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Title: Chemoprevention of cancer by targeting pro inflammatory cytokines
Authors: Rehan, Touseef
Keywords: Biological & Medical Sciences
Issue Date: 2020
Publisher: Quaid-i-Azam University, Islamabad.
Abstract: Leukemia, one of the major cancers, affects a large proportion of people around the world. Known causes of leukemia are ionizing radiations, certain cancer drugs and chemicals like benzene. Due to high mortality and morbidity rate of leukemia, its chemoprevention is desirable. Better treatment options for leukemia are required due to large number of side effects associated with current therapeutic regimens. Epidemiologic studies have demonstrated that consumption of medicinal herbs in diet may reduce the risk of various cancers. Due to these observations cancer research focus shifts towards isolation and characterization of chemopreventive agents present in medicinal herbs. Medicinal plants of family lamiaceae Ocimum basilicum (O. basilicum), Rosemarinus Officinalis (R. Officinalis) and Thymus Vulgaris (T. vulgaris) have been found to provide a good array of cytotoxic compounds. Current study was carried out to characterize the functional role of O. basilicum, R. officinalis and T. vulgaris in JNK and other apoptotic pathways. Apoptosis was induced in human leukemia cells and leukemia induced rats by screening out the pharmacologic and genetic approaches followed by these three plants. To elucidate the safety of the selected plants subacute toxicity profiling was carried out in Sprague dawley rats. Plant material of O. basilicum, R. officinalis and T. vulgaris was collected, dried and subjected to methanolic extract preparation. Crude extract was fractionated using column chromatography. N-hexane and ethyl acetate were used in different ratios as eluting solvent while silica gel was used as stationary phase. In plants safety study O. basilicum extract showed highest total phenols, flavonoids, antioxidant and antibacterial activities. T. vulgaris extract caused hypertrophy of liver while R. officinalis caused atrophy of spleen at both doses showing no significant histomorphological changes. T. vulgaris and O. basilicum extract significantly increased red blood cells, packed cell volume, haemoglobin and mean corpuscular volume at 1500 mg/kg body weight. To investigate the in vitro anticancer activity of these plants, extracts and fractions were applied on four types of leukemic cell lines U937, OCI-AML3, MV4-11and THP-1. The cell viability was assessed via Cell titer GLO assay and apoptosis was measured through Annexin V/ PI staining. Major Apoptotic markers like JNK and Caspase were analysed through western blotting while pro-inflammatory cytokines like TNFα, CCL2 and CXCL8 were analysed using QPCR. The most potent with lowest IC50 values among the fractions were BF2 (2:8 n-hexane:ethyl acetate) and BF3 (3:7 n-hexane:ethyl acetate), RF1(1:9 n hexane:ethyl acetate) , RF2 (2:8 n-hexane:ethyl acetate), TF2 (2:8 n-hexane:ethyl acetate), TF7 (3:7 n-hexane:ethyl acetate) and TF8 (8:2 n-hexane:ethyl acetate). Cytotoxicity was Abstract Chemoprevention of Cancer by Targeting Pro inflammatory Cytokines Page xv associated with apoptosis. Apoptosis was found caspase dependent and P-JNK activation was detected sustained. Significant increase in the level of TNF α and decrease in the level of CXCL8 was observed in BF2, BF3, RF1, RF2, TF2, TF7 and TF8 treated cells. To explore the in vivo anticancer activity of these plants, extracts and fractions of these plants were applied on leukemia induced Sprague Dawley rats. Leukemia induced Sprague Dawley rats were treated with each fraction and crude extract of each plant. Weight of liver, heart, kidneys and spleen was carried out after dissection. A differential white blood cell counting was also performed. No significant differences in weights of four organs and differential count of white blood cells were recorded in rats treated with crude extracts, DMSO, and extract fractions viz., BF2, BF3, RF1, RF2, TF2, TF7 and TF8, while in rats treated with other fractions weight of liver and spleen increased significantly. Significant increase was found in the level of TNF-α in serum samples of rats treated with fractions BF2, BF3, RF1, RF2, TF2, TF7 and TF8 and highest TNF-α concentration (199.85±4.6µg/ml) was recorded in serum samples of rats treated with basil extract fraction BF3. Selected fractions were subjected to Fourier-transform infrared spectroscopy analysis for functional group identification. The main functional groups were O-H (carboxylic/phenolic), C-H (SP2 , aromatic), C-H (SP3 , alkane), -C=C- (alkene), -C=O (carbonyl) and C-X (halides) which appeared in the range of 3300-3400, 3000-3100, 2720-2920, 1600-1630, 1710-1730 and 720-1300 cm-1 , respectively. Predominantly Epicatechin and cinnamic acid derivatives were found in these fractions as depicted from LC-MS data. Overall the study concluded that O. basilicum, R. officinalis and T. vulgaris were found to be rich sources of phenols and flavonoids and kill most cancer cells within very short time of treatment. Mechanism followed by O. basilicum for execution of apoptosis in cancer cells was found that TNF alpha is involved in activation of p-JNK that was found to be activated in a time dependent manner which in turn leads to the activation of caspases and cause cell death. Similarly enhanced expression of TNF alpha was also found in In vivo studies due to treatment with these plants. In this project an attempt has been made to characterize the plants and their extract that may be utilized in cancer treatment in Pakistan and in other countries as well. These plants can be used in diet and can be used for the treatment of cancer as recommended from this study. The research work presented in this thesis contributed in publication of the following articles
Gov't Doc #: 21198
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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