Apoptotic Fragmentation Research Articles

SONODYNAMIC EFFECT OF BERBERINE ON MACROPHAGES

ObjectivesAtherosclerosis (AS) is the major contributing factor that results in acute cardiovascular events, which leading to the high mortality of cardiovascular diseases, which. The pathophysiological mechanism of atherosclerosis remains unknown....

Induction of apoptosis in oral epithelial cells by Candida albicans

During infection, interactions between Candida albicans and oral epithelial cells result in oral epithelial cell death. This is clinically manifested by the development of oral mucosal ulcerations generally associated with discomfort. In vitro studies have shown that C.albicans induces early apoptotic alterations in oral epithelial cells; however, these studies have also shown that treatment of infected cells with caspase inhibitors does not prevent their death. The reasons for these contradictory results are unknown and it is still not clear if C.albicans stimulates oral epithelial signaling pathways that promote apoptotic cell death. Activation of specific death pathways in response to microbial organisms plays an essential role in modulating the pathogenesis of a variety of infectious diseases. The aim of this study was to (i) characterize C.albicans-induced apoptotic morphological alterations in oral epithelial cells, and (ii) investigate the activation of apoptotic signaling pathways and expression of apoptotic genes during infection. Candida albicans induced early apoptotic changes in over 50% of oral epithelial cells. However, only 15% of those showed mid-late apoptotic alterations. At the molecular level, C.albicans caused a loss of the mitochondrial transmembrane potential and translocation of mitochondrial cytochrome c. Caspase-3/9 activities increased only during the first hours of infection. Moreover, poly[ADP ribose] polymerase 1 was cleaved into apoptotic and necrotic-like fragments. Finally, five anti-apoptotic genes were significantly upregulated and two pro-apoptotic genes were downregulated during infection. Altogether, these findings indicate that epithelial apoptotic pathways are activated in response to C.albicans, but fail to progress and promote apoptotic cell death.

Increased Expression of Oxidation-Specific Epitopes and Apoptosis Are Associated With Haptoglobin Genotype: Possible Implications for Plaque Progression in Human Atherosclerosis

The purpose of this study was to test the hypothesis that increased oxidative stress is associated with apoptosis in human plaques with the haptoglobin (Hp) 2-2 genotype. Intraplaque hemorrhage releases free hemoglobin (Hb). Impaired Hb clearance induces oxidative stress leading to plaque progression. The binding of Hp to Hb attenuates iron-induced oxidative reactions. Twenty-six human aortic plaques were Hp genotyped. Hp2-2 plaques (n = 13) were compared with control (Hp1-1/2-1) (n = 13). The iron grade was measured by Perl's staining. Immunostaining was used to detect oxidation-specific epitopes (OSEs) reflecting oxidized phospholipids and malondialdehyde-like epitopes. The percentages of apoptotic cells and apoptotic morphological features were quantified. DNA fragmentation and active caspase-3 were measured by in situ end-labeling and immunohistochemistry, respectively. In Hp2-2 plaques, iron content was increased (1.22 ± 0.15 vs. 0.54 ± 0.08; p < 0.0001) along with expression of oxidized phospholipid- (78.9 ± 5.8 vs. 38.8 ± 3.8; p < 0.0001), and malondialdehyde-like OSEs (93.9 ± 7.9 vs. 54.7 ± 3.9; p < 0.0001). The total percentages of apoptotic cells (11.9 ± 0.44 vs. 3.5 ± 0.28; p < 0.0001), nuclear fragmentation (11.8 ± 0.50 vs. 3.3 ± 0.26; p < 0.0001), nuclear condensation (10.9 ± 0.58 vs. 3.4 ± 0.20; p < 0.0001), chromatin margination (14.2 ± 0.57 vs. 6.5 ± 0.37; p < 0.0001), cytoplasmic blebs (1.6 ± 0.28 vs. 0.8 ± 0.14; p < 0.002), and eosinophilia (10.8 ± 0.74 vs. 4.2 ± 0.27; p < 0.0001) were increased in Hp2-2 plaques. Furthermore, DNA fragmentation (119.9 ± 1.40 vs. 57.5 ± 0.80; p < 0.001), and active caspase-3 density (84.7 ± 7.62 vs. 50.6 ± 7.49; p < 0.004) were increased in Hp2-2 plaques. Logistic regression analysis identified correlation between the percentage of apoptotic cells and the density of OSEs (r = 0.56; p < 0.003). These findings provide insights into genetic predisposition to oxidative stress and the relationship between OSEs and macrophage apoptosis that may explain advanced atherosclerosis in human Hp2-2 plaques.

