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黄酮预防治疗结肠癌
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NUTRITION AND CANCER 57 1 28 37 CopyrightC 2007 Lawrence Erlbaum Associates Inc Zapotin a Phytochemical Present in a Mexican Fruit Prevents Colon Carcinogenesis Genoveva Murillo Wendy H Hirschelman Aiko Ito Robert M Moriarty A Douglas Kinghorn John M Pezzuto and Rajendra G Mehta Abstract Zapotin 5 6 2 6 tetramethoxyfl avone found in the tropical fruit zapote blanco Casimiroa edulis is con sumedinmanypartsoftheworld includingCentralAmerica and Asia Previously we have demonstrated in vitro chemo preventive activity of extracts derived from the seeds of C edulis Inthepresentstudy weexaminedtheeffectsofnatural and synthetic zapotin in SW480 SW620 and HT 29 colon cancer cell lines and on the generation of aberrant crypt foci ACF using mice Zapotin treatment IC50 2 74 10 7M resulted in a marked suppression of cell prolifer ation in the HT 29 cells Cell cycle analysis demonstrated a signifi cant accumulation of cells in the G2 M phase with a concomitant decrease of cells in the G0 G1phase after treatment with zapotin molecular weight 342 35 g mol 1 M for 18 24 and 48 h Zapotin treatment enhanced apoptosis in all of the colon cancer cell lines studied For the study of ACF 5 wk old CF 1 mice were given subcutaneous injections of azoxymethane AOM 10 mg kg body weight BW weekly for 2 wk and zapotin 5 or 10 mg kg BW 46 or 92 pmol kg BW or vehicle was administered intragastri cally 7 days wk The mean number of ACF for the control group was 14 0 2 3 whereas the mean numbers of ACF in the zapotin treated groups were 6 2 1 7 and 4 6 1 4 at doses of 5 0 and 10 0 mg kg BW respectively Loss of hex osaminidase a lysosomal enzyme active in normal colonic crypts but decreased in up to 95 of ACF was used as a sec ond biomarker for colon carcinogenesis Zapotin was found to signifi cantly P 50 inhibition of ACF number by the chemopre ventiveagentataconfi dencelevelof95 P 0 005 anda power of 0 80 The sample size calculation indicates that fi ve animals per group are needed for the ACF studies All data wereanalyzedusingGraphPadPrism version3 0 Treatment agents and schedules were compared with the AOM only group using one way analysis of variance ANOVA If a signifi cant difference P 0 05 was observed we used the Bonferroni t test as a multiple comparison test Results In Vitro Studies InhibitionofColonCancerCellGrowth Theantipro liferative properties of zapotin with cultured HT 29 cells are shown in Fig 2A The IC50was 212 ng ml for the pure isolated zapotin compared with 192 ng ml for the synthetic zapotin As shown in Fig 2B various concentrations of syn thetic zapotin 10 nM to 10 M were examined on HT 29 colon cancer cells Zapotin mediated growth inhibition in a dose dependent manner with 78 inhibition at a concentra tion of 1 M and an IC50of 2 74 10 7M Similar results were observed for the SW480 2 29 10 7M and SW620 5 27 10 7M cell lines The maximum antiproliferative response of zapotin was observed after 5 days of treatment Fig 2C Measurement of Cell Cycle Distribution The effects of zapotin on cell cycle progression were investigated For these experiments colon cancer cells were treated with za potin for 12 48 h and DNA content was analyzed by fl ow cytometry As shown in Fig 3 8 5 of control cells were in theG2phaseofthecellcyclecomparedwith28 ofthecells treated with zapotin for 18 h There was no difference in the distribution of control or zapotin treated cells in G1 and S phases of the cell cycle An arrest in the G2 phase remained evident after 48 h of treatment MeasurementofApoptosis Apoptosisincoloncancer cell lines was initially assessed by acridine orange ethidium bromide staining Treatment with zapotin induced features characteristic of apoptosis irregular fragmented nuclei and irregular cytoplasmic membranes The cells undergoing apoptosis in this procedure stained yellow whereas the necrotic cells stained orange red data not shown Fur thermore the Terminal Dexocyribonucleotide