Cell Apoptotic Bodies Research Articles

IR 820 dye encapsulated in polycaprolactone glycol chitosan: Poloxamer blend nanoparticles for photo immunotherapy for breast cancer.

In the present study, we have fabricated biocompatible and biodegradable monodisperse IR 820 encapsulated polycaprolactone (PCL) glycol chitosan (GC): Poloxamer blend nanoparticles (PP-IR NPs) for imaging and effective photo-immunotherapy. IR 820 has been used as an imaging and photothermal agent whereas glycol chitosan (GC) as an immunostimulatory agent. The combination of IR 820, poloxamer, and GC can be used effectively for photoimmunotherapy for cancer, drug-resistant and TNF-α resistant estrogen positive breast cancer. PP-IR NPs are stable in aqueous solution. The uniform size of 100-220 nm with a high zeta value of +38 ± 2 mV led them to accumulate in cancer cells. Laser treatment did not affect the morphology of PP-IR NPs as observed under the transmission electron microscope (TEM). In vitro cytotoxicity studies on MCF-7 cells showed enhanced toxicity upon laser treatment. Further, we validated the cell death by reactive oxygen species (ROS) production. Our studies thus showed that PP-IR NPs are effective in suppressing metastatic cancer as the combinational therapy leads to the formation of apoptotic bodies in MCF-7 cells.

MiR-124 Contributes to the functional maturity of microglia.

During early development of the central nervous system (CNS), a subset of yolk-sac derived myeloid cells populate the brain and provide the seed for the microglial cell population, which will self-renew throughout life. As development progresses, individual microglial cells transition from a phagocytic amoeboid state through a transitional morphing phase into the sessile, ramified, and normally nonphagocytic microglia observed in the adult CNS under healthy conditions. The molecular drivers of this tissue-specific maturation profile are not known. However, a survey of tissue resident macrophages identified miR-124 to be expressed in microglia. In this study, we used transgenic zebrafish to overexpress miR-124 in the mpeg1 expressing yolk-sac-derived myeloid cells that seed the microglia. In addition, a systemic sponge designed to neutralize the effects of miR-124 was used to assess microglial development in a miR-124 loss-of-function environment. Following the induction of miR-124 overexpression, microglial motility and phagocytosis of apoptotic cells were significantly reduced. miR-124 overexpression in microglia resulted in the accumulation of residual apoptotic cell bodies in the optic tectum, which could not be achieved by miR-124 overexpression in differentiated neurons. Conversely, expression of the miR-124 sponge caused an increase in the motility of microglia and transiently rescued motility and phagocytosis functions when activated simultaneously with miR-124 overexpression. This study provides in vivo evidence that miR-124 activity has a key role in the development of functionally mature microglia.

Induction of potent NK cell-dependent anti-myeloma cytotoxic T cells in response to combined mapatumumab and bortezomib

There is increasing evidence that some cancer therapies can promote tumor immunogenicity to boost the endogenous antitumor immune response. In this study, we used the novel combination of agonistic anti-TRAIL-R1 antibody (mapatumumab, Mapa) with low dose bortezomib (LDB) for this purpose. The combination induced profound myeloma cell apoptosis, greatly enhanced the uptake of myeloma cell apoptotic bodies by dendritic cell (DC) and induced anti-myeloma cytotoxicity by both CD8+ T cells and NK cells. Cytotoxic lymphocyte expansion was detected within 24 h of commencing therapy and was maximized when myeloma-pulsed DC were co-treated with low dose bortezomib and mapatumumab (LDB+Mapa) in the presence of NK cells. This study shows that Mapa has two distinct but connected modes of action against multiple myeloma (MM). First, when combined with LDB, Mapa produced powerful myeloma cell apoptosis; secondly, it promoted DC priming and an NK cell-mediated expansion of anti-myeloma cytotoxic lymphocyte (CTL). Overall, this study indicates that Mapa can be used to drive potent anti-MM immune responses.

Is Trichomonas tenax a Parasite or a Commensal?

