in cellular morphology and nuclear morphology. However, photoactivated ZnPcSmix resulted in a significant dose dependant decrease in viability and proliferation as well as an increase in cell membrane damage in MCTSs over time. ZnPcSmix photosensitization induces apoptotic cell death in MCTSs with a size of 500 µm and more resistantance when compared to monolayer cells and MCTSs with a size of 250 µm.

Capillin, a major constituent of Artemisia capillaris Thunb. flower essential oil, induces apoptosis through the mitochondrial pathway in human leukemia HL-60 cells

BackgroundNatural products are one of the most important sources of drugs used in pharmaceutical therapeutics. Screening of several natural products in the search for novel anticancer agents against human leukemia HL-60 cells led us to identify potent apoptosis-inducing activity in the essential oil fraction from Artemisia capillaris Thunb. flower. MethodsThe cytotoxic effects of extracts were assessed on human leukemia HL-60 cells by XTT assay. Induction of apoptosis was assessed by analysis of DNA fragmentation and nuclear morphological change. The plant name was checked with the plant list website (http://www.theplantlist.org). ResultsA purified compound from the essential oil fraction from Artemisia capillaris Thunb. flower that potently inhibited cell growth in human leukemia HL-60 cells was identified as capillin. The cytotoxic effect of capillin in cells was associated with apoptosis. When HL-60 cells were treated with 10−6 M capillin for 6 h, characteristic features of apoptosis such as DNA fragmentation and nuclear fragmentation were observed. Moreover, activation of c-Jun N-terminal kinase (JNK) was detected after treatment with capillin preceding the appearance of characteristic properties of apoptosis. Release of cytochrome c from mitochondria was also observed in HL-60 cells that had been treated with capillin. ConclusionCapillin induces apoptosis in HL-60 cells via the mitochondrial apoptotic pathway, which might be controlled through JNK signaling. Our results indicate that capillin may be a potentially useful anticancer drug that could enhance therapeutic efficacy.

The role of Beclin 1 in SDT-induced apoptosis and autophagy in human leukemia cells

Purpose: To prove the occurrence of autophagy after treatment by protoporphyrin IX (PpIX)-mediated sonodynamic therapy (SDT) of human chronic myelogenous leukemia K562 cells as well as its relationship with apoptosis.Materials and methods: The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenylter-trazolium bromide tetrazolium (MTT) assay was adopted to examine cytotoxicity of different treatments. Nuclear morphology changes were observed under a fluorescence microscopy with 4ʹ-6-Diamidino-2-Phenylindole (DAPI) staining. Western blotting was used to analyze the expression of caspase-3, Beclin 1 (BECN 1) and the conversion of LC3- phosphatidylethanolamine conjugate/a cytosolic form of LC3 (LC3 II/I). Fluorescence microscope was used to identify the formation of autophagic vacuoles (AVO) during autophagy.Results: Under optimal conditions, SDT was shown to induce autophagy in K562 cells, which caused the up-regulation of Beclin-1 and the formation of AVO. In addition, pre-treatment of cancer cells with Beclin 1-targeted short hairpin RNA (Beclin 1 shRNA) was shown to reduce the level of LC3-II accumulation and staining with punctate spots of monodansylcadaverine (MDC) staining. Besides, the cytotoxic effect of SDT was significantly increased by Beclin 1 shRNA. Furthermore, studies showed a marked effect on the apoptosis of cells by Beclin 1 shRNA to sonodamage with increased DAPI staining and caspase-3 cleavage.Conclusions: These results demonstrated that SDT significantly induced autophagy of K562 cells, probably to protect the K562 cells from sonodamage.

Autophagy and gap junctional intercellular communication inhibition are involved in cadmium-induced apoptosis in rat liver cells

