الفهرس 
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Abstract
Finding new natural products as new chemotherapeutic agents are well recognized nowadays and profound development has been achieved by researchers to deal with different molecular pathways of tumors. Marine natural products have been extracted and identified from marine organism and screened for several biological and medical applications However, the marine environment has been less explored for the production of safe and novel antitumor compounds.Marine bioactive extracts isolated from marine animals were examined for antitumor compounds against different human cancer cell lines in vitro as promising therapeutic drugs for several cancer cells. The current study conducted to evaluate the effect of hemolymph of Helix desertorum and Stramonita haemastoma snails as immune-modulatory and anticancer agents in both in vitro and in vivo studies. The two species of snails were collected from their own habitats and identified, and then their hemolymph was collected to run different experiment throughout this study. Morphology of hemocytes of Helix desertorum and Stramonita haemastoma snails using transmission electron microscopy. Cell-type populations were determined using SYBR Green I. All experiments in this study were performed using the breast tumor cell line Ehrlich Ascities Carcinoma (EAC). The results can be summarized as the following: The study reported that the UV-visible absorption spectra of Helix desertorum hemolymph (HD-H) and Stramonita haemastoma hemolymph(SH-H) was within almost at the same range at wave length λ max =290 nm. Furthermore, the results of this study reveal the occurrence of three types of hemocyte in the hemolynlph of Helix desertorum: hyalinocytes, granulocytes and agranulocytes. All these types are characterized by their pseudopodia that attached to substratum tightly. The hyalnocytes are agranular cells and present a central large nucleus surrounded by relatively small cytoplasm. The other two types of hemocyte cells; a granulocyte and granulocyte were distinguished by their eccentric nucleus and low nuclear cytoplasmic ratio and cytoplasmic vacuoles. The results of this study showed that the cytotoxic effect different concentration of HD-H and SH-H on EAC in vitro post 48 hours of treatment did not show any significant changes when compared to their control. However, treatment for 72 hours showed cytotoxic effect on EAC at concentration 100 μg/ml. Furthermore, there was no effect of different concentration of SHH on EAC for 72 hours exposure.Analysis of the apoptotic EAC cells post 48 or 72 hours of exposure to HD-H and SH-H in vitro showed that treatment with a concentration of 100 μg/ml of hemolymph of both snails significantly increased the apoptotic percentage. The results showed that among all concentrations of HD-H only treatment of EAC-cells in vitro with 100 μg/ml for 48 hours led to a significant increase in the percentage of G0/G1 and G2/M phases and. However, treatment with 100μg/ml and 500 μg/ml of HD-H for 72 hours in vitro showed significant decrease in decrease in the percentage of S phase with no significant changes in G0/G1 and G2/M phases. Furthermore, the results showed that all tested concentrations of SH-H did not show significant changes in the percentages of G0/G1 and S phases post 48 hours on EAC-cells when compared to the control. However, treatment with 100μg/ml and 500 μg/ml of SH-H for 48 hours led to significant decrease in G2/M cell cycle phases. However, post 72 hours of exposure to all tested concentration did not led to any significant changes G0/G1 phase. In addition, significant decrease in S phase and increase in G2/M phase post treatment with all concentration except 4 μg/ml. The data showed compared to the EAC tumor-bearing mice alone, treatment with Cis (40 mg), HD-H or SH-H (100 μg) led to decrease in the total body weight however, the group of mice, which treated with Cis (40 mg) showed the lowest body weight changes. Treatment with Cis (40 mg) post EAC-cells inoculation led to a significant decrease in the number of tumor cells when compared to group, however, treatment with HD-H or SH-H (100 μg) post EAC-cells inoculation also decreased the tumor cells count, but not that much as Cis treated group. Compared to tumor-bearing mice alone, treatment with Cis (40 mg) injection, HD-H or SH-H post EAC-cells inoculation increased the number of splenocytes significantly, however, the number of splenocytes in EAC-bearing mice treated with Cis (40 mg) was higher than this number in mice bearing tumor and treated with HD-H or SH-H (100 μg). The results showed that the number of EAC-apoptotic cells post treatment with Cis (40 mg) was higher than those of mice inoculated with EAC-cells alone.Treatment with HD-H (100 μg) post EAC inoculation showed an increase in the number of apoptotic cells. As compared to EAC-bearing mice, treatment with Cis or HD-H post tumor inoculation increased the percentages of early apoptotic EAC-cells; however, treatment with