Anti-proliferative properties of ethyl acetate extract of Phellinus linteus grown on ginseng (Panax ginseng) on HT-29 human colon carcinoma cells through inducing apoptosis

Phellinus linteus (PL) and ginseng (Panax ginseng, PG) have been widely used as traditional herbal medicines in East Asia. In this study, we evaluated whether the extract of P. linteus grown on ginseng (PGP) exhibited an more enhanced anti-proliferative activity on HT-29 human colon carcinoma cells than PL or PG extract. PGP EtOAc extract inhibited HT-29 cancer cell viability stronger than PG or PL EtOAc extract. FACS analysis revealed that PGP EtOAc extract increased sub-G1 (apoptotic) HT-29 cell population. The nuclear structures of HT-29 cells were disrupted, resulting in apoptotic fragments (45 kDa) of lamin B after treating PGP EtOAc extract. PGP EtOAc extract induced activation of caspase-8 and -9 in HT-29 cells. These results suggest that the PGP EtOAc extract has a direct anti-cancer effect through apoptosis and cell cycle blockade in HT-29 human colon carcinoma cells.

Receptor Fragments: Intracellular Signaling and Novel Therapeutic Targets

Many conventional GPCRs such as those associated with apelin, endothelin, prostaglandin E2, and angiotensin have also been localized to the intracellular space, principally the nucleus. These observations have involved a broad range of tissues, isolated primary cells, and cell lines and a variety of techniques including confocal microscopy, immunohistochemistry, immunocytochemistry, and western blotting. Some receptors are transported to nucleus as holoreceptors while other receptors have been shown to be cleaved with only a portion of the receptor trafficking to nucleus. Several studies from many different laboratories indicate that, depending on the cell type, the angiotensin II type 1 receptor can exist in nuclear membrane or nucleosol and that nuclear accumulation can be induced by ligand-treatment. Moreover, a population of the angiotensin receptor is cleaved in response to angiotensin II and the cytoplasmic carboxyterminal fragment trafficks to nucleus and is a potent apoptotic reagent. In this review, we discuss AT1R cleavage in light of several other receptor cleavage events which similarly produce apoptotic fragments; functionally active intracellular cleavage fragments represent novel targets for drug development.

Antitumor Activity of Extracts from Oxytropis falcata Bunge on human Hepatocellular Carcinoma SMMC-7721 Cells In Vitro and in H22 Bearing Mice In Vivo

Oxytropis falcata Bunge is widely used as one of the “three anti-inflamatory drugs” and is considered to be the “king of herbs” in Chinese Tibetan medicine. For thousands of years it has been used to treat inflammation, pyreticosis and bleeding [1]. In the present study, we investigated the antitumor effects of O. falcata extracts on human hepatocellular carcinoma SMMC-7721 cells in vitro and in transplanted murine H22 tumors in vivo. Cell proliferation, morphological changes, cell cycle distribution and apoptosis in SMMC-7721 cells were determined and tumor growth inhibition in H22 tumors was investigated. MTT assay revealed that essential oil fraction (EOOF) and total flavonoids fraction (TFOF) of O. falcata inhibited proliferation of SMMC-7721 cells in a dose-dependent manner. Growth inhibition in H22 solid tumors was significantly pronounced after being treated with TFOF. Hoechst 33258 staining of SMMC-7721 cells indicated that TFOF caused typical characteristics of apoptotic programmed cell death, such as cell shrinkage, apoptotic body formation and nuclear fragmentation. Cell cycle distribution was analyzed by flow cytometry with propidium iodide (PI) and annexin V-FITC/PI double staining that significant solid tumor inhibition occurred in H22 transplanted mice, cell cycle was arrested at G1 phase, and induction of apoptosis occurred in SMMC-7721 cells when subjected to TFOF. These results suggest that TFOF promotes apoptosis in SMMC-7721 cells and inhibits H22 tumor growth, resulting in a potential antitumor effect on hepatic cancer. Acknowledgements: Support for this research from the National Natural Science Foundation of China for Youth (Grant No.30902012) is gratefully acknowledged.