Cadmium (Cd) is known to induce hepatotoxicity, yet the underlying mechanism of how this occurs is not fully understood. In this study, Cd-induced apoptosis was demonstrated in rat liver cells (BRL 3A) with apoptotic nuclear morphological changes and a decrease in cell index (CI) in a time- and concentration-dependent manner. The role of gap junctional intercellular communication (GJIC) and autophagy in Cd-induced apoptosis was investigated. Cd significantly induced GJIC inhibition as well as downregulation of connexin 43 (Cx43). The prototypical gap junction blocker carbenoxolone disodium (CBX) exacerbated the Cd-induced decrease in CI. Cd treatment was also found to cause autophagy, with an increase in mRNA expression of autophagy-related genes Atg-5, Atg-7, Beclin-1, and microtubule-associated protein light chain 3 (LC3) conversion from cytosolic LC3-I to membrane-bound LC3-II. The autophagic inducer rapamycin (RAP) prevented the Cd-induced CI decrease, while the autophagic inhibitor chloroquine (CQ) caused a further reduction in CI. In addition, CBX promoted Cd-induced autophagy, as well as changes in expression of Atg-5, Atg-7, Beclin-1 and LC3. CQ was found to block the Cd-induced decrease in Cx43 and GJIC inhibition, whereas RAP had opposite effect. These results demonstrate that autophagy plays a protective role during Cd-induced apoptosis in BRL 3A cells during 6 h of experiment, while autophagy exacerbates Cd-induced GJIC inhibition which has a negative effect on cellular fate.

Pyrroloquinoline quinone against glutamate-induced neurotoxicity in cultured neural stem and progenitor cells

Pyrroloquinoline quinone (PQQ), as a well-known redox enzyme cofactor, has been proven to play important roles in the regulation of cellular growth and development in mammals. Numerous physiological and medicinal functions of PQQ have so far been reported although its effect on neural stem and progenitor cells (NS/PCs) and the potential mechanism were even rarely investigated. In this study, the neuroprotective effects of PQQ were observed by pretreatment of NS/PCs with PQQ before glutamate injury, and the possible mechanisms were examined. PQQ stimulated cell proliferation and markedly attenuated glutamate-induced cell damage in a dose-dependent manner. By observing the nuclear morphological changes and flow cytometric analysis, PQQ pretreatment showed its significant effect on protecting NS/PCs against glutamate-induced apoptosis/necrosis. PQQ neuroprotection was associated with the decrease of intracellular reactive oxygen species (ROS) production, the increase of glutathione (GSH) levels, and the decrease of caspase-3 activity. In addition, pretreatment with PQQ also significantly enhanced the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in the NS/PCs exposed to glutamate. These results suggest that PQQ can protect NS/PCs against glutamate toxicity associated with ROS-mediated mitochondrial pathway, indicating a useful chemical for the clinical application of NS/PCs.

Nuclear and Cell Morphological Changes during the Cell Cycle and Growth of the Toxic Dinoflagellate Alexandrium minutum

Elucidation of the cell cycle of dinoflagellates is essential to understand the processes leading to their massive proliferations, known as harmful algal blooms. In this study, we used imaging flow cytometry (IFC) to monitor the changes in DNA content and nuclear and cell morphology that occur during clonal growth of the toxic species Alexandrium minutum Halim. Our results indicate that the population was in S phase (C→2C DNA content) during the light period, whereas haploid cells with a C DNA content peaked only during a short interval of the dark period. The timing of the phases, identified based on the nuclear morphology and cytoplasmic-to-nuclear (CNR) ratio of the cells, suggests that the length of the G2/M phase is regulated by nutrient levels whereas the beginning of S phase is clock controlled. In addition we found that up to 7% of individual cells achieved a DNA content higher than 2C, indicative of either zygote formation and replication (homothallism), or of double-haploid cells able to divide (polyploid forms). Cells belonging to different cell cycle phases (G1-S-G2/M) could be readily discriminated based on nuclear size. Our study provides evidence of cell-cycle plasticity during clonal growth and unambiguously characterizes the cell-cycle phases of this dinoflagellate species.

β-elemene induces caspase-dependent apoptosis in human glioma cells in vitro through the upregulation of Bax and Fas/ FasL and downregulation of Bcl-2.