SH-H (100 μg) post tumor inoculation did not exhibited significant increase in the percentages of early apoptotic EAC-cells. Treated groups with Cis, HD-H or SH-H showed an increase in the percentages of the late apoptotic EAC-cells; however, the percentage of these cells was higher in HD-H and SH-H (100 μg) than those in EAC-bearing mice treated with Cis (40 mg). Upon the treatment of EAC-bearing mice with Cis (40 mg), the percentages of G0/G1 phase of the EAC-cell cycle was decreased while treatment with HD-H or SH-H (100 μg) did not show significant changes in the percentages of this phase compared with EAC-bearing mice alone. Treatment of EAC-mice with Cis alone or with HD-H (100 μg) post EAC-inoculation decreased the percentages of S phase of the EAC-cells, however, the treatment with SH-H (100 μg) post EAC inoculation did not show changes in this S phase when compared to EAC-bearing mice. Similar pattern was found in the G2/M phase as in the S phase post treatment with Cis (40 mg), HSH or SH-H (100 μg). Upon the treatment with Cis (40 mg) post EAC inoculation, the levels of ALT were increased significantly; however, treatment with HAA or SH-H did not change the levels of ALT when compared to the EAC-bearing mice alone. Treatment with Cis, HD-H or SH-H (100 μg) led to a significant decrease in the levels of AST enzymes. Compared to the EAC-bearing mice, the treatment with Cis (40 mg), HD-H or SH-H (100 μg) did not alter the number of the red blood cells (RBCs), however, treatment with SH-H post EAC inoculation showed a decrease in the levels of hemoglobin (Hb) and in the count of platelets. The present study extended to address the indirect effect of snails hemolymph on tumor cells in the EAC-bearing mice. The results showed that post 10 days of EAC-cells inoculation, the body weight of EAC-bearing mice decreased in all treated groups when compared to mice inoculated with EAC alone. As compared to EAC-bearing mice alone, 10 days post Cis (40 mg) injection, treatment with preconditioned splenocytes in vitro HD-H, SH-H, Il-2/Con-A alone or with non-activated splenocytes led to significant decrease in EAC-cells count. Groups of mice showed significant increase in splenocytes count when compared to EAC-bearing mice alone. The results showed that post 10 days of Cis (40 mg) injection, different treatments protocols of Cis/splenocytes activated with HD-H, Cis/splenocytes activated with SH-H, Cis/splenocytes activated with IL-2 and Con-A or Cis/splenocytes without activation led to significant increase in the percentage of apoptotic cells when compared to untreated EAC-bearing mice. Treatment with Cis/preconditioned splenocytesin vitro HD-H, SH-H, Il-2/Con-A alone or Cis/ non-activated splenocytes led to significant increase in the number of early apoptotic cells when compared to untreated EAC-bearing mice. The data of EAC-cells cycle analysis showed that Cis (40 mg) injection, different treatments conditions of Cis/splenocytes activated with HD-H, Cis/splenocytes activated with SH-H, Cis/splenocytes activated with IL-2 and Con-A or Cis/splenocytes without activation after 10 days of EAC-cells inoculation led to significant decrease in the percentage of G0/G1 phase when compared to untreated EAC-bearing mice. Similar pattern was obtained in the data of S-phase percentages. Groups treated with Cis (40 mg), Cis/preconditioned splenocytesin vitro with HD-H, SH-H or treated with Cis/non-activated splenocytes showed significant decrease in the percentage of G2/M phase. However, treatment with Cis/non-activated splenocytes led to non-significant decrease in the percentage of G2/M phase. All groups of mice treated with in vitro activated splenocytes with H. desertorum or S. haemastoma hemolymph showed non-significant changes in the level of alanine transaminases (ALT). However, the group of mice treated with Cis (40 mg) showed significant increase in ALT level when compared to groups of mice which inoculated only with EAC and other treated groups. Different treatment protocols led to significant decrease in the aspartate transaminases (AST) level when compared to untreated EAC-bearing mice. As compared to untreated EAC-bearing mice, all treated groups except group of mice which treated with Cis/splenocytes activated with HD-H showed non-significant changes in the total number of red blood cells (RBCs) and hemoglobin concentration. All treated groups except group of mice which treated with Cis alone showed significant increase in the total number of white blood cells (WBCs). No significant changes in the percentage of lymphocytes among all the experimental groups. However, group of mice which treated with Cis/splenocytes activated with HD-H, treated with Cis/splenocytes activated with SH-H or treated with Cis/splenocytes activated with IL-2/Con-A showed significant decrease in the percentage of monocytes. EAC-bearing mice treated with Cis alone or treated with Cis/non-activated splenocytes showed significant increase in the percentage of monocytes when compared to untreated EAC-bearing mice.