Trichomonas tenax is considered a commensal organism found under poor oral hygiene conditions. T. tenax presents morphological similarities with T. vaginalis, and there are doubts concerning whether this protist is a parasite and whether it is a genetic variant of T. vaginalis. This study aimed to investigate the capacity of T. tenax to cause mammalian cell damage and compare its cytotoxicity with that of T. vaginalis. Protozoan-host cell interaction assays were performed with Madin-Darby canine kidney, HeLa, and gum cells and 3D spheroids, which were examined by scanning electron and transmission electron microscopy. Cellular viability experiments were also performed. T. tenax attached and had different forms when interacting with mammalian cells and caused damage with time-dependent host-cell viability. We observed that T. tenax produced plasma membrane projections and phagocytosed portions of the mammalian cells. In addition, T. tenax caused membrane blebbing and apoptotic bodies in HeLa cells, thus inducing cell death. Spheroids were also used in interaction assays with T. tenax and they were damaged by these cells. This study shows that T. tenax fulfills the requisites of a parasite, causing damage to different mammalian cells and behaving similarly to T. vaginalis when in contact with target cells in vitro.

RNA interference-mediated knockdown of astrocyte elevated gene-1 inhibits growth, induces apoptosis, and increases the chemosensitivity to 5-fluorouracil in renal cancer Caki-1 cells.

Astrocyte elevated gene-1 (AEG-1) is a recently discovered oncogene that has been reported to be highly expressed in various types of malignant tumors, including renal cell carcinoma. However, the precise role of AEG-1 in renal cancer cell proliferation and apoptosis has not been clarified. In this study, we transfected the renal cancer cell line Caki-1 with a plasmid expressing AEG-1 short hairpin RNA (shRNA) and obtained cell colonies with stable knockdown of AEG-1. We found that AEG-1 down-regulation inhibited cell proliferation and colony formation and arrested cell cycle progression at the sub-G1 and G0/G1 phase. Western blot analysis indicated that the expression of proliferating cell nuclear antigen (PCNA), cyclin D1 and cyclin E were significantly reduced following AEG-1 down-regulation. In addition, AEG-1 knockdown led to the appearance of apoptotic bodies in renal cancer cells, and the ratio of apoptotic cells significantly increased. Expression of the anti-apoptotic factor Bcl-2 was dramatically reduced, whereas the pro-apoptotic factors Bax, caspase-3 and poly (ADP-ribose) polymerase (PARP) were significantly activated. Finally, AEG-1 knockdown in Caki-1 cells remarkably suppressed cell proliferation and enhanced cell apoptosis in response to 5-fluorouracil (5-FU) treatment, suggesting that AEG-1 inhibition sensitizes Caki-1 cells to 5-FU. Taken together, our data suggest that AEG-1 plays an important role in renal cancer formation and development and may be a potential target for future gene therapy for renal cell carcinoma.

Cytotoxicity, DNA damage, and apoptosis induced by titanium dioxide nanoparticles in human non-small cell lung cancer A549 cells.

Concerns about the risk of titanium dioxide nanoparticles (TiO2 NPs) to human health and environment are gradually increasing due to their wide range of applications. In this study, cytotoxicity, DNA damage, and apoptosis induced by TiO2 NPs (5nm) in A549 cells were investigated. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays revealed the time- and concentration-dependent cytotoxic effects of TiO2 NPs in a concentration range of 50 to 200μg/mL. A statistically significant (p < 0.05) induction in DNA damage was observed by the comet assay in cells exposed to 50 to 200μg/mL TiO2 NPs for 48h. A significant (p < 0.05) induction in micronucleus formation determined by 4,6-diamino-2-phenylindole (DAPI) staining was also observed at the above concentrations. Typical apoptotic morphological feature and apoptotic bodies in A549 cells induced by TiO2 NPs at the above concentrations were observed by scanning electron micrographs. Flow cytometric analysis demonstrated that the cells treated with TiO2 NPs at concentrations of 100 and 200μg/mL showed a significant G2/M phase arrest and a significant increased proportion of apoptotic cells. TiO2 NPs also disrupted the mitochondrial membrane potential evaluated by rhodamine 123 staining. Further analysis by quantitative real-time PCR (qRT-PCR) indicated that the expression of caspase-3 and caspase-9 messenger RNA (mRNA) was increased significantly at the concentrations of 100 and 200μg/mL TiO2 NPs for 48h. Taken together, these findings suggest that TiO2 NPs can inhibit A549 cell proliferation, cause DNA damage, and induce apoptosis via a mechanism primarily involving the activation of the intrinsic mitochondrial pathway. The assay data provide strong evidence that TiO2 NPs can induce cytotoxicity, significant DNA damage, and apoptosis of A549 cells, suggesting that exposure to TiO2 NPs could cause cell injury and be hazardous to health.

