Expression, Down-regulation and Function of CCRL2 on Three Human Cancer Cell Lines

Abstract C-C chemokine receptor like 2 (CCRL2) is a member of the atypical chemokine receptor family; it is a hepta helical transmembrane receptor, expression of which has been shown on almost all human hematopoietic cells. CCRL2 were previously considered to be orphan receptor and as a receptor presenting its chemo attractant ligand to functional receptors. The function and expression of CCRL2 in cancer is not understood at present. Here, we investigated the expression of CCRL2 as well as the effects on cellular proliferation resulting from their knockdown in three cancer cell lines include: human cerebral glioblastoma multi form (ANGM, at passages 75-84), human cervical cancer (HeLa, at 70 passages), and human pelvic rhabdomyosarcoma (RD, at 75 passages) cell lines. In addition, all cell lines were screened for mRNA expression of CCRL2 by reverse transcription polymerase chain reaction (RT-PCR). Cell lines with detectable expression were used for knockdown experiments; and the respective influence after transfection with small interfering RNA (siRNA) concentrations (2,3,4,5,6,7 and 8) ρmol for (24 , 48 and 72) hour were determined for both CCRL2 gene and the house keeping gene GAPDH as control. The Knockdown of CCRL2 was highly successful; the expression of CCRL2 was down-regulated by over 76.0%, 89.6% and 80.7% after transfection for 48 hour to (ANGM, HeLa and RD) cell lines respectively. The results also indicated that in the CCRL2 absence there was a significant decrease in the cell proliferation, suggesting a pro-tumoral effect of CCRL2. The potential roles of CCRL2 as a novel therapeutic target and biomarker warrant further investigations

The Cytotoxic Effect of 2-Deoxy-D-Glucose Combination with 5-Fluorourasil and NO-Aspirin on Mammary Adenocacinoma Cell Line

2-deoxy-D-glucose (2DG) and NO-Aspirin (NO-ASA) are new anticancer agents that are under intense clinical investigation for their remarkable cytotoxic activity. Combining 2DG, which targets glucose metabolism, with other agents mainly the DNA- and the mitochondrial-damaging agents represent a promising chemotherapeutic strategy.  In this study, we investigated the cytotoxic effects of 2DG, 5-fluorourasil (5FU), and NO-ASA on AMN3 breast cancer model, in addition to the cytotoxic effects of 2DG combination with 5FU and NO-ASA on the same cells. The cytotoxic activity of 2DG, 5FU, and NO-ASA was measured by using the MTT assay at 24, 48, and 72 hr. Then 2DG was combined with 5FU and NO-ASA in a constant concentration ratio based on their corresponding IC50s and the inhibition of cell growth was measured by MTT assay at 72hr. Median effect analysis was conducted to determine the cytotoxic activity of the combinations. 2DG, 5FU, and NO-ASA were found to exert a significant dose- and time-dependant growth inhibition on AMN3 cells. The mean combination index values reveal an additive effect for both combinations. This study demonstrated that 2DG and NO-ASA are capable of inhibiting the breast tumor growth effectively. It also shows that 2DG/5FU and 2DG/NO-ASA combinations result in mean additive effects with good dose reduction index values that have the advantages of reducing the toxicity, adverse effects, and the drug resistance in cancer patients. Key words: 2-deoxy-D-glucose (2DG), NO-Aspirin (NO-ASA), 5-fluorourasil (5FU), Glucose metabolism, Median effect analysis, Breast cance

The effect of aqueous and ethanolic extracts of Artemisia herba alba on human laryngeal carcinoma and murine mammary adenocarcinoma cell lines

The present study was carried out to evaluate the cytological effects of aqueous (AE) and ethanolic (EE) extracts of Artemisia herba alba on human laryngeal carcinoma (Hep-2) cell line and murine mammary adenocarcinoma (AMN-3) cell line in vitro. The cytological study performed simultaneously with cell growth assay. The results of study revealed concentration-dependent cytological changes like patchy growth inhibition, loss of confluent feature and cellular degeneration after exposure to the lowest concentrations (156.25 and 312.5 μg/ml). The early findings of cytolysis were seen after exposure to 625 μg/ml. While the highest concentrations (1250, 2500 and 5000 μg/ml) caused severe growth inhibition with marked cytolytic features including loss of cellular outlines, large numbers of dead cells and high content of cellular debris. In conclusion, the results of this study revealed the high cytological effect of Artemisia herba alba extracts on Hep-2 and AMN-3 cell lines in vitro