Innate and adaptive immune responses to dead and dying cells

Under steady state conditions, billions of dead and dying cells are removed by extrusion from epithelial surfaces as well as by phagocytosis. Cells such as macrophages and dendritic cells have specialized receptors that directly recognize altered protein or lipids on apoptotic cells or opsonins that bind to the dying cell. Once engulfed, phagosomes containing apoptotic cells are rapidly acidified and the contents degraded by proteases and nucleases in lysozymes. During necrosis, cellular material is released prior to engulfment and extracellular nucleases as well as intracellular sensors dictate the inflammatory potential of the cellular debris. The outcome may be release of TNF-a, IL-1-b or interferon (IFN)-a depending upon the type of phagocyte, molecular nature of the cellular particle and the intracellular sensor engaged. In addition to responses by cells of the innate immune system, we have recently defined a link between processing of apoptotic cells and their debris to T cell activation [1]. MFG-E8 is an opsonin (bridging protein) that binds to phosphatidylserine on apoptotic cells and facilitates their removal through interaction with integrins on phagocytes. Mice deficient in MFG-E8 develop lupus like autoimmunity associated with accumulation of apoptotic cells in vivo. We observed that older MFG-8-/- mice spontaneously developed a dermatitis associated with CD8 T cell infiltration and striking activation of effector memory CD8 T cells. T cell responses to both exogenous and endogenous apoptotic cell associated antigens were enhanced in MFG-E8 deficient mice and transfer of ovalbumin (OVA) reactive OT-I CD8 T cells caused accelerated diabetes in MFG-E8-/- RIP-mOVA mice and skin disease in kmOVA transgenic mice. The enhanced CD8 T cell response was attributed to increased cross-presentation by dendritic cells (DCs) associated with increased detection of antigen peptide MHCI complexes. Investigation of intracellular trafficking revealed that, whereas intact apoptotic cells ingested by wild type DC rapidly fused with lysosomes, in the absence of MFG-E8, smaller apoptotic cell fragments persisted in endosomal compartments and failed to fuse with lysosomes. These observations suggest that in addition to altering the rate of clearance of apoptotic cells, MFG-E8 deficiency promotes immune responses to self antigens by altered intracellular processing leading to enhanced antigen presentation. Thus, handling of dead and dying cells impacts both innate and adaptive immune responses to self antigens.

EXTRACELLULAR DNA AND THE LEVEL OF ITS METHYLATION IN DIFFERENT RHEUMATIC DISEASES

Система комплексной поддержки и сопровождения научного журнала Elpub позволяет запустить двуязычный сайт журнала со встроенной системой электронной редакции в соответствии с лучшими издательскими практиками, а так же предусматривает техническую и методическую поддержку со стороны специалистов НЭИКОН.