The synergistic effect of organic silicone quaternary ammonium salt and 5-fluorouracil on hepatocellular carcinoma in vitro and in vivo

Hepatocellular carcinoma (HCC) is the third most common type of cancer worldwide, causing over 370,000 deaths per year, with approximately half of them in China. Chemotherapy is the optimal treatment for patients with advanced HCC, although chemoresistance has become a significant obstacle to successful liver cancer surgery. In this paper, we have assessed the characteristics of drugs to explore the effects of individual and combined action of organic silicone quaternary ammonium salt (Jieyoushen) and 5-fluorouracil (5-FU). The results of MTT assays showed that single and combined action of Jieyoushen and 5-FU can inhibit the proliferation of liver carcinoma cell lines in a dose-dependent and time-dependent manner, respectively. Electron microscopy and Hoechst 33342 staining showed characteristic apoptotic bodies in apoptotic cells treated with Jieyoushen and 5-FU. Flow cytometry results indicated that the percentage of cells at G0/G1 phase gradually increased, whereas it gradually decreased during the S phase after treatment. Taken together, these results suggest that the combination of Jieyoushen with 5-FU exerts a synergistic anticancer effect on HCC growth and that targeted therapeutic strategies may improve HCC sensitivity to chemotherapy.

Anticancer activity of 28-Oxoallobetulin on HT-29 human colon cancer cells

Many successful therapeutic drug, including several anticancer agents, have been isolated for natural products. In a recent screen of methanolic extracts from several natural products, we isolated an active compound, from the methanolic extract of persimmon calyx, which promoted cytotoxicity and apoptosis in HT-29 human colon cancer cells. This compound was identified as 28-oxoallobetulin, a prototypical compound, by UV-MS analysis and various NMR spectroscopic methods. The highly cytotoxic effects of 28-oxoallobetulin on HT-29 cells were demonstrated using MTT reduction, LDH release, and colony formation assays. Furthermore, we were able to detect apoptotic bodies in HT-29 cells by Hoechst staining as well as flow cytometric analysis for the presence of sub-G1 DNA peaks, indicative of apoptotic cells. Finally apoptosis in HT-29 cells following 28-oxoallobetulin treatment was also confirmed by the presence of activated caspase-3 and specific proteolytic cleavage of poly (ADP-ribose) polymerase.

The reduced autophagic response by oxidative stress in angiotensin II-induced hypertrophic H9C2 cells causes more apoptotic cell death.

Autophagy is an important process in the pathogenesis of cardiovascular diseases, and angiotensin II (Ang II) plays a causative role in the induction of cardiomyocyte autophagy. The purpose of this study was to explore whether, under conditions of oxidative stress, levels and types of cell death were different in untreated and Ang II-treated cardiomyocytes (H9C2 cells). Treatment with 20 µM Ang II induced cardiac hypertrophy in H9C2 cells, with increased expression of the hypertrophic markers c-Fos, ß-myosin heavy chain, atrial natriuretic factor (ANF), and brain natriuretic factor (BNF). Under normal conditions, there was no difference in the levels of autophagic vacuoles and apoptotic bodies in untreated and Ang II-treated H9C2 cells. However, oxidative stress generated by 100 µM H₂O₂ triggered autophagy in untreated control cells, but had a reduced effect in Ang II-induced hypertrophic cells, resulting in more cell death, and this was associated with a decrease in connexin 43 expression. Blocking this autophagic response with 3-methyladenine resulted in a significant increase in cell death and apoptosis of H9C2 cells but did not significantly affect the response of Ang II-treated cells. The autophagic response to 100 µM H₂O₂ provides a survival advantage for cells and this is reduced by Ang II treatment.

Metformin inhibits the proliferation of A549/CDDP cells by activating p38 mitogen-activated protein kinase.