Transferase mediated dUTP Nick End Labeling TUNEL method was used to quantitate the number of apoptotic cells Zapotin treatment 1 M 48 h resulted in a signifi cant P 0 001 increase in the percentage of apoptotic cells of all three cell lines InHT 29cells zapotintreatmentresultedin48 apop totic cells compared with 5 in the control cells Similarly there were 35 and 29 of cells undergoing apoptosis in SW620 and SW480 cells respectively compared with 7 apoptotic cells in their corresponding controls Fig 4A DNA fragmentation was used to further verify apoptosis in the colon cancer cells treated with zapotin Figure 4B shows that treatment of HT 29 cells with 1 M zapotin induced DNA fragmentation after 72 h Thus these data demonstrate that zapotin is a potent inducer of apoptosis in colon cancer cells In Vivo Studies Aberrant Crypt Foci Studies The effects of zapotin on the development of AOM induced ACF are presented in Fig 5 Treatment of animals with AOM resulted in a 100 incidence of ACF whereas no ACF were identifi ed in saline treated animals The distribution of the ACF was greatest in the distal colon with the fewest ACF found in the Vol 57 No 131 Figure 2 A Dose response effects of natural and synthetic zapotin on HT 29 cell proliferation Cells were treated for 5 days B Dose response effects of synthetic zapotin on HT 29 cells treated for 5 days C Time course studies of zapotin treatment for cell lines HT 29 SW620 and SW480 proximalcolon Therewasnoevidenceoftoxicityinanimals treated with either chemopreventive agents or carcinogen BWs of the mice were monitored at Day 0 and at the time of termination and no signifi cant differences P 0 123 were observed All mice were active and healthy during the 6 wk experimental period In the dose response study a reduction of ACF by 56 and 67 by zapotin at doses of 5 0 and 10 0 mg kg BW respectively was evident Fig 5A The occurrence of ACF was signifi cantly lower in groups treated with zapotin either at 5 0 P 0 05 or 10 0 P 0 01 mg kg BW compared with the control group However no signifi cant differences 32Nutrition and Cancer 2007 Figure 3 Effects of zapotin on HT 29 cell cycle distribution Cell cycle analysis was performed as described in Materials and Methods Representative results from three experiments are presented A Control 18 h B Zapotin 18 h Ht 29 cells were treated with either control ethanol or zapotin 1 M for appropriate time points were observed between 5 0 and 10 0 mg kg BW suggesting nodoseeffectandfurthermoresuggestingthatadministration of 5 0 mg zapotin per kilogram BW is suffi cient to signifi cantly suppress ACF in rodents Because studies have shown that ACF composed of four or more crypts per focus correlate more closely with sub sequent development of colon cancer 30 the number of multicrypt 4 clusters per focus was evaluated The mean SE number of large foci in the control animals was 2 4 0 37 vs 0 4 0 02 and 0 8 0 06 at doses of 5 0 and 10 0 mg kg BW respectively Thus zapotin treatment resulted in a signifi cant P 0 001 reduction in the number of large ACF by 87 and 67 at doses of 5 0 and 10 0 mg kg BW respectively Fig 5C Hexosaminidase Determination Following evalua tion for ACF the colons of each animal were stained for hex osaminidase activity The appearance of crypts that lacked hexosaminidase activity hexNeg stained intensely green whereas those with normal levels of activity stained reddish brown Inthepresentstudy adecreaseinenzymeactivitywas present in both the crypts with abnormal morphology and in those appearing to have normal morphology In the dose response study zapotin treatment signifi cantly P 0 001 suppressed the appearance of hexNegcrypts The mean num ber of hexNegcrypts in the control animals was 37 6 4 7 100 compared with 16 4 1 9 43 6 and 16 2 2 2 43 0 in the 5 0 and 10 0 mg zapotin per kilogram BW groups respectively Fig 5B Thus the reduction in hexNeg lesions by 56 is comparable with that described for ACF However theoverallnumberofhexNeglesionsisgreaterthan that for ACF These