A 24-hour fermentation of the optimized synbiotic composed of B. longum and EPR was performed to give a cell-free fermentation broth (S24). S24 was co-cultured with two colon cancer cell lines (Caco-2 and SW620) and normal colon cells (FHC). S24 significantly inhibited cell proliferation for both colon cancer cells but promoted FHC cell growth by 10-25% as shown by MTT and BrdU arrays. Primary DNA damage analysis by alkaline comet assay showed S24 caused DNA damage to a comparable extent as the positive control of 10 mM H2O2 (treated for 1 hour) for both cancer cells. Dynamic analysis on DNA damage-associated DNA repair showed the two colon cancer cells had different response pattern to S24. Flow cytometric analysis showed that both Caco-2 and SW620 when treated with S24 (IC 50=3.66 mM of acetate) were arrested initially at G2/M and subsequently at S phase accompanied with large sub-G1 peaks. Dual staining with PI/AnnexinV further proved the appearance of apoptosis. Live cell imaging analysis on Caco-2 cells treated with S24 showed the following events: mitochondria were rapidly destroyed within the first two-hour treatment, the cells bubbled and the nucleus condensed after the mitochondrial had shrunken, followed by apoptosis. / Despite active research on synbiotic on anti-carcinogenesis of colon cancer by synbiotics, the underlying mechanism still remains unclear. This study investigated a novel synbiotic composed of non-digestible carbohydrates (NDCs) extracted from mushroom sclerotia as prebiotics and Bifidobacteria as probiotics. Preliminary results on incubation of two probiotics ( Bifidobacterium longum and Lactobacillus brevis) and one pathogenic bacterium (Clostridium celatum) separately with 3 NDCs extracted from mushroom sclerotia [Poria cocos (PC), Polyporus rhinocerus (PR) and Pleurotus tuber-regium (PT)] indicated that the growth of B. longum and L. brevis was stimulated more preferentially than C. celatum after 72-hour fermentation. The short-chain fatty acid (SCFA) profile was dominated by acetate (> 98% of total SCFAs) with very little butyrate (< 2.0% of total SCFAs) and the organic matter disappearance (OMD) during fermentation was consistent with the bacterial growth. Among the synbiotic combinations, NDC from PR and B. longum gave the largest amount of acetate (2.47+/-0.232 mmol/g of organic matter disappearance). / Results obtained from human pathway finder RT2 Profiler(TM) PCR Array indicated that S24 could modulate the proliferation of colon cancer cells mainly by various pathways such as cell cycle and DNA damage repair, apoptosis and cell senescence, etc. In SW620 cells, PCR Array of Human Cell Cycle further revealed that the modulated genes mainly belonged to the gene cluster of S phase and DNA replication as well as G2 and G2/M transition. While for Caco-2 cells, the cell-cycle modulated genes mainly belonged to the cluster of G2 and G2/M transition. Immuno-blotting on the pivotal upstream regulators showed that phosphorylation of ATM at Serine 1981 was significantly increased in both cancer cells. Site-specific phosphorylation of pRB was decreased and phosphorylation of Chk1 was increased in both cancer cells while Chk2 were increased in SW620 cells. Cdc25A was phosphorylated at serine17 in both cancer cells. It can be proposed that the blockage of DNA synthesis or DNA damage was due to the down-regulation of some pivotal DNA replication related proteins such as RPA3, PCNA and MCMs, detected by ATM-Chk1/Chk2-Cdc25A pathway. This would cause the prolonged staying of cells at the G1/S checkpoint which further moved on to S phase arrest for SW620 cells. Moreover the sharply up-regulated p21, an important inhibitor of Cdk2 would further hinder the cells passing the G1/S checkpoint in SW620 cells. / The tumor suppressor p53 was detected phosphorylated at various sites in SW620 but not in Caco-2 cells. In SW620 cells, G2/M arrest was caused by the inhibition of CDK1/CDC2 due to increased expression of GADD45A and p21 and phosphorylation of Cdc25A, while for Caco-2, the G2/M arrest was caused by degradation of Cdc25A due to the absence of p53-activated GADD45A and p21 expression as shown in the pathway finder results. Some apoptosis-related proteins of Bax, Apaf-1 and PARP were modulated as shown by immuno-blotting in both colon cancer cells. (Abstract shortened by UMI.) / Gao, Shane. / Adviser: Peter Chi-Keung Cheung. / Source: Dissertation Abstracts International, Volume: 72-11, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 55-94). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_344583 |
| Date | January 2009 |
| Contributors | Gao, Shane., Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, theses |
| Format | electronic resource, microform, microfiche, 1 online resource (xix, 95 leaves : ill. (some col.)) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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