Atif M. El-Naggar

Anticancer activities of selected species of North American lichen extracts.

Cancer is the second leading cause of human deaths in the USA. Despite continuous efforts to treat cancer over the past 50 years, human mortality rates have not decreased significantly. Natural products, such as lichens, have been good sources of anticancer drugs. This study reports the cytotoxic activity of crude extracts of 17 lichen species against Burkitts lymphoma (Raji) cells. Out of the 17 lichen species, extracts from 14 species showed cytotoxicity against Raji cells. On the basis of IC50 values, we selected Xanthoparmelia chlorochroa and Tuckermannopsis ciliaris to study the mechanism of cell death. Viability of normal lymphocytes was not affected by the extracts of X. chlorochroa and T. ciliaris. We found that extracts from both lichens decreased proliferation, accumulated cells at the G0/G1 stage, and caused apoptosis in a dose‐dependent manner. Both lichen extracts also caused upregulation of p53. The T. ciliaris extract upregulated the expression of TK1 but X. chlorochroa did not. We also found that usnic, salazinic, constictic, and norstictic acids were present in the extract of X. chlorochroa, whereas protolichesterinic acid in T. ciliaris extracts. Our data demonstrate that lichen extracts merit further research as a potential source of anticancer drugs. Copyright

Investigating the role of macrophages in tumor formation using a MaFIA mouse model

Tumor-associated macrophages (TAMs) interact with tumors in their development, growth and metastatic activities. Using a transgenic mouse model that allows for the selective depletion of macrophages we were able to access the macrophages potential to facilitate metastasis. In the MaFIA (Macrophage Fas-Induced Apoptosis) mouse, transgene-expressing cells of the myeloid lineage undergo death by apoptosis in the presence of the drug AP20187. Enhanced green fluorescent protein (EGFP) was fused to the suicide gene to allow identification of transgene-expressing cells. Tumor induction was accomplished by subdermal and intravenous injections of B16-F10 melanoma cells. Metastasis in mice with depleted macrophages was compared to metastasis in normal control mice. The lungs and kidneys were examined for metastatic cells. The macrophage-depleted groups showed significantly less metastasis (P>0.001) compared to the control groups. We theorize that macrophages may aid the metastatic process by fusing with melanoma cells. Using appropriate cell markers and fluorescence-activated cell sorting, we were able to detect a small population of double-positive cells. We confirmed cell fusion by microscopic analysis, visualizing the cells morphology by both immunohistochemistry and immunofluorescence. The presence of double-positive cells suggests macrophage/cancer cell fusion could be a possible mechanism for metastasis.

DNA damage caused by inorganic particulate matter on Raji and HepG2 cell lines exposed to ultraviolet radiation

Epidemiological studies have correlated exposure to ultraviolet-irradiated particulate matter with cardiovascular, respiratory, and lung diseases. This study investigated the DNA damage induced by two major inorganic particulate matter compounds found in diesel exhaust, ammonium nitrate and ammonium sulfate, on Burkitts lymphoma (Raji) and hepatocellular carcinoma (HepG2) cell lines. We found a dose-dependent positive correlation of accumulated DNA damage at concentrations of ammonium nitrate (25 μg/ml, 50 μg/ml, 100 μg/ml, 200 μg/ml, 400 μg/ml) with ultraviolet exposure (250 J/m(2), 400 J/m(2), 600 J/m(2), 850 J/m(2)), as measured by the comet assay in both cell lines. There was a significant difference between the treated ammonium nitrate samples and negative control samples in Raji and HepG2 cells (p<0.001). Apoptosis was shown in Raji and HepG2 cells when exposed to high concentrations of ammonium nitrate (200 μg/ml and 400 μg/ml) for 1h in samples without ultraviolet exposure, as assessed by the comet assay. However, the level of apoptosis greatly diminished after ultraviolet exposure at these concentrations. Over a 24h period, at intervals of 1, 4, 8, 12, 18, and 24h, we also observed that ammonium nitrate decreased viability in Raji and HepG2 cell lines and inhibited cell growth. Ammonium sulfate-induced DNA damage was minimal in both cell lines, but there remained a significant difference (p<0.05) between the ultraviolet radiation treated and negative control samples. These results indicate that the inorganic particulate compound, ammonium nitrate, induced DNA strand breaks at all concentrations, and indications of apoptosis at high concentrations in Raji and HepG2 cells, with ultraviolet radiation preventing apoptosis at high concentrations. We hypothesize that ultraviolet radiation may inhibit an essential cellular mechanism, possibly involving p53, thereby explaining this phenomenon. Further studies are necessary to characterize the roles of apoptosis inhibition induced by DNA damage caused by inorganic particulate matter.

The morphology and histopathology of Nephridiacanthus major (Acanthocephala: Oligacanthorhynchidae) from hedgehogs in Iran

The morphology of Nephridiacanthus major (Bremser 1811 in Westrumb 1821) Golvan, 1962 collected from the long-eared hedgehog Hemiechinus auritus (Gmelin 1770) and the Eastern European hedgehog Erinaceus concolor Martin, 1838 (Erinaceidae) is described using SEM for the first time. This acanthocephalan was previously described from hedgehogs in Europe, Asia, and Africa. Measurements of specimens from Iran, Bulgaria, Germany, Central Asia, Morocco, and Egypt show considerable variations in the size of the trunk, proboscis, proboscis hooks and receptacle, and eggs. The SEM studies add new perspectives to its morphology. Features observed for the first time include the near terminal position and shape of the female gonopore and orifice, among others. Histopathological studies for this species are reported for the first time. Tissue sections show extensive damage near the proboscis with hemorrhaging and formation of collagenous connective tissue, compression of the intestinal mucosa, obstruction of intestinal lumen, and extensive necrosis of host epithelial tissue.