β-elemene, extracted from herb medicine Curcuma wenyujin has potent anti-tumor effects in various cancer cell lines. However, the activity of β-elemene against glioma cells remains unclear. In the present study, we assessed effects of β-elemene on human glioma cells and explored the underlying mechanism. Human glioma U87 cells were used. Cell proliferation was determined with MTT assay and colony formation assay to detect the effect of β-elemene at different doses and times. Fluorescence microscopy was used to observe cell apoptosis with Hoechst 33258 staining and change of glioma apoptosis and cell cycling were analyzed by flow cytometry. Real-time quantitative PCR and Western-blotting assay were performed to investigated the influence of β-elemene on expression levels of Fas/FasL, caspase-3, Bcl-2 and Bax. The experiment was divided into two groups: the blank control group and β-elemne treatment group. With increase in the concentration of β-elemene, cytotoxic effects were enhanced in the glioma cell line and the concentration of inhibited cell viability (IC50) was 48.5 μg/mL for 24h. β-elemene could induce cell cycle arrest in the G0/G1 phase. With Hoechst 33258 staining, apoptotic nuclear morphological changes were observed. Activation of caspase-3,-8 and -9 was increased and the pro-apoptotic factors Fas/FasL and Bax were upregulated, while the anti-apoptotic Bcl-2 was downregulated after treatment with β-elemene at both mRNA and protein levels. Furthermore, proliferation and colony formation by U87 cells were inhibited by β-elemene in a time and does- dependent manner. Our results indicate that β-elemene inhibits growth and induces apoptosis of human glioma cells in vitro. The induction of apoptosis appears to be related with the upregulation of Fas/FasL and Bax, activation of caspase-3,-8 and -9 and downregulation of Bcl-2, which then trigger major apoptotic cascades.

Antiproliferative and Pro-Apoptotic Activity of Diarylheptanoids Isolated from the Bark of Alnus japonica in Human Leukemia Cell Lines.

Alnus japonica Steud is a tree that grows in damp areas of mountain valleys and has been used as a traditional medicine in Asia. We investigated the antiproliferative activity of hirsutanone (Hir) and oregonin (Ore) in human cancer cell lines and elucidated their mechanisms of action. A cytotoxicity study using a panel of 12 human cancer and 4 normal cell lines indicated that Hir exhibited potent antiproliferative activity against 4 leukemia (Jurkat, U937, THP-1, and HL-60) and 2 colon cancer cell lines (HCT-15 and Colo205). Although Ore suppressed the cell growth of Jurkat and THP-1, its inhibitory potency was weaker than that of Hir. The IC50 values of Hir and Ore in Jurkat were 11.37 μM and 22.16 μM, respectively. Further analysis on Jurkat cells demonstrated that Hir caused a sequence of events involved in apoptosis, including nuclear morphological changes and accumulation of cells with sub-G1 DNA content. Hir led to the cleavage of poly(ADP-ribose) polymerase (PARP) and activation of caspase-3, -8, and -9. In addition, Hir-induced PARP cleavage was completely abolished by specific inhibitors to these caspases. Our data suggested that Hir is a potent antiproliferative compound against the 4 leukemia cell lines and the 2 colon cancer cell lines tested. Furthermore, Hir exerts antiproliferative actions via caspase-dependent apoptotic cell death.

Rhein Elicits In Vitro Cytotoxicity in Primary Human Liver HL-7702 Cells by Inducing Apoptosis through Mitochondria-Mediated Pathway.

Objective. To study rhein-induced apoptosis signaling pathway and to investigate its molecular mechanisms in primary human hepatic cells. Results. Cell viability of HL-7702 cells treated with rhein showed significant decrease in dose-dependent manner. Following rhein treatment (25 μM, 50 μM, and 100 μM) for 12 h, the detection of apoptotic cells was significantly analyzed by flow cytometry and nuclear morphological changes by Hoechst 33258, respectively. Fatty degeneration studies showed upregulation level of the relevant hepatic markers (P < 0.01). Caspase activities expressed significant upregulation of caspase-3, caspase-9, and caspase-8. Moreover, apoptotic cells by rhein were significantly inhibited by Z-LEHD-FMK and Z-DEVD-FMK, caspase-9 inhibitor, and caspase-3 inhibitor, respectively. Overproduction of reactive oxygen species, lipid peroxidation, and loss of mitochondrial membrane potential were detected by fluorometry. Additionally, NAC, a ROS scavenger, significantly attenuated rhein-induced oxidative damage in HL-7702 cells. Furthermore, real-time qPCR results showed significant upregulation of p53, PUMA, Apaf-1, and Casp-9 and Casp-3 mRNA, with no significant changes of Fas and Cytochrome-c. Immunoblotting revealed significant Cytochrome-c release from mitochondria into cytosol and no change in Fas expression. Conclusion. Taken together, these observations suggested that rhein could induce apoptosis in HL-7702 cells via mitochondria-mediated signal pathway with involvement of oxidative stress mechanism.

Xanthohumol induces growth inhibition and apoptosis in ca ski human cervical cancer cells.