In Vitro Cytotoxic Study for Purified Resveratrol Extracted from Grape Skin Fruit Vitis vinifera

This study  was  conducted  with the  aim to  extract and  purify  a  polyphenolic  compound  â€œ Resveratrol†from the skin of black grapes Vitis vinifera cultivated in Iraq. The purified resveratrol is obtained after ethanolic extraction with 80% v/v solution for fresh grape skin, followed by acid hydrolysis   with   10%   HCl   solution then   the  aglycon   moiety   was  taken  with   organic   solvent ( chloroform). Using silica gel G60 packed glass column chromatography with mobile phase benzene: methanol: acetic acid  20:4:1 a partial purified resveratrol was obtained then preperative thin layer chromatography technique yielded pure crystals identified as resveratrol (mixture of two isomers cis and trans) in relation  to resveratrol standard (35 mg resveratrol crystals / 0.5 kg fresh grape skin was obtained as a result of these processes ). The study was also employed an in vitro evaluation the cytotoxic effect of pure resveratrol on some cell line including : the murine mammary adenocarcinoma AMN-3 cell line, the human laryngeal carcinoma (Hep-2) cell line and the Rat embryo fibroblast (Ref) cell line, at different concentrations and different expousure time. The cytotoxic effect of the pure resveratrol was studied in comparison with trans- resveratrol standard in concentrations of (12.5, 25, 50 and 100) µg/ml for both purified resveratrol and the standard, also the comparism included methotrexate drug in concentrations  (0.05 , 0.1 , 0.2 and 0.4)  µg/ml toward the growth effects of the three types of cell lines and at three exposure times (24, 48 and 72) hours. The cytotoxic inhibition effect for the purified extracted resveratrol revealed that the highest significant effect (P<0.01) was achieved after 24 hr of exposure on both AMN-3 and Ref cell lines. Hep-2 cell line responded to extracted resveratrol in different manners. Keywords: Resveratrol , grape skin , polyphenols, cytotoxic stud

PHYSIOLOGICAL AND CYTOGENETICAL STUDY FOR EMPLOYEES AT HIGH VOLTAGE ELECTRIC STATIONS

This research was conducted on 75 blood samples randomly selected from 25 persons directly exposed to electromagnetic field and 25 persons indirectly exposed in addition to 25 persons not exposed to that electromagnetic field(as a control group). The aim of this study is to know the effect of electromagnetic field (resulted from electric station) on some physiological blood parameters, and the genetic materials for workers at these electricity station .The physiological results indicated that there is a significant decrease in the hemoglobin concentration average at the probability P field as compared to control group; there was a decrease in the compact red blood cell volume average, in the total number of white blood cells and the percent of lymphocytes, monocytes and oesinophils, However there was a significant increase in the red blood cell sedimentation ratio and in the neutrophils and basophiles percents .Statistical results did not indicate any significant difference in the blood platelets ratio among the blood sample of the three various groups under study .The cytogenetic tests showed no chromosomal number changes within the samples of the three groups, but there was 14 qesses of structural chromosomal aberrations in the directly exposal group, seven of which as chromosome fragmentation ,one as chromosome deletion ,three as dicentric chromosomes, one as a centric chromosomes and two as ring chromosome as compared to four chromosomal fragmentation in the control group .There was decrease in the cell mitotic index in the persons how are directly exposed to the electromagnetic field while there was an increased effect on the lymphocytes with the advance of age and time exposure.In conclusion, the electromagnetic field has a direct and indirect effect on some physiological blood parameters and the genetic materials through the chromosomal aberration which case disease and deformation

EFFECT OF CRUDE EXTRACTS OF DATE PITS (PHOENIX DACTYLIFERA L. CV. ZAHDI) ON SOME CANCER CELL LINES IN VITRO AND THEIR EFFECT ON HUMAN LYMPHOCYTES AND TREATMENT OF TRANSPLANTED MAMMARY ADENOCARCINOMA IN MICE