OC-33 Non-invasive diagnosis of non-alcoholic steatohepatitis by combined cytokeratin-18 apoptotic fragment and elastometry

OC-33 Non-invasive diagnosis of non-alcoholic steatohepatitis by combined cytokeratin-18 apoptotic fragment and elastometry

Evaluation of antiobesity and cardioprotective effect of Gymnema sylvestre extract in murine model

Objective:Obesity plays a central role in the insulin resistance syndrome, which is associated with hyperinsulinemia, hypertension, hyperlipidemia, type 2 diabetes mellitus, and an increased risk of atherosclerotic cardiovascular disease. The present study was done to assess the effect of Gymnema sylvestre extract (GSE) in the high fat diet (HFD)-induced cellular obesity and cardiac damage in Wistar rats.Materials and Methods:Adult male Wistar rats (150–200 g body weight) were used in this study. HFD was used to induce obesity. Body mass index, hemodynamic parameters, serum leptin, insulin, glucose, lipids, apolipoprotein levels, myocardial apoptosis, and antioxidant enzymes were assessed. Organ and visceral fat pad weights and histopathological studies were also carried out.Results:Oral feeding of HFD (20 g/day) for a period of 28 days resulted in a significant increase in body mass index, organ weights, visceral fat pad weight, cardiac caspase-3, cardiac DNA laddering (indicating apoptotic inter-nucleosomal DNA fragment), and lipid peroxide levels of cardiac tissues of rats. Further, mean arterial blood pressure, heart rate, serum leptin, insulin, LDH, LDL-C, total cholesterol, triglycerides, and apolipoprotein-B levels were enhanced significantly, whereas serum HDL-C, apoliporotein-A1 levels, and cardiac Na+ K+ ATPase, antioxidant enzymes levels were significantly decreased. Furthermore, treatment with standardized ethanolic GSE (200 m/kg/p.o.) for a period of 28 days resulted in significant reversal of above mentioned changes in the obese Wistar rats.Conclusion:The present study has demonstrated the significant antiobesity potential of GSE in murine model of obesity.

Caspase-mediated apoptosis of trophoblasts in term human placental villi is restricted to cytotrophoblasts and absent from the multinucleated syncytiotrophoblast

Human placental villi are surfaced by a multinucleated and terminally differentiated epithelium, the syncytiotrophoblast, with a subjacent layer of mononucleated cytotrophoblasts that can divide and fuse to replenish the syncytiotrophoblast. The objectives of this study were i) to develop an approach to definitively identify and distinguish cytotrophoblasts from the syncytiotrophoblast, ii) to unambiguously determine the relative susceptibility of villous cytotrophoblasts and syncytiotrophoblast to constitutive and stress-induced apoptosis mediated by caspases, and iii) to understand the progression of apoptosis in villous trophoblasts. Confocal microscopy with co-staining for E-cadherin and DNA allowed us to clearly distinguish the syncytiotrophoblast from cytotrophoblasts and identified that many cytotrophoblasts are deeply interdigitated into the syncytiotrophoblast. Staining for specific markers of caspase-mediated apoptosis indicate that apoptosis occurs readily in cytotrophoblasts but is remarkably inhibited in the syncytiotrophoblast. To determine if an apoptotic cell or cell fragment was from a cytotrophoblast or syncytiotrophoblast, we found co-staining with E-cadherin along with a marker for apoptosis was essential: in the absence of E-cadherin staining, apoptotic cytotrophoblasts would easily be mistaken as representing localized regions of apoptosis in the syncytiotrophoblast. Regions with perivillous fibrin-containing fibrinoid contain the remnants of trophoblast apoptosis, and we propose this apoptosis occurs only after physical isolation of a region of the syncytium from the main body of the syncytium. We propose models for the progression of apoptosis in villous cytotrophoblasts and for why caspase-mediated apoptosis does not occur within the syncytium of placental villi.