We investigate induction of apoptosis by xanthohumol on Ca Ski cervical cancer cell line. Xanthohumol is a prenylated chalcone naturally found in hop plants, previously reported to be an effective anticancer agent in various cancer cell lines. The present study showed that xanthohumol was effective to inhibit proliferation of Ca Ski cells based on IC50 values using sulforhodamine B (SRB) assay. Furthermore, cellular and nuclear morphological changes were observed in the cells using phase contrast microscopy and Hoechst/PI fluorescent staining. In addition, 48-hour long treatment with xanthohumol triggered externalization of phosphatidylserine, changes in mitochondrial membrane potential, and DNA fragmentation in the cells. Additionally, xanthohumol mediated S phase arrest in cell cycle analysis and increased activities of caspase-3, caspase-8, and caspase-9. On the other hand, Western blot analysis showed that the expression levels of cleaved PARP, p53, and AIF increased, while Bcl-2 and XIAP decreased in a dose-dependent manner. Taken together, these findings indicate that xanthohumol-induced cell death might involve intrinsic and extrinsic apoptotic pathways, as well as downregulation of XIAP, upregulation of p53 proteins, and S phase cell cycle arrest in Ca Ski cervical cancer cells. This work suggests that xanthohumol is a potent chemotherapeutic candidate for cervical cancer.

Effects of Selenium Yeast on Oxidative Stress, Growth Inhibition, and Apoptosis in Human Breast Cancer Cells

Recent evidence suggests that selenium (Se) yeast may exhibit potential anti-cancer properties; whereas the precise mechanisms remain unknown. The present study was aimed at evaluating the effects of Se yeast on oxidative stress, growth inhibition, and apoptosis in human breast cancer cells. Treatments of ER-positive MCF-7 and triple-negative MDA-MB-231 cells with Se yeast (100, 750, and 1500 ng Se/mL), methylseleninic acid (MSA, 1500 ng Se/mL), or methylselenocysteine (MSC, 1500 ng Se/mL) at a time course experiment (at 24, 48, 72, and 96 h) were analyzed. Se yeast inhibited the growth of these cancer cells in a dose- and time-dependent manner. Compared with the same level of MSA, cancer cells exposure to Se yeast exhibited a lower growth-inhibitory response. The latter has also lower superoxide production and reduced antioxidant enzyme activities. Furthermore, MSA (1500 ng Se/mL)-exposed non-tumorigenic human mammary epithelial cells (HMEC) have a significant growth inhibitory effect, but not Se yeast and MSC. Compared with MSA, Se yeast resulted in a greater increase in the early apoptosis in MCF-7 cells as well as a lower proportion of early and late apoptosis in MDA-MB-231 cells. In addition, nuclear morphological changes and loss of mitochondrial membrane potential were observed. In conclusion, a dose of 100 to 1500 ng Se/mL of Se yeast can increase oxidative stress, and stimulate growth inhibitory effects and apoptosis induction in breast cancer cell lines, but does not affect non-tumorigenic cells.

Methyl pyropheophorbide-a-mediated photodynamic therapy induces apoptosis in human ovarian cancer cells

Objective To investigate the mechanism by which methyl pyropheophorbide-a-mediated photodynamic therapy(Mppa-PDT)inhibit cell viability and induce apoptosis in human ovarian cancer cell line SKOV3.Methods Human ovarian cancer cells SKOV3 at the logarithmic growth phase were divided into Mppa-PDT treated group(both Mppa and PDT treated group)and control groups(the blank group,the only Mppa treated group and only PDT treated group).After Mppa-PDT treatment,the cell viability was examined with CCK-8assay;cell apoptosis was detected by flow cytometry with Annexin Ⅴ-FITC/PI;and nuclear morphological changes during cell apoptosis was detected by DAPI staning.Moreover,the celluar reactive oxygen species(ROS) were detected by DCFH-DA staining;DNA damage was observed by single cell gel electrophoresis;and the protein expression of p53,Caspase-3,Bax,and Bcl-2 were assessed by Western blotting analysis.Results(1)Mppa-PDT could greatly suppress the cell viability of human ovarian cancer cells SKOV3 in a dose-dependent manner.(2)The cell apoptosis rate of Mppa-PDT treated group was significanlty higher than those of three control groups(blank group,Mppagroup and PDT group)(P0.05),and there was no difference among the three control groups(P0.05).(3)After treating with Mppa-PDT,DAPI staining showed strongly stained nuclei of the apoptotic cells;DCFH-DA staining displayed higher level of ROS than those of the three control groups;single cell gel electrophoresis showed greater DNA damage than those of the three control groups;and Western blotting analysis showed that the expression of p53,Caspase-3and Bax protein was increased and Bcl-2protein was decreased(P0.05).Conclusion Mppa-PDT can significantly suppress cell viability and induce apoptosis in human ovarian cancer cell SKOV3,accompanied by DNA damage and the activation of mitochondrial apoptosis pathway.