The present investigation represents a preliminary study of the effect of crude extracts (aqueous, ethanolic and hexanic) of date pits (Phoenix dactylifera L. cv. Zahdi) on two malignant cell lines (human laryngeal carcinoma-Hep2 and murine mammary adenocarcinoma-AMN3) and one normal cell line (rat embryo fibroblast-REF). The study also includes evaluation of the effect of these extracts on several cytogenetic parameters such as mitotic index (MI%), blast index (BI%) and chromosomal aberrations (CA) after in vitro culture of peripheral blood lymphocytes. This work also includes a study of the therapeutic potential of one of these extracts in the treatment of transplanted murine mammary adenocarcinoma in mice.The in vitro cell growth assay showed that there was time- and concentration-dependent cytotoxic effects of crude extracts of date pits on Hep2 and AMN3 cell lines. The highest significant effect of these extracts was achieved after 72 hrs of exposure with the highest concentration (10000 μg/ml). Ethanolic extract of pits (EP) caused growth inhibition percentage 89.4% , 93.4% for Hep2 and AMN3 respectively. However, 72 hrs exposure to EP at concentration of 10000 μg/ml caused slight inhibitory effect on REF cell line, reaching 17.7%.On the other hand, the crude extracts of pits caused significant reduction in the mitotic index and blast index of peripheral human lymphocytes, but without any structural or numerical chromosomal aberrations. Also these extracts neither replaced phytohemagglutinin (PHA) as mitogenic agent, nor colcemide as mitotic arresting agent at metaphase.The therapeutic doses of EP were determined according to LD50 in mice. The results indicated high effectiveness of this extract in a dose- and time-dependent manner. The highest therapeutic doses of EP (1 gm/kg B.wt.) showed the best therapeutic effect by reducing the tumor volume in mice to about 83.8%.The comparison of relative tumor volumes of different groups revealed highly significant differences among all treated groups and those of untreated (control) group

CYTOTOXIC EFFECT OF SYNTHESIZED SILVER NANOPARTICLES BY PULSE LASER ABLATION ON BREAST CANCER CELL LINE (AMJ13) FROM IRAQI PATIENT AND NORMAL HUMAN LYMPHOCYTES

 Objective: Synthesized silver nanoparticles (AgNPs) in liquids were investigated as anticancer cells in the present study. Cytotoxic activities of six different concentrations 0.78, 1.56, 3.125, 6.25, 12.5, and 25 μg/ml of AgNPs against human breast cancer cell line (AMJ13) and lymphocytes were assessed with MTT assay.Methods: A Q-Switched Nd: YAG pulsed laser (λ=1064 nm, 800 mJ/pulse) was used for ablation of a pure silver plate to synthesis AgNPs in the polyvinylpyrrolidone and deionize distilled water. Ultraviolet-visible spectroscopy confirmed the synthesis of AgNPs and zeta potential was evaluated. Morphology and size were analyzed by transmission electron microscope. AgNPs concentrations were determined by atomic absorption spectroscopy. Possibilities of apoptosis induction were confirmed using mitochondrial membrane potential assay, DNA fragmentation assay, and glutathione (GSH) assay.Results: The results indicated that AgNPs were able to induce an inhibition of AMJ13 cells compared their damaging effect toward normal lymphocytes were at minimal according to viability with MTT assay.. Furthermore, these results suggested that AgNPs-induced mitochondrial-mediated apoptosis cause DNA fragmentation, but no significant change in GSH level in AMJ13 cells.Conclusions: The overall results indicated that the physically synthesized AgNPs were exhibited dose-dependent cell death in AMJ13 breast cancer cell line, while the effect of AgNPs on lymphocytes was very low, suggesting that physically synthesized AgNPs might be a potential alternative agent for human breast cancer therapy

CYTOTOXIC EFFECT OF SYNTHESIZED SILVER NANOPARTICLES BY PULSE LASER ABLATION ON BREAST CANCER CELL LINE (AMJ13) FROM IRAQI PATIENT AND NORMAL HUMAN LYMPHOCYTES