Withaferin A-Induced Apoptosis in Human Breast Cancer Cells Is Mediated by Reactive Oxygen Species

Withaferin A (WA), a promising anticancer constituent of Ayurvedic medicinal plant Withania somnifera, inhibits growth of MDA-MB-231 and MCF-7 human breast cancer cells in culture and MDA-MB-231 xenografts in vivo in association with apoptosis induction, but the mechanism of cell death is not fully understood. We now demonstrate, for the first time, that WA-induced apoptosis is mediated by reactive oxygen species (ROS) production due to inhibition of mitochondrial respiration. WA treatment caused ROS production in MDA-MB-231 and MCF-7 cells, but not in a normal human mammary epithelial cell line (HMEC). The HMEC was also resistant to WA-induced apoptosis. WA-mediated ROS production as well as apoptotic histone-associated DNA fragment release into the cytosol was significantly attenuated by ectopic expression of Cu,Zn-superoxide dismutase in both MDA-MB-231 and MCF-7 cells. ROS production resulting from WA exposure was accompanied by inhibition of oxidative phosphorylation and inhibition of complex III activity. Mitochondrial DNA-deficient Rho-0 variants of MDA-MB-231 and MCF-7 cells were resistant to WA-induced ROS production, collapse of mitochondrial membrane potential, and apoptosis compared with respective wild-type cells. WA treatment resulted in activation of Bax and Bak in MDA-MB-231 and MCF-7 cells, and SV40 immortalized embryonic fibroblasts derived from Bax and Bak double knockout mouse were significantly more resistant to WA-induced apoptosis compared with fibroblasts derived from wild-type mouse. In conclusion, the present study provides novel insight into the molecular circuitry of WA-induced apoptosis involving ROS production and activation of Bax/Bak.

Antitumor and apoptosis-promoting properties of emodin, an anthraquinone derivative from Rheum officinale Baill, against pancreatic cancer in mice via inhibition of Akt activation

Pancreatic adenocarcinoma is one of the most common malignancies worldwide. Gemcitabine is currently the standard first-line chemotherapeutic agent for pancreatic cancer. However, gemcitabine can induce activation of Akt and nuclear factor-κB (NF-κB), which is associated with its chemoresistance. It has been reported that gemcitabine combination therapies result in improved survival outcomes in pancreatic cancer. Therefore, agents that can either enhance the effects of gemcitabine or overcome chemoresistance to the drug are needed for the treatment of pancreatic cancer. Emodin is an active component of Chinese medicinal herbs and can inhibit the activation of Akt and NF-κB. In this study, we investigated whether emodin could enhance the anticancer effect of gemcitabine on pancreatic cancer in vivo. We demonstrated that treatment of gemcitabine combined with emodin efficiently suppressed tumor growth in mice inoculated with pancreatic tumor cells. This treatment paradigm promoted apoptotic cell death and mitochondrial fragmentation. Furthermore, it reduced phosphorylated-Akt (p-Akt) level, NF-κB activation and Bcl-2/Bax ratio, increased caspase-9 and -3 activation, Cytochrome C (CytC) release occurred in combination therapy. Collectively, emodin enhanced the activity of gemcitabine in tumor growth suppression via inhibition of Akt and NF-κB activation, thus promoting the mitochondrial-dependent apoptotic pathway. Therefore, our findings may provide new insights into understanding the pharmacological regulation of emodin on gemcitabine-mediated proapoptosis in pancreatic cancer and may aid in the design of new therapeutic strategies for the intervention of human pancreatic cancers.

Synergistic Apoptosis-Inducing Antileukemic Effects of Arsenic Trioxide andMucuna macrocarpaStem Extract in Human Leukemic Cells via a Reactive Oxygen Species-Dependent Mechanism

The objective of this study was to examine the potential of enhancing the antileukemic activity of arsenic trioxide (ATO) by combining it with a folk remedy, crude methanolic extract of Mucuna macrocarpa (CMEMM). Human leukemia cells HL-60, Jurkat, and Molt-3 were treated with various doses of ATO, CMEMM, and combinations thereof for 24 and 48 h. Results indicated that the combination of 2.5 μM ATO and 50 μg/mL CMEMM synergistically inhibited cell proliferation in HL-60 and Jurkat cell lines. Apoptosis triggered by ATO/CMEMM treatment was confirmed by accumulation of cells in the sub-G1 phase in cell cycle analyses, characteristic apoptotic nuclear fragmentation, and increased percentage of annexin V-positive apoptotic cells. Such combination treatments also led to elevation of reactive oxygen species (ROS). The antioxidants N-acetyl cysteine (NAC), butylated hydroxytoluene, and α-tocopherol prevented cells from ATO/CMEMM-induced apoptosis. The ATO/CMEMM-induced activation of caspase-3 and caspase-9 can be blocked by NAC. In summary, these results suggest that ATO/CMEMM combination treatment exerts synergistic apoptosis-inducing effects in human leukemic cells through a ROS-dependent mechanism and may provide a promising antileukemic approach in the future.