results suggest that it may be important todetermineacombination ofboththelargeACFandhexNeg characteristics of the ACF Discussion Consumption of fruits and vegetables has been associ ated with a reduced incidence of colon cancer 31 32 The benefi cial effects have been attributed among other things to the high content of bioactive compounds 33 Studies conducted in the last 2 decades have shown that these agents haveimportantrolesinthepreventionofchronicdiseases in cluding cancer diabetes and hypercholesterolemia Natural substances are widely being evaluated as potential cancer chemopreventive agents for several diseases including can cer 34 Noteworthy examples of plant derived bioactive substances that have been shown to reduce experimental colon carcinogenesis are indole 3 carbinol from cruciferous Vol 57 No 133 Figure 4 A Percentage of apoptotic cells by TUNEL HT 29 SW620 and SW480 were plated on eight chamber slides and cultured either with control ethanol or zapotin 1 M for 48 h Results represent the mean SE of three different fi elds Asterisks show values signifi cantly different from those in the control group P 0 01 and P 0 001 B DNA fragmentation in HT 29 cells treated with zapotin 1 M for 24 72 h DNA was isolated as described in Materials and Methods C control cells treated with ethanol Zap zapotin 1 M treated cells vegetables such as Brussels sprouts and broccoli 35 genis tein from soybeans 36 resveratrol from red wine 37 cur cumin from the root of Curcuma 38 and epigallocatechin gallate from tea 39 Several of these agents are currently being investigated in clinical trials 40 41 Many of the plant derived chemopreventive agents are fl avonoids and they have been extensively studied for their effectsoncellcyclecheckpointsandapoptosis Inthepresent study zapotin treatment in colon cancer cells resulted in an increase of the G2 M population suggesting that the strong growth inhibition observed with treatment could in part be due to a change in cell cycle progression Accordingly our observationsareinagreementwithpreviousstudiesthathave found that fl avones inhibit cell proliferation via cell cycle ar rest Moreover we observed that zapotin is a potent inducer of apoptosis This arrest was associated at least in part with inhibited activity of p34 cdc2 kinase and reduced accu mulation of p34 cdc2 and cyclin B1 proteins Our results are consistent with the fi ndings reported for other fl avonoids 42 43 suchasgenisteinandapigenin bothwhichhavebeen shown to induce cell cycle arrest at G2 M and apoptosis in many cancer cell lines 44 Given the substantial activity of zapotin in colon can cer cell lines we investigated the in vivo effects of this 34Nutrition and Cancer 2007 Figure 5 A Mean SE number of aberrant crypt foci ACF for control vs zapotin at dose 5 0 and 10 0 mg kg body weight BW B Mean SE number of crypts negative for hexosaminidase activity C Analysis of ACF in the colon that are four or more crypts per focus large crypts The mean number SE of large crypts is shown for the control and zapotin that is 5 0 and 10 0 mg kg BW treated groups compound using the ACF assay in CF 1 mice Several lines of evidence strongly suggest that ACF are good intermediate biomarkers of colon cancer both in rodents 45 and in hu mans 29 Morphologically ACF are distinguishable from normal crypts by their increased size and the more ellipti cal shape of the luminal opening with a thicker lining of epithelial cells 28 The ACF contain elements of dyspla sia evident by alterations in enzyme activity and express Vol 57 No 135 mutations in the APC gene and the ras oncogene which suggests that they are part of the pathway leading to colon cancer 45 46 Longitudinal studies have shown that the areas where ACF appear correlate with tumor appearance suggesting that these are the preferred sites for tumorigene sis 44 Furthermore ACF have been identifi ed as one of the earliest recognizable lesions on the colonic mucosal surface of rodents treated with carcinogens It has also been demon strated that