Metformin (Met) has been widely used in hypoglycemic therapy, and it is also able to reduce the incidence of tumors and tumor-related mortality. The present study investigated whether Met could inhibit the proliferation of lung cancer cells and enhance the sensitivity of a cisplatin-resistant lung cancer A549/CDDP cell line to cisplatin. It was found that Met treatment inhibited the proliferation of different lung cancer cells. Met inhibited the cell cycle of the A549/CDDP cells and induced apoptosis. Upon Met treatment, the A549/CDDP cells were arrested at the G1 phase. The apoptosis of the A549/CDDP cells was confirmed by the appearance of apoptotic bodies in cells stained with Hoechst 33258, and by the cleavage of BH3 interacting-domain death agonist and poly (ADP-ribose) polymerase. Furthermore, results showed that the phosphorylation level of p38 mitogen-activated protein kinase (MAPK) was increased after Met treatment. The p38 MAPK inhibitor, SB203580, significantly blocked Met-induced apoptosis in the A549/CDDP cells. It was further demonstrated that Met could enhance the sensitivity of the A549/CDDP cells to cisplatin. In summary, the present study identified Met as a drug sensitizer that could improve the treatment effect of cisplatin in cisplatin-resistant lung cancers.

Analysis of the misregulation of RhoA and RhoH in autoimmune mice. (HUM7P.302)

Abstract When cultured in the presence of apoptotic cells, cell bodies or lipids, RhoA is misregulated in macrophages from all 6 autoimmune mouse strains but none of the 7 normal strains tested. Misregulation of RhoA results in defective morphology, altered cytokine production, and increased adhesion. The same macrophages cultured without apoptotic cell lipids had properly regulated RhoA and behaved normally. Normal mouse macrophages cultured with the Rho inhibitor C3 toxin, had defective macrophage morphology and behavior. Therefore, we proposed that autoimmunity is due to an apoptotic cell recognition signaling defect and that normal macrophage function may be restored by a chemical reversal of the defect. Our new data demonstrate that lupus strain, MRL, macrophages cultured in the presence of C3 no longer had defects in morphology and function. The fact that a RhoA inhibitor restored RhoA function indicates that the misregulation in autoimmune macrophages involves a RhoA regulator also affected by C3 and not RhoA itself. RhoH is a known inhibitor of RhoA and it is only expressed in hematopoietic cells. Experiments comparing RhoH expression in BALB and MRL macrophages suggest at least four different sizes of RhoH protein are expressed in normal macrophages in response to lipid exposure and that lupus macrophages do not express the multiple forms of RhoH even in the presence of lipids. A lack of proper RhoH expression could be behind the autoimmune macrophage defect.

Oleanolic acid derivatives induce apoptosis in human leukemia K562 cell involved in inhibition of both Akt1 translocation and pAkt1 expression.

Oleanolic acid (OA) derivatives exhibit numerous pleiotropic effects in many cancers. The present study aimed to investigate the molecular mechanisms of 5'-amino-oleana-2,12-dieno[3,2-d]pyrimidin-28-oic acid (compound 4) and oleana-2,12-dieno[2,3-d]isoxazol-28-oic acid (compound 5) inducing apoptosis in human leukemia K562 cell. We investigated the effects of the compounds on K562 cell growth, apoptosis and cell cycle. The compounds showed strong inhibitory effects on K562 cell viability in a dose-dependent manner determined by the 3-(4,5-dimethylthiazoyl)-2,5-diphenyltetrazolium bromide assay and significantly increased chromatin condensation and apoptotic bodies in K562 cells. Flow cytometry assay suggested that the compounds induced inhibition of K562 cell proliferation associated with G1 phase arrest. In addition, the compounds inhibited Akt1 recruiting to membrane in CHO cells which express Akt1-EGFP constitutively and down-regulated the expression of pAkt1 in K562 cell. These results suggested that the compounds can efficiently inhibit proliferation and induce apoptosis perhaps involved in inactivation of Akt1. The OA derivatives may be potential chemotherapeutic agents for the treatment of human cancer.