Sonodynamic Effect of an Anti-Inflammatory Agent – Emodin on Macrophages

Emodin has been used as an anti-inflammatory agent and inflammation is a crucial feature of atherosclerosis. Here, we investigated the sonodynamic effect of emodin on macrophages, the pivotal inflammatory cells in atherosclerotic plaque. THP-1 derived macrophages were cultured with emodin and exposed to ultrasound. Six hours later, unlike the cells treated for 5 and 10 min, the viability of cells treated for 15 min decreased significantly and the cells showed typical apoptotic chromatin fragmentation. The percentage of apoptotic and necrotic cells in the sonodynamic therapy (SDT) group was higher than that in the ultrasound group. Two hours after treatment for 15 min, the cytoskeleton lost its original features as the filaments dispersed and the cytoskeletal proteins aggregated. The percentage of cells with disturbed cytoskeletal filaments in the SDT group was higher than that in the ultrasound group. These results suggest emodin has a sonodynamic effect on macrophages and might be used as a novel sonosensitizer for SDT for atherosclerosis.

Phloroglucinol (PG) purified from Ecklonia cava attenuates radiation-induced apoptosis in blood lymphocytes and splenocytes

Phloroglucinol (PG), a polyphenol compound of Eckloniacava known as brown algae abundant in Jeju island, has been proposed to exert the antioxidative and cytoprotective effects against oxidative stress. In this study, we confirmed that PG protected mice from damages caused by ionizing radiation and investigated its protection mechanism in detail. The result showed that PG increased the viability of splenocytes without cytotoxicity. Also, PG significantly enhanced the proliferation of splenocytes by limiting the increment of sub-G1 DNA contents via the inhibition of reactive oxygen species production in 2Gy-irradiated splenocytes. In addition, PG significantly decreased DNA damage and the number of apoptotic fragments in lymphocytes against oxidative stress. Also, PG increased the counts of endogenous spleen CFUs, compared with only ionizing radiation-irradiated mice. These results demonstrate the multi-faceted protection mechanisms of PG in mice against oxidative stress caused by ionizing radiation, providing the benefit of inhibiting apoptosis and strengthening hematopoiesis.

UVB를 조사한 HaCaT 세포의 세포사멸과 p53 및 GADD45 유전자 발현에 대한 파프리카 추출물 및 성분들의 효과

In the present study, the effects of paprika extract and its components including vitamin C, lycopene and beta-carotene on cell death in ultraviolet B (UVB)-exposed HaCaT cells were investigated. The cell viability upon treatment for 24 hr with either paprika extract or vitamin C alone was similar to or greater than that of the untreated control. However, the viability of the cells treated with lycopene or beta-carotene decreased to about 20% of that in the untreated control. When UVB-exposed cells were post-incubated for 24 hr in medium containing paprika extract or vitamin C, cell viability increased in a concentration dependent manner as compared to those post-incubated in a normal growth medium. In contrast, post-incubation of UVB-exposed cells with lycopene or beta-carotene decreased cell viability in a concentration dependent manner as compared to those post-incubated in a normal growth medium. The nuclear fragmentation analysis showed that paprika extract or vitamin C decreases UVB-induced apoptosis. The apoptotic nuclear fragmentation resulting from UVB exposure was also protected by the paprika extract or vitamin C post-incubation. However, the UVB-induced apoptotic nuclear fragmentation of the cells treated with lycopene or beta-carotene increased in a concentration dependent manner. Western blot analysis showed that either paprika extract or vitamin C treatment alone did not significantly change the level of p53 and GADD45 protein. Interestingly, post-incubation of UVB-exposed cells with paprika extract or vitamin C decreased the p53 and GADD45 protein level as compared to those post-incubated in a normal growth medium. In contrast, incubation of UVB-exposed or non-irradiated cells with lycopene or beta-carotene increased the p53 and GADD45 protein levels in a concentration dependent manner as compared to those incubated in a normal growth medium. All these results suggest that paprika extract and vitamin C help the survival of the UVB-exposed cells, while lycopene and beta-carotene potentiate the apoptotic death of UVB-exposed cells, in accordance with the respective changes in p53 and GADD45 protein levels.

