Investigation Of The Preventive Effects Of Low Molecular Weight Apple Polysaccharides On Colitis Associated Colorectal Cancer And The Possible Mechanisms

Background:Epidemiologic studies show that: Colorectal cancer (CRC) is one of the mostcommon cancers, staying on top both in prevalence and in lethality. In the pastdecade, many efforts have been put into the area of new drug discovery and newtreatment strategy exploration, however, the mortality of CRC still remains high.Thus, looking for preventive targets and effectively preventive drugs areurgently needed. The role of diet in the development and progression of CRCare being paid more attention. The World Health Organization has identifieddiet as a critical risk factor in the development and progression of CRC andpointed out the protective role of high level of fruit and vegetable consumption.Apple is proved to have beneficial effects for intestine. It possesses the second highest level of anti-oxidative power among all fruits and is correlated with thealteration of the risk of CRC. Many ingredients of apple have been proved tohave anti-tumor potency as well, such as polyphenols and flavones. However,both apple polyphenols and flavonoes mainly exist in apple peel, which wouldincrease the cost and limit the output when being put into production. And appleflesh and residue are rich in polysaccharides. It is reported that cloudy applejuice decreases DNA damage, hyperproliferation and aberrant crypt focidevelopment in the distal colon of DMH-initiated rats. One component thatmakes the apple juice cloudy is polysaccharides. We then wondered ifpolysaccharides itself have anti-tumor effect as well. If polysaccharidesextracted from apple are verified having anti-CRC potency by in vitro and invivo experiments, then, the use of apple in CRC prevention seems to bepromising. Botanical polysaccharides have multiple biological functions, likeanti-inflammation and immunological regulation. The mechanisms of action ofbotanical polysaccharides are complicated. Some people managed to elucidatethe possible mechanisms using high-flux screening methods, e.g. c-DNA arrayand protein array. Considering that botanical polysaccharides often containgalactose and/or galacturonic acid, galectin-3 (gal-3) may become a potentialtarget. Gal-3 is a beta-galactoside-binding protein whose expression has beencorrelated with progression and metastasis of colon cancer. It is involved in cellgrowth, adhesion, differentiation, apoptosis and tumor progression andmetastasis mainly through binding to glycoproteins. Gal-3 is not only thought tobe a pro-inflammatory factor, but also viewed as a potent prognostic marker ofcancer. And its alteration is associated with the development and metastasis ofCRC: the expression of gal-3 increases in the cytoplasm of colon cancer cellscompared with that of normal cells. And gal-3 concentrations in the serum havebeen found to be higher in CRC patients with metastasis than in patients withlocalized tumors. If apple polysaccharides could influence the expression andfunction of gal-3, then, it will be helpful to elucidate the possible mechanisms of apple polysaccharides and lay a solid foundation for it becoming a drug.Methodsethods:Human colon carcinoma cells: SW-1116, HT-29 and SW-620 and a colitisassociated colorectal cancer model (CACC) were used to evaluate the CRCpreventive effects of low molecular weight apple polysaccharides (LMWAP,MW: 1,000-3,000) and the possible mechanisms. The experiment was dividedinto three parts.First, the CRC preventive effect of LMWAP was evaluated using a CACCmouse model. A total of 120 male ICR mice were randomly divided into fivegroups: model group, 3 LMWAP-treated groups and control group (except formodel group having 40 mice, others were all 20). The mice in model group and3 LMWAP-treated groups were given a single intraperitoneal (i.p.) injection of 1,2-Dimethylhydrazine (DMH) at the dose of 15 mg/kg body weight. One weeklater, the mice were treated with 2% (w/v) dextran sodium sulfate (DSS) in theirdrinking water for 1 week. This was followed no further treatment by 2 weeks.After another 1 week of 2% DSS treatment, normal water was given for anadditional 15 weeks. On week 9, the mice in LMWAP-treated groups were thenmaintained on the basal diets mixed with different concentrations of LMWAP(2.5%, 5% and 10% w/w) for 12 weeks. The control group was injected withsaline at the beginning and untreated till the end of the experiment. All micewere sacrificed on week 20 by ether overdose and colon sample was thencollected for histopathological examinations and western-blot. All the processeswere handling in ice bath.Second, immunofluorescence and immunohistochemistry were employed toobserve the expression of gal-3 and toll like receptor 4 (TLR-4) in the mucosa ofICR mice and CRC patients. A human carcinoma cell expressing moderate gal-3and TLR-4, namely, SW-620 was chose. And laser confocal andimmunoprecipitation and RNA interference were used to find the relationshipbetween gal-3 and TLR-4. Third, human colon carcinoma HT-29 cells were cultured in DMEMcontaining 0.1 mg/mL LMWAP for 2 days. Then, total RNA was isolated for c-DNA array analysis. Genes expression over cut-off values (>or=2-fold) wereconsidered to be statistically significant. And western blot was employed toconfirm the results.Resultsesults:1. LMWAP prevented colon tumor development induced by DMH/DSS inICR mice. On week 20, the tumors appeared as flat nodular polypoid orcaterpillar-like tumors in the middle and/or distal colon in the mice treated withDMH/DSS. LMWAP significantly inhibited tumorigenesis and made the colonof the mice treated with DMH/DSS in an inflammatory state. H.E. stainingshowed that the incidence of tumor formation was 90% in the mice treated withDMH/DSS without LMWAP; it then decreased to 25% (5/20) with DMH/DSSplus 2.5% MAP, 15% (3/20) with DMH/DSS plus 5% MAP, and 5% (1/20) withDMH/DSS plus 10% LMWAP. And pathological examination of the colonsshowed the lymphocytic infiltrate in the mucosa and edema in the submucosa ofthe colon.2. Results of immunofluorescence and immunohistochemistry showed thatgal-3 and TLR-4 co-exist on the cell surface of the epithelium cells of mouseand human mucosa. Laser confocal demonstrated that moderate gal-3 and TLR-4 co-exist on the cell surface of SW-620 cells. Immunoprecipitation indicatedthat there may be a molecular partnership between gal-3 and TLR-4. And downregulationof gal-3 by small interfering RNA could decrease the expression ofTLR-4; down-regulation of TLR-4 by RNAi, however, had little effect on gal-3,which implies that gal-3 may help TLR-4 recognize its ligands. And LMWAPmay affect the expression and function of TLR-4 through influencing theexpression of gal-3.3. Results of micro-array from Agilent Company which contains 44,000 geneprobes demonstrated that treatment of HT-29 with LMWAP for 2 days resulted in 333 genes expression over cut-off values (>or=2-fold): the expression of 30genes increased, whereas 303 genes decreased. Further analysis indicated thatpathways of cell cycle were mainly affected. FCA showed that at theconcentrations from 0.001 to 0.1 mg/mL, LMWAP arrested the cell cycle inG0/G1 phase in a dose dependent way. And western blot analysis for cells andmucosa of mice showed that this effect was p53 independent.Conclusiononclusion:1. LMWAP could protect ICR mice against colitis associated colorectal cancereffectively.2. There may be a molecular partnership between gal-3 and TLR-4; LMWAPmay play the CRC preventive role, at least in part, through mediating theexpression and function of gal-3 and TLR-4.3. LMWAP inhibited the transition from G0/G1 to S phase in HT-29 cells in ap53 independent manner.