الفهرس 
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Abstract
Cancer is one of the leading causes of death in the globe which affecting millions of people every year. Free radicals were known to be one of the main causes for the conversion of a normal cell to cancerous cells. They impair vital biological molecules like DNA, proteins and lipids resulting in many degenerative disorders including cancer. Previous studies indicated that autophagy can prevent cells from apoptosis by helping tumor cells to survive under stress conditions. Therefore, autophagy inhibition contributes to apoptosis which elevated the potency of antineoplastic drugs. Hydroxychloroquine (HCQ) is a commonly used autophagy inhibitor in clinical trials and exhibited anticancer effects against various malignancies. Natural products which possess antioxidant, chemopreventive and therapeutic effects without causing deleterious effects on normal tissues remain important for the health and vitality of humans. Nowadays, there is a great interest to use natural products to inhibit cancer development. Indole-3-carbinol (I3C) is one of these natural compounds which is produced by cruciferous vegetables and possessed antineoplastic properties in different experimental models. This work aimed to isolate I3C from some cruciferous vegetables, and studying its free radical scavenging activity as well as its cytotoxic effect against various cancer cell lines in vitro. Moreover, this study was designed to assess the antineoplastic effect of I3C and/or HCQ on EAC-bearing mice with a focus on autophagy and apoptosis. The study included: I. Isolation, characterization and evaluation of the antioxidant activity of I3C Indole-3-carbinol was extracted from different cruciferous vegetables followed by characterization using thin-layer chromatography (TLC), Fourier transform of infrared spectrum (FTIR), UV wavelength scanning and high-performance liquid chromatography (HPLC). Moreover, I3C antioxidant activity was measured by monitoring its free radical scavenging activity against both 2, 2 diphenyl-1-picrylhydrazyl (DPPH) and 2, 2 azino-bis3-ethylbenthiazoline-6-sulfonic acid (ABTS) radicals. II. In vitro studies The cytotoxicity of I3C against human breast (MCF-7 and MDA-MB-231), colorectal (HCT-116) and hepatocellular (HepG2) cancer cell lines together with Ehrlich ascites carcinoma (EAC) and human amniotic normal (WISH) cells was studied by MTT assay. III. In vivo studies III.1. The effects of I3C and/or HCQ on EAC cells viability as well as serum, hepatic and renal biochemical parameters in mice. A total of eighty adult female Swiss albino mice were used in this study. The mice were divided into six groups: control group, I3C group, EAC group and groups of EAC-bearing mice treated with I3C, HCQ and I3C combined with HCQ. All groups were injected intraperitoneally with (1x10 Summary and conclusion III.2. Molecular studies: III.2.a. The effects of I3C and/or HCQ on the percentage of LC3B in EAC cells via flow cytometry. III.2.b. The effects of I3C and/or HCQ on LC3B, p62, cathepsin D and LAMP1 gene expression in EAC cells via RT-PCR. III.2.c. The effects of I3C and/or HCQ on DNA fragmentation, a marker of apoptosis in EAC cells by agarose gel electrophoresis. III.2.d. The effect of I3C and/or HCQ on apoptotic (cleaved caspase-3 and Bax) and anti-apoptotic (Bcl-2) proteins expression in EAC cells by western blotting. III.3. Histopathological examination of liver and kidney tissues. The results of the present study were as the following: • Broccoli gave the highest yield of I3C followed by cauliflower, green cabbage and red cabbage. • The Rf value of standard and isolated I3C using TLC was 0.7 and 0.73 respectively. The IR spectrum also indicated the presence of functional groups of I3C. In addition, I3C detection was carried out at an absorption wavelength of 279 nm using a standard solution in ethanol. Regarding HPLC, standard and isolated I3C retention time was 5.632 and 5.650 min respectively and the peak areas were 176458 and 116788 μV*sec respectively. • IC50 values of I3C against DPPH and ABTS radicals were 0.236 mg/ml and 4.842 μg/ml respectively. • IC50 values of I3C against two breast (MCF-7 and MDA-MB-231), colorectal (HCT116), liver (HepG2) cancer cell lines, Ehrlich tumor cells and normal cells (WISH) treated with I3C for 48 h were 45.07, 27.71, 22.29, 42.16, 18.9 and 73.47μg/ml respectively. EAC cells were more sensitive to I3C cytotoxic effect. However, I3C exhibited less cytotoxicity to the normal cell line when compared with cancer cell lines. • The results showed that I3C alone and combined with HCQ exhibited antineoplastic activity against EAC cells in mice as shown by decreasing the ascitic volume and viable tumor cell count and elevating dead cell count. Moreover, I3C and/or HCQ prolonged the survival of EAC-bearing mice. • Serum ALT, AST and ALP activities as well as creatinine and urea concentrations were significantly elevated while total protein and albumin levels were significantly reduced in EAC-bearing mice in comparison with control group which indicated the toxic effect of EAC cells on the liver and kidney tissues. In contrast, the administration of I3C alone or combined with HCQ significantly reduced serum ALT, AST and ALP activities as well as creatinine and urea levels while increased total protein and albumin levels when compared with EAC group. These findings indicated the capability of I3C and/or HCQ to diminish the toxic effects of EAC cells. • Hepatic and renal TBARS levels were significantly elevated while GSH level as well as GPx, GST and CAT activities were significantly declined in EAC-bearing mice in comparison with normal control group. In this regard, the oxidative stress induced by EAC cells resulted in lipid peroxidation and the consumption of enzymatic and non-enzymatic antioxidants. In contrast, TBARS level was significantly reduced and GSH level as well as GPx, GST and CAT activities were significantly elevated in groups treated with I3C either alone or with HCQ as compared to EAC group. These findings indicated the antioxidant activities of I3C and/or HCQ which inhibited free radical formation. • The percentage of LC3B increased significantly in EAC cells of mice treated with I3C either alone or with HCQ when compared with that of untreated mice. In addition, I3C group revealed a significant up-regulation of LC3B and down-regulation of p62 gene expression in EAC cells in comparison to that of untreated mice which indicated induction of autophagy. On the other hand, HCQ inhibited autophagy in EAC cells as showed by the elevation of %LC3B as well as the up-regulation of both LC3B and p62 gene expression. • The administration of I3C and/or HCQ significantly down-regulated both cathepsin D and LAMP1 gene expression in EAC cells as compared to EAC group which could result in reducing the metastatic potential of EAC cells. • The treatment of EAC mice with I3C and/or HCQ induced apoptosis in EAC cells which appeared as DNA fragmentation as well as a significant up-regulation of the apoptotic cleaved caspase-3 and Bax and down-regulation of the anti-apoptotic Bcl-2 protein expression. However, the highest apoptotic effect was observed in the combinatorial group. These data collectively indicated the antineoplastic activity of I3C and the potency of HCQ to augment I3C anticancer effect.