Abstract 3235: Improved in vitro efficacy study of RGD grafted docetaxel encapsulated solid lipid nanoparticles on breast cancer cells

Abstract DC- encapsulated solid lipid nanoparticles (SLN) were prepared using Supercritical fluid technology using CO2 as an anti-solvent. RGD-conjugation was done by carbodiimide coupling method. The SLN were characterized for particle size and zeta potential by zeta sizer (Nano-ZS), entrapment efficiency and in-vitro drug release profile using HPLC assay, and RGD-conjugation efficiency by amino acid analysis method. The possibility of drug-lipid -RGD interaction was ascertained using a differential scanning colorimetry and X-ray diffraction techniques. To establish antiproliferative effect, the RGD-conjugated SLN of DC(RGD-SLN-DC), unconjugated SLN(DC-SLN) and equal amounts of the free drug were studied on MDA-MB-231 cells by CCK-8 assay method. DNA content analysis was done on flowcytometry by propadium iodide staining. The mode of cell death at different time and concentration was determined by FITC-Annexine V assay. Results: The DC encapsulated RGD conjugated and unconjugated SLN were found to be nanosized with optimum drug entrapment. The in-vitro release profiles of unconjugated and conjugated SLN were found to be similar except the fast drug release was observed in case of RGD conjugated pegylated SLN due to the fast hydration process of PEG molecules on the surface of the particles. The cytotoxicity indicated by IC50 values suggests that RGD conjugated SLN at 72hrs are 2.1 and 6.3 times more cytotoxic than unconjugated SLN and drug solution for for MDA-MB-231 cells. The results shows that the RGD conjugated SLN at the concentration of 4nM showed 97% G2 phase arrest as compared to 65% G2 phase arrest with unconjugated SLN at the same concentration at 48hrs. Two types of mode of cell death were found during the FITC-Annexin V assay. At 48 hrs, the treatment of 4nM of drug solution with cells resulted in 23.5% &amp; 2.8 % necrotic &amp; apoptotic cell fragments respectively. While at similar drug equivalent concentration, the RGD-SLN-DC and DC-SLN showed 2.9% &amp; 76.3% and 3.4% &amp; 42.3% necrotic &amp; apoptotic cell fragments respectively. With increase in time and concentration the mode of cell death by apoptosis was found to be increasing. Conclusions: To conclude, RGD conjugation to SLN improved antiproliferative activity when assessed in vitro in breast cancer cells compared to free drug and unconjugated SLN. The DNA content analysis depicted the cell cycle arrest in G2 phase was more even at lower drug equivalent concentration of RGD-SLN-DC. The mode of cell death was found to be mainly by necrosis at low drug equivalent concentration (1nM) and by apoptosis at high drug equivalent concentration (10nM) of RGD conjugated drug encapsulated SLN for breast cancer cells. Hence, it may be concluded that drug nanocarriers once conjugated with RGD can provide prolonged drug release to cytoplasm and can affectively target breast cancer and can probably reduce the limitations associated with breast cancer chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3235. doi:10.1158/1538-7445.AM2011-3235