carcinogens for example AOM inducing ACF also induce colon cancer in rodents 47 As a result numer ous studies have used the ACF assay to assess the effi cacy of chemopreventive compounds 48 In our laboratory the ACF assay has been used to test po tential chemopreventive agents using CF 1 mice induced by the colon carcinogen AOM 49 Potential chemopreventive agents have been tested during the initiation or the postini tiation period By studying the precancerous lesions grossly and genetically it may be possible to learn more about the causes of colon carcinogenesis In addition by testing new compounds through the ACF assay it is possible not only to fi nd potentially new chemopreventive compounds but also to study potential mechanisms of action Given the value of the ACF assay an online chemoprevention database has been created by Corpet et al to better evaluate and com pare the effi cacy of chemopreventive agents in ACF studies on rodents 48 50 51 Using this database the suppression in ACF number by zapotin ranks 50th out of 339 agents as evaluated by Corpet et al 48 50 51 Until now zapotin had not been associated with the pre vention or treatment of colon cancer The pronounced in vitro and in vivo activity suggests that zapotin has potent anticancer effects and results in both cell cycle arrest and apoptosis The indications of the in vivo studies will need to beconfi rmedwithcoloncarcinogenesisstudies but asnoted previously agoodcorrelationexistsbetweenthemultiplicity ofACFandincidenceofcoloncarcinogenesis Anadditional aspect worth considering is the ramifi cations of consuming zapotin in the diet These data provide support for the pro tective effects of zapotin present in fruit against cancer Acknowledgments and Notes ThisworkwassupportedinpartbytheNationalCancerInstituteProgram Project P01 CA48112 Part of this work has been previously reported in the San Francisco 2002 American Association of Cancer Research meeting and published in the Proceedings of AACR 2002 Address correspondence to R G Mehta Carcinogenesis and Chemopre vention Division IIT Research Institute 10 West 35th Street Chicago IL 60616 Phone 312 567 4970 FAX 312 567 4931 E mail Rmehta iitri org Submitted 29 June 2006 accepted in fi nal form 22 September 2006 References 1 Jemal A Siegal R Ward E Murray T Xu J et al Cancer statistics CA Cancer J Clin 57 43 66 2007 2 Raju R and Cruz CorreaM Chemopreventionof colorectal cancer Dis Colon Rectum 49 113 124 2006 3 Finley JW Reduction of cancer risk by consumption of selenium enriched plants enrichment of broccoli with selenium increases the anticarcinogenic properties of broccoli J Med Food 6 19 26 2003 4 Mason JB Nutritional chemoprevention of colon cancer Semin Gas trointest Dis 13 143 153 2002 5 Lamprecht SA and Lipkin M Chemoprevention of colon cancer by calcium vitamin D and folate molecular mechanisms NatRev Cancer 3 601 614 2003 6 HoenschHPandKirchW Potentialroleoffl 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methanolic extracts in rats and guinea pigs J Ethnopharmacol 64 35 44 1999 18 Garzon De la Mora P Garcia Lopez PM Garcia Estrada J Navarro Ruiz A and Villaneuva Michel T Casimiroa edulis seed extracts show anticonvulsivepropertiesinrats JEthnopharmacol68 275 282 1999 19 Ito A Shamon LA Yu B Mata Greenwood E and Lee SK Antimu tagenic constituents of Casimiroa edulis with potential cancer chemo preventive activity J Agric Food Chem 46 3509 3516 1998 20 Mata Greenwood E Ito A Westenburg H Cui B and Mehta RG Discovery of novel inducers of cellular differentiation using HL 60 promyelocytic cells Anticancer Res 21 1763 1770 2001 21 DepeintF GeeJM WilliamsonG andJohnsonIT Evidenceforconsis tent patterns between fl avonoid structures and cellular activities Proc Nutr Soc 61 97 103 2002 22 Barnes S Boersma B Patel R Kirk M and Darley Usmar VM Isofl avonoids and chronic disease mechanisms of action Biofactors 12 209 215 2000 23 Lin JK Cancer chemoprevention by tea polyphenols through mod ulating 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