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العنوان
Evaluating and Comparing the Anti-tumour Effects of Alcoholic Extract and Some Bioactive Components of Sweet Marjoram (Origanum majorana L.) on Colon Cancer Cell Line /
المؤلف
Zahra, Mai Mamdouh Hassan Hosni.
هيئة الاعداد
باحث / Mai Mamdouh Hassan Hosni Zahra
مشرف / Nadia Mohamed Abd El-Aziz El-Beih
مشرف / Gamal Ramadan Shebl Ramadan
مناقش / Khaled Mahmoud Mohamed Hanafi
تاريخ النشر
2018.
عدد الصفحات
190p. :
اللغة
الإنجليزية
الدرجة
الدكتوراه
التخصص
علم الحيوان والطب البيطري
تاريخ الإجازة
1/1/2018
مكان الإجازة
جامعة عين شمس - كلية التمريض - علم الحيوان
الفهرس
Only 14 pages are availabe for public view

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from 190

Abstract

Increasing incidence of death from colorectal cancer (CRC), despite the huge production in the Western therapy, has directed researchers’ attention towards improvement of the anticancer strategies by using the natural products. Phytochemicals apply their anticancer activity through several mechanisms including modulation of cellular signalling pathways and gene expression to inhibit cell proliferation and/or induce apoptosis. Sweet marjoram (Origanum majorana L.) is one of the popular beverages and spices consumed in the world. It contains phenolic terpenoids (such as carvacrol, CA) and acids (such as ursolic acid, UA). Our previous study showed that marjoram leaves modulated most clastogenic and genotoxic effects of cyclophosphamide, an alkylating chemotherapy that widely used in the treatment of many neoplastic diseases. Other reports suggested the anticancer activity of marjoram extracts and their bioactive components, although the molecular pathways that led to alterations in cancer cell behaviour remain mostly unclear. Therefore, the present study aimed to in vitro assess and compare the anticancer activity of different concentrations of methanolic extract of Egyptian sweet marjoram (ME) and some of its bioactive components (CA and UA) on colon cancer cell line (HCT116), as well as monitoring the mechanisms of their action with special reference to the inflammatory-apoptotic pathway.
The data of the present study indicated that ME, CA and UA induced cytotoxic effects on the HCT116 cells in a concentration and time-dependent manner, where the IC50 values (half maximum inhibitory concentration, which caused death of 50% of cells) were approximately 229.1, 209.7 and 42.7 g/ml for ME; 36.2, 22.2 and 17.0 g/ml for CA; and 21.0, 22.4 and 7.8 g/ml for UA after 24, 48 and 72 hours, respectively, of cell culture. In addition,
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incubation of non-cancerous human immortalized foreskin fibroblast cell line (BJ-1) with ME, CA and UA induced lower cytotoxic effects (higher IC50 values) compared with the cancerous HCT116 cell line. The above findings indicated that ME, CA and UA were selectivity toxic for HCT116 cells, where the selectivity index (SI) values were 1.7, 2.2 and 7.1 for ME; 3.1, 5.1 and 6.2 for CA and 5.9, 6.3 and 14.1 for UA after 24, 48 and 72 hours, respectively (selective toxicity for cancer cells was achieved when the SI was ≥ 2). Based on the IC50 and SI data of ME, CA and UA, three concentrations (0.25 IC50, 0.5 IC50 and 1.0 IC50 at 48 hours of cell culture incubation) were chosen for further investigations to evaluate the molecular base of the possible anticancer mechanisms of ME, CA and UA on the HCT116 cells, with special reference to the antiproliferative and proapoptotic pathways.
Morphological changes associated with apoptosis were observed in a concentration-dependent manner after treatment of HCT116 cells with ME, CA and UA for 48 hours including: (a) The reduction of cell to cell contact through enhancing cell shrinkage (with short or fewer filopodia). (b) chromatin condensation and degradation of DNA with formation of membrane blebbing and apoptotic bodies that led to a cellular destruction. Also, the decrease shown in the present study in the adhesion potential and migration ability of the HCT116 cells after the treatment with ME, CA and UA indicating the important role of these natural products in decreasing the acquisition of invasive/metastasis properties of cancer cells. Moreover, ME, CA and UA were able to reduce the volume of the HCT116 spheroids, reflecting the diffusion/penetrating ability of the marjoram components through the cancer multilayers.
ME, CA and UA induced oxidative stress in the HCT116 cells by decreasing their overall antioxidant capacity. In addition, they increased the DNA fragmen-
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tation (a hallmark of cells undergoing apoptosis) and the percentage of apoptotic cells (hypodiploid cells in sub-G1 phase as estimated by flow cytometry) in the HCT116 cells. These results pointed out to some possible mechanisms of death-induction in the colon cancer cells by sweet marjoram and its components.
The present study also investigated the expression of some markers associated with the extrinsic and intrinsic apoptotic pathways by using ELISA, Western blot analysis, and quantitative (real-time) polymerase chain reaction (qPCR), which can yield a more complete and in-depth insight into the mode of anticancer activity of sweet marjoram and its bioactive components. Our results showed that ME, CA and UA increased significantly, in a concentration-dependent manner, the tumour suppressor p53 protein and caspase 3 (which could trigger cell growth arrest and apoptosis, respectively), caspase 8 (a crucial mediator of the extrinsic pathway of apoptosis), caspase 9 (a crucial mediator of the intrinsic pathway of apoptosis) in the HCT116 cells after 48 hours of the treatment. In addition, they enhanced the release of mitochondrial cytochrome C (a caspase 9-activating factor) into the cytosol through increasing the Bcl-2 associated X protein (Bax, an apoptosis promoter)/B-cell lymphoma-2 (Bcl-2, an apoptosis inhibitor) ratio in the HCT116 cells. Moreover, they increase the expression of CD95 (a Fas death receptor that triggers the extrinsic pathway of apoptosis after binding with its Fas ligand) and the tumour necrosis factor receptor-1 (TNFR1, a member of death receptors that triggers the extrinsic pathway of apoptosis after binding with TNF-) in the HCT116 cells. In conclusion, our data provided insight into the molecular mechanisms of ME, CA and UA-induced apoptosis in the human HCT116 colon cancer cells. In addition, the toxicity of these natural compounds was specific for cancer cells and less effective in the normal
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non-cancerous cells, suggesting their biosafety for treating cancer patients. In general, the highest anticancer activity was achieved by UA followed by CA, rendering these compounds may become promising anticancer agents in treating CRC patients in the near future