Abstract 1790: Mutagenic effects of inorganic particulate matter on Raji and HepG2 cell lines exposed to ultraviolet radiation.

Epidemiological studies have correlated exposure to ultraviolet-irradiated particulate matter with cardiovascular, respiratory, and malignant lung diseases. This study investigated the DNA damage induced by two major inorganic particulate matter compounds found in diesel exhaust, ammonium nitrate and ammonium sulfate on Burkitt9s lymphoma (Raji) and hepatocellular carcinoma (HepG2) cell lines. We found a dose-dependent positive correlation of accumulated DNA damage in concentrations of ammonium nitrate and ammonium sulfate (25 μg/ml, 50 μg/ml, 100 μg/ml, 200 μg/ml, 400 μg/ml) with ultraviolet exposure (250 J/m 2 , 400 J/m 2 , 600 J/m 2 , 850 J/m 2 ), as measured by the Comet Assay in both cell lines. There was a significant difference between the treated ammonium nitrate samples and negative control samples in Raji and HepG2 (p Citation Format: Michael Xiao, Atif El-Naggar, Albert V. Helsing, Philip M. Lynch, Melissa M. Alegre, Richard A. Robison, Kim L. O9Neill. Mutagenic effects of inorganic particulate matter on Raji and HepG2 cell lines exposed to ultraviolet radiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1790. doi:10.1158/1538-7445.AM2013-1790

Abstract 1288: The potential of Resveratrol and Nagami kumquat extracts in facilitating DNA repair in Raji cells.

Cancer is the second biggest killer in the United States. It has long been recognized that people differ in their susceptibility to many different types of cancer. Some of these differences are the heritable traits that modify the effects of environmental exposures. Genetic instability, which drives tumorigenesis, is itself fuelled by DNA damage and often by errors made by the DNA repair machinery. DNA repair, using natural sources of antioxidants, is a new developing area of cancer prevention. The aim of the current study was to investigate DNA repair capacity and DNA damage of Burkitt9s lymphoma (Raji) cells exposed to Resveratrol and different extracts of Nagami kumquat (Fortunella margarita). Nagami kumquat is known as “the little gold gem of the citrus family,” and its pulp has been recently highlighted as one containing the most potent antioxidant and high phenolic contents among citrus fruits. Kumquat skin had the highest vitamin C content (3,069.183 μg/g) when compared to both the seed (506.14 μg/g) and pulp (2,440.448 μg/g). Recent evidence has shown that diets rich in antioxidants, such as those including kumquats, may be effective in lowering an individual9s risk for cancer. We measured the DNA repair rate of cells by the Comet Assay, taken at several time points throughout the course of 1 hour. DNA damage was induced by exposing the cells to 4mM H2O2 for 15 minutes. This damage was then measured by the comet assay. We measured the amount of DNA damage at 0, 6, 15, 30 and 60 minutes, in order to determine the DNA repair rate. The results were analyzed with Tri-Tek Comet Score Software Version 1.5. The DNA Repair after one hour, using the kumquat Pulp was highly significant (P Citation Format: Albert V. Helsing, Atif El-Naggar, Philip M. Lynch, Michael Xiao, Madison Ramsden, B Fabrizio Alegre, Richard A. Robison, Kim L. O9Neill. The potential of Resveratrol and Nagami kumquat extracts in facilitating DNA repair in Raji cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1288. doi:10.1158/1538-7445.AM2013-1288

Abstract 4775: Compound S2AMN: A potential non-toxic anti-cancer therapy

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL S2AMN is a natural compound; garlic, onion and green pepper extracts that demonstrate apoptotic potential against a variety of cancer cell lines. In this study, we examined specifically whether treatment with S2AMN would affect the viability of several human cell lines, including Burkitts lymphoma (Raji cell), melanoma cell lines (MDA-MB-435), human breast adenocarcinoma cell line (MCF-7) and human colon carcinoma (HT29). It was determined by flow cytometry that S2AMN works by arresting cell-cycle progression in the G1 phase. Using RT-PCR, the p53 gene was shown to be up regulated in the cells during G1 phase, suggesting that S2AMN is p53 dependent. Using DNA extraction and agarose gel electrophoresis the DNA of cells treated with S2AMN was found partially degraded after 24 hours and completely degraded after 48 hours. Viability analysis showed a dose-dependent growth curve; 24 hours after treatment there was a marked decrease in viability and within 48 hours there was complete cell death (control viability > 94%). Results from viability analyses supported the same conclusion as the DNA electrophoresis data, namely that within 48 hours there was complete DNA degradation and apoptosis. We also examined the proliferation rate at 24 and 48 hours, and found that proliferation decreased at 24 hours and completely stopped at 48 hours. One of the more striking characteristics of S2AMN is its cancer-specific cytotoxicity. After treating human lymphocytes induced with PHA (normal cells) with the same concentrations of S2AMN as shown above, cell viability, proliferation, cell cycle flow cytometry, and RT-PCR experiments were performed. Results showed that there was no significant change between treatment and control cells. In conclusion, S2AMN seems to be selectively cytotoxic by inducing apoptosis in cancer cells but not in normal cells. S2AMN could potentially be used as a non-toxic anti-cancer therapy, though more research is necessary. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4775. doi:1538-7445.AM2012-4775