| Colon cancer is one of leading causes of cancer death, and in recent years, alongwith diet and lifestyle changes makes its incidence increased. Therefore studies oncolon cancer get more and more attention of scholars. Curcumin is extracted from theginger family, on people,s daily life, it is indicated that curcumin has some effect ontreatment of cancer, studies have shown that curcumin can inhibit carcinoma througha variety of mechanisms. However, curcumin has some disadvantages such as poorwater solubility, poor stability and low utilization, so we designed and synthesized sixpyrimidine-substituted curcumin analogues in order to make up for the shortcomingsof curcumin. In this thesis we studied the mechanism of these compounds oninhibiting the growth of colon cancer cells systematically. We hope to determine thefunctional efficacy of these compounds and provide support for future drug design.We also studied the effects of these compounds on the glycosaminoglycan of cellsurface. Glycosaminoglycan as an important biological macromolecules can play animportant regulatory role on the growth of cells, immersed, angiogenesis andmetastasis of cells through combining with a variety of growth factors. Therefore weextracted glycosaminoglycan of cells, analyzed the structure and detected the activityin order to determine what changed after drug treatment. Through analysis, we wantto further explain the relationship between glycosaminoglycans and cancer, and byanalyzing the activity of these glycosaminoglycans to identify those biologicalchanges caused by glycosaminoglycan is a beneficial tumor suppressor role moreintuitively.1. Significant inhibitory effects of pyrimidine-substituted curcumin analogues oncolon cancer cellsDepending on the chemical structure of these compounds we divided them intothree groups, Three curcumin analogues with hydroxyl group were4b,4d, and4f andtheir corresponding curcumin analogues without hydroxyl group were4a,4c, and4e,respectively. We compared the inhibitory effects of the six analogues on two coloncancer cell lines using resazurin assay. Analogues with hydroxyl group showed much stronger grow inhibition effects than their corresponding curcumin analogues withouthydroxyl group. It may indicate that hydroxyl is essential for the inhibitory effect ofthese compounds.2.4b induces cell cycle arrest on colon cancer cells significantlyWe thus tested how the curcumin analogues affect cell cycles in both HCT116and HT29cells.4b at the concentration of20μM increased G2/M cell population to2.05-fold of that of the control cells, while4a at the same concentration increased theG2/M cell population only to1.54-fold. We assessed cell cycle signaling checkpointproteins, including p21WAF-1/CIP-1, CDK2, CDK4, Cyclin D1, Cyclin E1, and Rbsystematically in HCT116cells after treating the cells with or without the analogues.4a at20μlM and4b at5,10, and20μM concentrations induced significantp21WAF-1/CIP-1expression. Analogue4a at20μM did not induce significant changes inall other proteins tested. In contrast,4b inhibited expression of Cyclin D1, Cyclin E1,and Rb in a concentration-dependent manner even though4b did not inducesignificant changes in CDK2and CDK4proteins at all concentrations tested.3.4b can change the expression of proteins involved in apoptosisWe first examined the expression levels of activated caspase3and cleavedPARP because PARP is a substrate of caspase3after treating the HCT116coloncancer cells with or without the analogues. The results showed that4a at20lMconcentration did not cause any noticeable change in both caspase3and PARP. Incontrast,4b caused activated caspase3and cleaved PARP in a concentration-dependent manner.4b also decreased the levels of HSP90and HSP70expressions in aconcentration-dependent manner. Compound4a decreased slightly the level of anti-apoptotic Bcl-2expression but increased the level of pro-apoptotic protein Baxexpression. Compound4b had a stronger effect on the expression of Bcl-2and Baxand the effect was concentration-dependent.4.4b can change the structure and content of cell surface glycosaminoglycansAfter treatment with or without the analogues, we extracted glycosaminoglycansof every group and then we determine the content of uronic acid using carbazole. Wealso detected the content of glucosamine and galactosamine of every group by High Performance Liquid Chromatography. The results show that4b(10μM) increased thelevel of uronic acid, glucosamine an glactosamine,4a under the same although canincrease the concentration of their expression, but increased the amount is limited.However the trend of ratio of glucosamine and galactosamine after treatment withboth of4a and4b is same, when compared with control group the number is lower.5. The biological of glycosaminoglycans which was affected by4a or4bBaF3is a growth factor dependent cell line and it can not express endogenousgrowth factors, while only additional exogenous growth factors andglycosaminoglycans to contain normal growth. Detected the glycosaminoglycanswhich was extracted from cells with or without treatment with4a or4b, we can findthat samples extracted from4b group can promote BaF3cell to grow more strongly.We also find that when we add FGF1into medium the cells grow more quickly thanFGF8, it indicate that the samples bind with FGF1and FGFR1to promote the growthof BaF3cells more easily. While the samples which was extracted from cells whichwas treated with4a can not promote the growth of BaF3cell. Meanwhile, by directlydetecting the binding ability of these growth factors with cell surfaceglycosaminoglycans by flow cytometry can be more intuitive discovery4b aftertreatment with growth factors glycosaminoglycan binding capacity was significantlyenhanced, while after treatment with4a can not achieve the same effect, suggestingthat after the drug treatment the change of cell surface glycosaminoglycans isdifferent. We also find that at the same concentration of4b (10μM), although canmake cells conjunction move to high energy direction, but its value is still low. Theresult is same with the ability to promote BaF3cell to grow, both of these two resultsindicate that4b plays its role more likely to through FGF1signaling pathway. |
