الفهرس 
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Abstract
Colorectal cancer (CRC) is the third most commonly diagnosed cancer. In Egypt, It is the 6th ranked cancer representing about 4% of total cancers in both sexes compared to the 3rd rank and about 11% in USA.
CRC is an obesity-related cancer; several pathophysiological mechanisms linking obesity and the development of CRC have been elucidated, including imbalance of adipokines, including leptin; that can promote development and progression of CRC.
A multistep genetic model of colorectal carcinogenesis (adenoma-carcinoma sequence) involving mutation of the APC,DCC, K-ras, and p53 genes have been established suggesting to play a major role in dysregulation of cell cycle progression, evasion of apoptosis, induction of genetic instability and enhanced invasiveness and metastasis.
Current treatment options such as surgical intervention and adjuvant chemotherapy have several One of the primary modes of preventing CRC therefore, will be increasing the consumption of food containing anticancer compounds
The use of natural products as an alternative therapy in CRC treatment has become a growing interest among researchers. Ellagic acid (EA) has been known for its chemopreventive activity.
The aim of the present study was to investigate the potential anti-cancerous effect and the mode of action of EA against two human colon cancer cell lines with different genetic status HCT-116 (p53+/Ras -) and CaCo-2 (p53-/Ras+), and to evaluate the impact of genetic variation on response to treatment.
A set of experiments were carried out on the cell lines to investigate the possible anti-tumorigenic effect of different EA concentrations (0.0-200µg/ml) for 72 hours on cell proliferation and apoptosis.
Active human leptin was added (200ng/ml) in addition to various concentrations of EA parallel to treatment with EA alone. Subsequent alterations in colon cancer cell lines were assessed in presence and absence of leptin by set of assessment including:
• Cell proliferation using (MTS) assay.
• Morphological examination by inverted microscope
• (PCNA) expression by immunocytochemistry.
• Cell cycle analysis by flow cytometer.
• Colorimetric assay of Caspase -8 activity.
• Determination of Bax protein in cell fractions using Western blotting.
EA elicited a dose-dependent inhibition of CaCo-2 and HCT-116 cell proliferation. This inhibitory effect was significant when compared to untreated cells (100% proliferation). The anti-proliferative influence was highly significant at the highest concentrations (p≤ 0.001).In contrast Leptin showed a significant proliferative influence on both cell types, which appeared to be maximum at 200 ng/ml(p≤ 0.001).
The anti-proliferative influence of EA was similar to that reported in treatment with EA alone. No significant difference between responses of HCT-116 and CaCo-2 cell lines.
Microscopic examination was performed for two cell lines under study following incubation of cells with EA showed dramatic changes from epithelial-like adherent monolayer cells to rounded cells with membrane blebbing, cytoplasmic and nuclear condensation, easily detached from culture plate and float in the medium, and the most severe change was more visible in treatment with 100 EA µg/ ml +/- leptin after 72 hrs incubation, indicating the pro-apoptotic effect of EA. Similar pattern of morphological change in two cell types in absence and presence of leptin was observed. Treatment with leptin alone resulted increase in the density of cells monolayer.
PCNA expression was assessed by immunocytochemistry following the incubation of cells with EA for 72hrs. Results showed that incubation of both cell types with EA, in the presence or absence of leptin (200 ng /ml) resulted in a significant reduction in PCNA expression when compared to the untreated cells (P <0.01). A similar, but more significant effect was observed when cells incubated with EA (100 µg/ ml +/- leptin). There was no significant difference in cellular responses between the two cell types .Treatment with leptin alone significantly increased the PCNA expression.
The EA treated cells were stained with PI and the DNA contents were then assessed by flow cytometery to investigate the effect of EA on cell cycle progression. Results revealed cell cycle arrest at the G0/G1 phase in response to treatment with increase in percentage of sub G1 (apoptotic cells), meanwhile the cell percentages in the G2/M and S phases were progressively decreased for both cell lines .Treatment with leptin alone was associated with increase in cell population in both S and M phases indicating its proliferative effect.
The ability of EA to induce the extrinsic pathway of apoptosis was determined by measuring the caspase-8 activity in cell lysates of two cell lines after treatment with the two different concentrations for incubation time intervals at 24, 48, 72 hours using colorimetric method. Activity of Caspase-8 was measured as a fold increase relative to EA-untreated cells. Treatment with leptin alone failed to induce the caspase-8, while its activity showed significant and progressive increase in a time and dose dependent manner when compared with corresponding untreated cells(P <0.001).This result was obtained in the two studied cell lines when treated with EA in presence as well as absence of leptin without significant difference indicating that EA has ability to activate caspase-8.
Translocation of pro-apoptotic protein Bax from cytoplasm to mitochondria, inducing mitochondrial outer membrane permeabilization is a key limiting step in mitochondrial apoptotic pathway. Bax was determined by western blotting in cytosolic and mitochondrial fractions of two cell lines after treatment with the two different concentrations; (25 and 100 µg /ml EA +/- 200 ng/ml leptin) and treatment with leptin alone for 72 hours.
The densitometric analysis of bands resulted at 21 KDa was performed by the image j software revealed that treatment with leptin alone could not increase the Bax protein expression, meanwhile there was a dose dependent increase in Bax expression and this increase was associated with increased expression in mitochondrial fraction when treated with EA in presence and absence of leptin indicating its pro-apoptotic effect.
In conclusion, the results of our investigation revealed that:
• EA has an inhibitory effect on the cell proliferation of the two types of colon cancer cell lines K-ras mutated HCT-116 (p53+/Ras-), and p53 mutated CaCo-2 (p53-/Ras+) regardless to their genetic status.
• Obesity hormone leptin showed strong proliferative and mitotic effects on both cancer cell lines HCT-116, CaCo-2.
• EA has cytostatic effect; it can overcome the proliferative effect of leptin even at high level in cell culture environment for HCT-116, CaCo-2,
• EA exerted its pro-apoptotic effect even in presence of high leptin level by inducing both apoptotic pathways; the extrinsic pathway by progressive increase in caspase-8 activity and intrinsic pathways via induction of expression and translocation of pro-apoptotic protein Bax to mitochondria.
• Based on these finding, EA is tempting to be suggested as potential therapeutic agent in treatment of CRC in obese as well as non-obese patients.
The following recommendations should be taken in consideration:
• Early screening is highly recommended especially for individuals at high risk due to chronic IBD, ulcerative colitis, or family history of CRC.
• Low fat diet, low in red meat, high in fibers can be used as a protective strategy against obesity which considered as a major risk factor in CRC.
• Further in vitro and in vivo studies are required for more specification of molecular targets of EA, a dose adjustment, and route of administration.
• Poor absorp¬tion, low systemic bioavailability, and short retention time of ETs and their metabolites including EA may undermine their full chemopre¬ventive potential. So encapsulation of EA into biocompatible and biodegradable nanoparticles (NPs) may overcome their suscep¬tibility to gastrointestinal hydrolysis, to enhance its bio-efficacy.