Fe3 O4 nanoparticle redox system modulation via cell-cycle progression and gene expression in human mesenchymal stem cells.

The use of engineered nanoparticles (NPs) across multiple fields and applications has rapidly increased over the last decade owing to their unusual properties. However, there is an increased need in understanding their toxicological effect on human health. Particularly, iron oxide (Fe3 O4 ) have been used in various sectors, including biomedical, food, and agriculture, but the current understanding of their impact on human health is inadequate. In this investigation, we assessed the toxic effect of Fe3 O4 NPs on human mesenchymal stem cells (hMSCs) adopting cell viability, cellular morphological changes, mitochondrial transmembrane potential, and cell-cycle progression assessment methodologies. Furthermore, the expression of oxidative stress, cell death, and cell-cycle regulatory genes was assessed using quantitative polymerase chain reaction. The Fe3 O4 NPs induced cytotoxicity and nuclear morphological changes in hMSCs by dose and time exposure. Cell-cycle analysis indicated that Fe3 O4 NPs altered the cell-cycle progression through a decrease in the proportion of cells in the G0 -G1 phase. The hMSC mitochondrial membrane potential loss increased with an increase in the concentration of Fe3 O4 NPs exposure. The observed expression levels of the CYP1A, TNF3, TNFSF10, E2F1, and CCNC genes were significantly upregulated in hMSCs in response to Fe3 O4 NPs exposure. Our findings suggest that Fe3 O4 NPs caused metabolic stress through altered cell cycle, oxidative stress, and cell death regulatory gene expression in hMSCs. The results of this investigation revealed that Fe3 O4 NPs exhibited moderate toxicity on hMSCs and that Fe3 O4 NPs may have biomedical applications at low concentrations. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 901-912, 2016.

Mechanisms of cell death induced by infusion sets leachables in in vitro experimental settings

Leachable materials that are released from infusion sets during their use can induce local and systemic toxic effects. We studied the mechanisms and kinetics of cell death induced by infusion sets leachates in vitro using L-929 and bEnd. 3 cells. Changes in cell morphology and metabolic activity were determined using light microscopy and the MTT test, respectively. Detailed analysis of the mechanisms of cell death was performed using membrane integrity and caspases 3 and 7 activity tests, annexin V-FITC/7-AAD analysis by FACS, and DAPI nuclear staining followed by confocal microscopy. Infusion sets released toxic leachables and induced toxic effects. Latex flashball was the most toxic part of the studied infusion sets, and it potently induced cell oncosis via increased permeability of the cell membrane. Latex-induced decrease in cells metabolic activity and cell death were not accompanied by activation of caspases 3 and 7, changes in nuclear morphology, or substantial annexin V-FITC cell staining. Leachables from the tube part of the infusion sets were less toxic, and induced some biochemical changes without altering the cells morphology. Further studies are needed to reveal the in vivo toxicity of infusion sets and its correlation with the results of in vitro toxicity studies.

Theranostic properties of a survivin-directed molecular beacon in human melanoma cells.

Survivin is an inhibitor of apoptosis overexpressed in different types of tumors and undetectable in most terminally differentiated normal tissues. In the current study, we sought to evaluate the in vitro theranostic properties of a molecular beacon-oligodeoxynucleotide (MB) that targets survivin mRNA. We used laser scanning confocal microscopy to study MB delivery in living cells and real-time PCR and western blot to assess selective survivin-targeting in human malignant melanoma cells. We further assess the pro-apoptotic effect of MB by measuring internucleosomal DNA fragmentation, dissipation of mitochondrial membrane potential (MMP) and changes in nuclear morphology. Transfection of MB into A375 and 501 Mel cells generated high signal intensity from the cytoplasm, while no signal was detected in the extracellular environment and in survivin-negative cells (i.e., human melanocytes and monocytes). MB time dependently decreased survivin mRNA and protein expression in melanoma cells with the maximum effect reached at 72 h. Treatment of melanoma cells with MB induced apoptosis by significant changes in MMP, accumulation of histone-complexed DNA fragments in the cytoplasm and nuclear condensation. MB also enhanced the pro-apoptotic effect of standard chemotherapeutic drugs tested at clinically relevant concentrations. The MB tested in the current study conjugates the ability of imaging with the pharmacological silencing activity against survivin mRNA in human melanoma cells and may represent an innovative approach for cancer diagnosis and treatment.