Tea polyphenols induced apoptosis of breast cancer cells by suppressing the expression of Survivin

To study the mechanism of tea polyphenols (TP)-induced apoptosis of breast cancer cells. Proliferation of MCF-7 and SK-BR-3 cells was evaluated by MTT assays. Cellular ultrastructure was examined by electron microscopy. Apoptosis was detected by TUNEL. PCNA、 Cyclin D1、 Cyclin E and Survivin expression was measured by Western blot. Cell proliferation was significantly inhibited by TP. Spindle and round cells were loosely distributed with increased particles after TP treatment. Increased cell size, frequent nuclear atypia and a collapse of apoptosis were observed. The nucleus was pushed towards one side, while the cytoplasm was rich in free ribosome. The membrane of mitochondria was thickening, and the cell apoptotic body was observed. TP treated cells experienced significantly enhanced apoptosis compared with 5-Fu treated or control groups. The expression of survivin was downregulated by TP. To conclude, TP can inhibit cell growth and induce apoptosis through downregulating the expression of survivin in breast cancer.

Curcumin-loaded nanoparticles enhance apoptotic cell death of U2OS human osteosarcoma cells through the Akt-Bad signaling pathway

Curcumin has potential anticancer activity and has been shown to be involved in several signaling pathways including differentiation and apoptosis. Our previous study showed that water-soluble PLGA curcumin nanoparticles (Cur-NPs) triggered apoptotic cell death through regulation of the function of MDR1 and the production of reactive oxygen species (ROS) in cisplatin-resistant human oral cancer CAR cells. In this study, we investigated the anti-proliferative effects of Cur-NPs on human osteosarcoma U2OS cells. The morphology of Cur-NPs showed spherical shape by TEM analysis. The encapsulation efficiency of curcumin in Cur-NPs prepared by single emulsion was 90.5 ± 3.0%. Our results demonstrated that the curcumin fragments on the mass spectrum of Cur-NPs and the peaks of curcumin standard could be found on the Cur-NPs spectrum by 1H-NMR spectra analysis. Cur-NPs induced anti-proliferative effects and apoptosis in U2OS cells. Compared to the untreated U2OS cells, more detectable amount of Cur-NPs was found inside the treated U2OS cells. Cur-NPs induced DNA fragmentation and apoptotic bodies in U2OS cells. Both the activity and the expression levels of caspases-3/-7 and caspase-9 were elevated in the treated U2OS cells. Cur-NPs upregulated the protein expression levels of cleaved caspase-3/caspase-9, cytochrome c, Apaf-1 and Bad and downregulated the protein expression level of p-Akt in U2OS cells. These results suggest Cur-NPs are effective in enhancing apoptosis in human osteosarcoma cells and thus could provide potential for cancer therapeutics.

Caspase‐generated neoepitopes as markers of axonal degeneration in neural development and injury

Neuronal death, axonal degeneration, and synapse loss are hallmarks of central nervous system development and injury. Commonly employed markers of apoptosis are TUNEL and immunohistochemistry for activated caspases, but these often fail to report axonal degeneration associated with somatic apoptosis. Axonal degeneration is classically detected by silver degeneration stains, but these have limitations (non‐selectivity, minimal reproducibility and incompatibility with co‐labeling). We demonstrate that antibodies to caspase‐cleaved protein substrates beta‐actin (cleaved by caspase 3 at D244) and alpha‐tubulin (cleaved by caspase 6 at D438) enable superior visualization of neurite degeneration. These antibodies label apoptotic cell bodies and neurites during embryonic and postnatal developmental pruning. In addition, they reveal injured neurons and neurites after induction of apoptosis in a mouse model of fetal alcohol syndrome and in sympathetic neuron cultures induced to undergo apoptosis after nerve growth factor withdrawal. Finally, the markers may have clinical utility, as they highlight degenerating neurites in neonatal hypoxic/ischemic injury and in adult patients with multiple sclerosis. Ongoing work aims to identify novel compartment‐specific neoepitopes that will allow monitoring of somatodendritic, axonal, and synaptic remodeling and degeneration. Supported by NS075869.