 Objective: Synthesized silver nanoparticles (AgNPs) in liquids were investigated as anticancer cells in the present study. Cytotoxic activities of six different concentrations 0.78, 1.56, 3.125, 6.25, 12.5, and 25 μg/ml of AgNPs against human breast cancer cell line (AMJ13) and lymphocytes were assessed with MTT assay.Methods: A Q-Switched Nd: YAG pulsed laser (λ=1064 nm, 800 mJ/pulse) was used for ablation of a pure silver plate to synthesis AgNPs in the polyvinylpyrrolidone and deionize distilled water. Ultraviolet-visible spectroscopy confirmed the synthesis of AgNPs and zeta potential was evaluated. Morphology and size were analyzed by transmission electron microscope. AgNPs concentrations were determined by atomic absorption spectroscopy. Possibilities of apoptosis induction were confirmed using mitochondrial membrane potential assay, DNA fragmentation assay, and glutathione (GSH) assay.Results: The results indicated that AgNPs were able to induce an inhibition of AMJ13 cells compared their damaging effect toward normal lymphocytes were at minimal according to viability with MTT assay.. Furthermore, these results suggested that AgNPs-induced mitochondrial-mediated apoptosis cause DNA fragmentation, but no significant change in GSH level in AMJ13 cells.Conclusions: The overall results indicated that the physically synthesized AgNPs were exhibited dose-dependent cell death in AMJ13 breast cancer cell line, while the effect of AgNPs on lymphocytes was very low, suggesting that physically synthesized AgNPs might be a potential alternative agent for human breast cancer therapy

HLA Class I and Class II Polymorphisms and Anti-nuclear Antibodies in Hyperprolactinaemic Iraqi Females with Primary Infertility

Background: The study was conducted to investigate the association between hyperprolactinaemia and markers of human leukocyte antigen (HLA) system in a sample of Iraqi infertile females, together with the profile anti-nuclear antibodies (ANA). Objectives: One hundred and seventy five female patients (age range: 20 -40 years) were recruited in this study. They were attending the Institute for Embryo Research and Infertility Treatment (Al-Nahrain University) during the period January 2005 - September 2006. Results:After clinical and laboratry evaluations, it was found that 100 patients were hyperprolactinaemic, whereas the other 75 patients were euprolactinaemic, therefore, they were considered as a control group. Based on serum level of prolactin (22-29, 30-39 and ≥ 40 ng/ml), the total hyperprolactinaemic patients were divided into three groups; I (35 patients), II (40 patients) and III (25 patients), respectively. The HLA antigens showed significant variations between patients (total and groups) and controls. In total patients, B8 (25.0 vs. 9.3%), DR3 (48.0 vs. 17.3%) and DR4 (39.0 vs. 13.3%) showed significant increased frequencies, while B35 showed a significant decreased frequency (7.0 vs. 24%). The latter decrease was also observed (5.7 vs. 24.0%) in group I of patients, which also showed a significant increased frequency of DR3 (54.3 vs. 17.3%). In groups II and III of patients, only DR3 (45.0 and 56.0, respectively vs. 17.3%) and DR4 (37.5 and 56.0, respectively vs. 13.3%) showed significant increased frequencies. Autoantibody evaluation by ANA test revealed that 22% of the total patients was positive, while all control subjects were negative, and such positivity paralleled the increased level of serum prolactin

In Vitro Synergistic Enhancement of Newcastle Disease Virus to 5-Fluorouracil Cytotoxicity against Tumor Cells

Background: Chemotherapy is one of the antitumor therapies used worldwide in spite of its serious side effects and unsatisfactory results. Many attempts have been made to increase its activity and reduce its toxicity. 5-Fluorouracil (5-FU) is still a widely-used chemotherapeutic agent, especially in combination with other chemotherapies. Combination therapy seems to be the best option for targeting tumor cells by different mechanisms. Virotherapy is a promising agent for fighting cancer because of its safety and selectivity. Newcastle disease virus is safe, and it selectively targets tumor cells. We previously demonstrated that Newcastle disease virus (NDV) could be used to augment other chemotherapeutic agents and reduce their toxicity by halving the administered dose and replacing the eliminated chemotherapeutic agents with the Newcastle disease virus; the same antitumor activity was maintained. Methods: In the current work, we tested this hypothesis on different tumor cell lines. We used the non-virulent LaSota strain of NDV in combination with 5-FU, and we measured the cytotoxicity effect. We evaluated this combination using Chou–Talalay analysis. Results: NDV was synergistic with 5-FU at low doses when used as a combination therapy on different cancer cells, and there were very mild effects on non-cancer cells. Conclusion: The combination of a virulent, non-pathogenic NDV–LaSota strain with a standard chemotherapeutic agent, 5-FU, has a synergistic effect on different tumor cells in vitro, suggesting this combination could be an important new adjuvant therapy for treating cancer