Assessing the Mechanism of Cytarabine-Induced Killing in Acute Leukemia

The nucleoside analogue cytarabine is part of standard therapy for acute myelogenous leukemia (AML) and is also widely used in various acute lymphocytic leukemia (ALL) regimens. During a 7-day infusion at 100 mg/m2/d, which is part of the “7 + 3” AML induction regimen, steady-state cytarabine concentrations in the 100-200 nM range are achieved. Previous literature suggesting the mechanism of cytarabine-induced killing in AML examined cytarabine concentrations in the micromolar range, which might be less pertinent to routinely achieved drug exposure. To better understand the mechanism of cytarabine-induced killing, AML and ALL cell lines were exposed to varying cytarabine concentrations (25-200 nM) for 24-48 hours and assayed for cell death using a variety of assays. Dose- and time-dependent induction of apoptosis was observed in both AML and ALL cell lines, as detected by phosphatidylserine externalization (Annexin V staining), DNA fragmentation (propidium iodide staining for subdiploid cells), nuclear morphological changes, and cleavage of caspase substrates. Jurkat T cell ALL variants lacking critical components of the death receptor pathway (FADD or procaspase 8) underwent apoptosis with kinetics indistinguishable from parental cells, arguing against involvement of the death receptor pathway. Immunoblotting for Bcl-2 family members in AML and ALL cell lines revealed upregulation of the BH3-only proteins Bim, Puma and Noxa across multiple lines. In further experiments, small interfering RNA (siRNA)-mediated knockdown of Bim, Puma, and Noxa or the mitochondrial proteins Bax and Bak markedly diminished the apoptotic response to cytarabine. These results suggest that cytarabine at nanomolar concentrations kills cells through the mitochondrial apoptotic pathway but, in contrast to more targeted agents, does so by upregulating multiple BH3-only Bcl-2 family members simultaneously. Additional studies to determine the mechanisms of Bim, Puma, and Noxa upregulation are underway. (Supported in part by R01 CA166741 and predoctoral fellowships from the Mayo Foundation for Education and Research.) DisclosuresNo relevant conflicts of interest to declare.

Identification of titanium dioxide nanoparticles in food products: Induce intracellular oxidative stress mediated by TNF and CYP1A genes in human lung fibroblast cells

Food grade TiO2 (E171) is a synthetic additive, and widely used as a coloring agent in many foods, pharmaceutical and personal care products. A few reports have highlighted that insoluble particulates (less than 200nm) of food grade TiO2 are found in many foods and confectionary products. However, information regarding the physico-chemical properties (i.e., size and shape)-based food grade TiO2 nanotoxicity related human health issues are limited. The main goal of this study is to examine the presence of nano-sized particulates and its structural characteristics of food grade- TiO2 materials and to assess the acute cellular uptake and metabolic stress induced by these particulates in human lung fibroblast (WI-38) cells. The results of transmission electron microscopy, energy dispersive X-ray spectroscopy, and X-ray diffraction studies indicated that about food grade TiO2 sample contains spherical shaped particulate forms in the nano-scale range, <100nm. The intracellular oxidative stress in human lung fibroblast cells (WI-38) was assessed through studies investigating the cellular uptake of the particles, changes in nuclear and cytoplasmic morphology, intracellular ROS, mitochondrial trans-membrane potential, the cell cycle and the expression of genes linked to metabolic stress markers. Altogether our data clearly indicate that primary metabolic stress indicators such as changes in the intracellular ROS, the dose-dependent loss of the mitochondrial membrane potential, alterations in cell cycle progression (G2/M>S>G0/G1) and changes in the TNF and CYP1A gene expression pattern are linked to cellular stress. Thus, food grade TiO2 as nano-scaled contaminants could not only be potential human health risk factors, suggesting that safety considerations with special respect to a few crucial factors such as size, and shape should be considered and regulated by food regulators.