Inhibition of topoisomerase II α activity and induction of apoptosis in mammalian cells by semi-synthetic andrographolide analogues

Topoisomerase II α enzyme plays a critical role in DNA replication process. It controls the topologic states of DNA during transcription and is essential for cell proliferation. Human DNA topoisomerase II α (hTopo II α) is a promising chemotherapeutic target for anticancer agents against a variety of cancer types. In the present study, andrographolide and its structurally modified analogues were investigated for their inhibitory activities on hTopo II α enzyme. Five out of nine andrographolide analogues potently reduced hTopo II α activity and inhibited cell proliferation in four mammalian cell lines (Hela, CHO, BCA-1 and HepG2 cells). IC50 values for cytotoxicity of analogues 3A.1, 3A.2, 3A.3, 1B and 2C were 4 to 7μM. Structure-activity relationship studies revealed that both core structure of andrographolide and silicon based molecule of functional group were important for the inhibition of hTopo II α activity whereas position C-19 of analogues was required for anti-proliferation. In addition, the analogue 2C at 10μM concentration inhibited hTopo II α, and induced apoptosis with nuclear fragmentation and formation of apoptotic bodies in HepG2 cells. The analogue 2C may, therefore, have a therapeutic potential as effective anticancer agent targeting the hTopo II α functions.

Colon cancer cell chemosensitisation by fish oil emulsion involves apoptotic mitochondria pathway

Adjuvant use of safe compounds with anti-tumour properties has been proposed to improve cancer chemotherapy outcome. We aimed to investigate the effects of fish oil emulsion (FOE) rich in n-3 PUFA with the standard chemotherapeutic agents 5-fluorouracil (5-FU), oxaliplatin (OX) or irinotecan (IRI) on two human colorectal adenocarcinoma cells with different genetic backgrounds. The HT-29 (Bax+/+) and LS174T (Bax-/-) cells were co-treated for 24-72 h with 1 μm-5-FU, 1 μm-OX or 10 μm-IRI and/or FOE dilution corresponding to 24 μm-EPA and 20·5 μm-DHA. Soyabean oil emulsion (SOE) was used as isoenergetic and isolipid control. Cell viability, apoptosis and nuclear morphological changes were evaluated by cytotoxic colorimetric assay, flow cytometry analysis with annexin V and 4',6'-diamidino-2-phenylindole staining, respectively. A cationic fluorescent probe was used to evaluate mitochondrial dysfunction, and protein expression involved in mitochondrial apoptosis was determined by Western blot. In contrast to SOE, co-treatment with FOE enhanced significantly the pro-apoptotic and cytotoxic effects of 5-FU, OX or IRI in HT-29 but not in LS174T cells (two-way ANOVA, P <0.01). These results were confirmed by the formation of apoptotic bodies in HT-29 cells. A significant increase in mitochondrial membrane depolarisation was observed after the combination of 5-FU or IRI with FOE in HT-29 but not in LS174T cells (P <0.05). Co-administration of FOE with the standard agents, 5-FU, OX and IRI, could be a good alternative to increase the efficacy of chemotherapeutic protocols through a Bax-dependent mitochondrial pathway.

Development of ramified microglia from early macrophages in the zebrafish optic tectum

Microglia, the resident macrophage precursors of the brain, are necessary for the maintenance of tissue homeostasis and activated by a wide range of pathological stimuli. They have a key role in immune and inflammatory responses. Early microglia stem from primitive macrophages, however the transition from early motile forms to the ramified mature resident microglia has not been assayed in real time. In order to provide such an assay, we used zebrafish transgenic lines in which fluorescent reporter expression is driven by the promoter of macrophage expressed gene 1 (mpeg1; Ellet et al. [2011]: Blood 117(4): e49-e56,). This enabled the investigation of the development of these cells in live, intact larvae. We show that microglia develop from highly motile amoeboid cells that are engaged in phagocytosis of apoptotic cell bodies into a microglial cell type that rapidly morphs back and forth between amoeboid and ramified morphologies. These morphing microglia eventually settle into a typical mature ramified morphology. Developing microglia frequently come into contact with blood capillaries in the brain, and also frequently contact each other. Up to 10 days postfertilization, microglia were observed to undergo symmetric division. In the adult optic tectum, the microglia are highly branched, resembling mammalian microglia. In addition, the mpeg1 transgene also labeled highly branched cells in the skin overlying the optic tectum from 8-9 days postfertilization, which likely represent Langerhans cells. Thus, the development of zebrafish microglia and their cellular interactions was studied in the intact developing brain in real time and at cellular resolution.