Abstract 2083: The stress oncoprotein LEDGF/p75 attenuates oxidative stress-induced necrosis but not apoptosis in prostate cancer cells

Abstract The lens epithelium-derived growth factor p75 (LEDGF/p75) is emerging as a stress response oncoprotein in human cancer. Our group and others showed previously that this transcription coactivator is overexpressed in major human cancers, including prostate cancer, and protects against lysosomal cell death and chemotherapeutic drugs. Although LEDGF/p75 is considered a novel anti-apoptotic protein due to its ability to attenuate lysosomal cell death, previous studies from our group demonstrated its inactivation by caspases during apoptosis. We hypothesized that LEDGF/p75 promotes resistance to stress-induced caspase-independent cell death, but not to apoptosis, in cancer cells. To test this hypothesis, we established PC3 and RWPE-2 prostate cancer cell lines stably overexpressing LEDGF/p75, and evaluated its ability to protect these cells against non-apoptotic and apoptotic insults. LEDGF/p75 significantly attenuated cytotoxicity induced by tert-butyl hydroperoxide (TBHP), a strong inducer of oxidative stress. Characterization of TBHP-induced cell death showed that it was caspase-independent since it could not be inhibited with the broad caspase inhibitor z-VAD-fmk and did not involve caspase-3 activation. Furthermore, TBHP-treated cells displayed a necrotic morphology characterized by extensive cytoplasmic fragmentation and shrunken, non-fragmented nuclei. Necrosis was also confirmed by the appearance in immunoblots of a 45 kD cleavage fragment of DNA topoisomerase that was previously established by our group as a marker of necrosis. Overexpression of LEDGF/p75 attenuated the generation of reactive oxygen species (ROS) in TBHP-treated cells. Interestingly, LEDGF/p75 also attenuated ROS in cells treated with the classical apoptosis inducers staurosporine and TRAIL, but failed to protect against these apoptotic insults. These results indicated that the ability of LEDGF/p75 to attenuate ROS is not sufficient for its pro-survival function. Analysis of LEDGF/p75 integrity by immunoblotting analysis showed that the protein remained intact in cells treated with TBHP, but was cleaved into its signature 65 kd apoptotic fragment in cells treated with staurosporine and TRAIL. Our group has shown previously that this caspase-generated fragment lacks pro-survival activity. Taken together, these results strongly suggest that overexpression of LEDGF/p75 can protect tumor cells from caspase-independent cell death processes in which this protein remains structurally intact. This study provides the basis for investigating the contribution of LEDGF/p75 to prostate cancer resistance to therapeutic modalities that induce caspase-independent cell death, as well as its potential as a novel therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2083. doi:10.1158/1538-7445.AM2011-2083

Increased expression of apoptotic genes in cancer cells by heat-processed crude saponin

Heat treatment of Panax ginseng C.A. Meyer at a temperature higher than that applied to the conventional preparation of Korea ginseng has been reported to yield a mixture of saponins with potent cytotoxic properties. In an effort to understand the mechanism of cytotoxicity, we studied the effect of heat-processed crude saponin (H-CS) on the viability, nuclear morphology and apoptotic gene induction of human colorectal carcinoma cells (HCT-15 cells) and human glioma carcinoma cells (U87MG). Treatment of the carcinoma cells with a final concentration of up to 1 mg/mL of H-CS resulted in a dose-dependent decrease in viability. In comparison with non-heatprocessed crude saponin (NH-CS), H-CS exhibited increased cytotoxicity. In addition, H-CS induced more apoptotic nuclear fragments in HCT-15 cells than in NH-CS. An analysis of apoptotic gene expression using quantitative real-time PCR revealed high expression levels of the genes encoding caspase 7, caspase 9, cytochrome C, and Bax at RQ values of 26.87, 17.39, 4.55, and 8.51, respectively. Collectively, our results suggest that H-CS has higher anti-proliferative effects on carcinoma cells by inducing apoptosis of the mitochondrial pathway.