Isolation of a Glucosamine Binding Leguminous Lectin with Mitogenic Activity towards Splenocytes and Anti-Proliferative Activity towards Tumor Cells

A dimeric 64-kDa glucosamine-specific lectin was purified from seeds of Phaseolus vulgaris cv. “brown kidney bean.” The simple 2-step purification protocol involved affinity chromatography on Affi-gel blue gel and gel filtration by FPLC on Superdex 75. The lectin was absorbed on Affi-gel blue gel and desorbed using 1M NaCl in the starting buffer. Gel filtration on Superdex 75 yielded a major absorbance peak that gave a single 32-kDa band in SDS-PAGE. Hemagglutinating activity was completely preserved when the ambient temperature was in the range of 20°C–60°C. However, drastic reduction of the activity occurred at temperatures above 65°C. Full hemagglutinating activity of the lectin was observed at an ambient pH of 3 to 12. About 50% activity remained at pH 0–2, and only residual activity was observed at pH 13–14. Hemagglutinating activity of the lectin was inhibited by glucosamine. The brown kidney bean lectin elicited maximum mitogenic activity toward murine splenocytes at 2.5 µM. The mitogenic activity was nearly completely eliminated in the presence of 250 mM glucosamine. The lectin also increased mRNA expression of the cytokines IL-2, TNF-α and IFN-γ. The lectin exhibited antiproliferative activity toward human breast cancer (MCF7) cells, hepatoma (HepG2) cells and nasopharyngeal carcinoma (CNE1 and CNE2) cells with IC50 of 5.12 µM, 32.85 µM, 3.12 µM and 40.12 µM respectively after treatment for 24 hours. Flow cytometry with Annexin V and propidum iodide staining indicated apoptosis of MCF7 cells. Hoechst 33342 staining also indicated formation of apoptotic bodies in MCF7 cells after exposure to brown kidney bean lectin. Western blotting revealed that the lectin-induced apoptosis involved ER stress and unfolded protein response.

Curcumin abrogates bile-induced NF-κB activity and DNA damage in vitro and suppresses NF-κB activity whilst promoting apoptosis in vivo, suggesting chemopreventative potential in Barrett’s oesophagus

Curcumin has been suggested to possess anti-neoplastic properties. As oesophageal adenocarcinoma (OA) and Barrett's oesophagus (BO) represent a neoplastic series, we postulated that curcumin supplementation may slow neoplastic progression at this site. Our aim was to investigate the effects of curcumin in vitro and in vivo on markers of oesophageal cancer progression. We investigated the in vitro ability of curcumin to prevent bile acid-induced DNA damage using micronucleus assay and nuclear factor-kappaB (NF-κB) activity in the oesophageal cell lines (OE33) using real-time PCR of the extracted RNA. We also analysed NF-κB p65 activation in curcumin-pre-treated OE33 cells exposed to deoxycholic acid (DCA) using ELISA. In another pilot study, BO patients took a daily 500 mg curcumin tablet for 7 days prior to their endoscopy. In biopsies collected from these patients (n=33, 16 curcumin, 17 control), we examined NF-κB-driven gene expression (interleukin (IL)-8, inhibitor- kappaB (I-κB)) using real-time PCR of the extracted RNA from the biopsy sample. The apoptotic frequency was assessed by counting the number of apoptotic bodies in the epithelial cells from the Barrett's tissue with and without curcumin. In vitro, curcumin (50 μM) significantly abrogated DNA damage and NF-κB activity induced by bile. Pretreating OE33 cells with curcumin (50 μM) completely abolished the ability of DCA (300 μM) to activate NF-κB. In vivo, IL-8 expression was non-significantly suppressed in the curcumin-supplemented patients compared to the squamous control tissue, whilst also showing a doubling in the apoptotic frequency compared to non-supplemented control patients. Curcumin abrogated bile-driven effects in vitro. The in vivo data also suggests that curcumin supplementation had beneficial effects (increased apoptosis, potentially reduced NF-κB activity) in the Barrett's tissues themselves, despite poor delivery of the curcumin to the oesophagus.