過去很多研究指出益生菌具有抗大腸直腸癌之潛能，但不同的益生菌菌種可能會影響其抗癌和抗腫瘤的活性。因此，本研究的目的為探討本土所研發之雙岐桿菌(Bifidobacterium longu)與乳酸桿菌(Lactobacillus gasseri)是否能預防DMH誘發大腸直腸腫瘤之生成。實驗選取6週大的ICR公鼠，每週以肌肉注射DMH(20 mg/kg)一次，共10週，接著分為15週中長期預防惡性病灶(ACF)及24週長期預防大腸直腸腫瘤(colon tumor)的動物模式來評估其抗腫瘤活性及進行相關機制之探討。結果顯示：灌食雙岐桿菌和乳酸桿菌會顯著減少小鼠惡性病灶及大腸直腸腫瘤的數目，並且可能是藉著減少小鼠腸黏膜有絲分裂細胞數目、稍微增加腸道中不正常細胞之apoptosis及顯著減少不正常細胞之proliferation。此外，利用MTT方法發現乳酸桿菌可增加巨噬細胞(RAW 264.7)的生長，進一步利用BrdU cell cycle analysis assay 分析細胞DNA合成之情形，發現RAW 264.7細胞在加入乳酸桿菌後S phase有增加之現象，而於西方墨點法分析細胞週期相關蛋白中，Cyclin A及PCNA表現量有上升之趨勢。由目前的實驗數據指出，雙岐桿菌及乳酸桿菌可能於大腸直腸腫瘤之預防上具有潛在抗腫瘤之能力。

　Probiotics are thought to be protective against colon cancer. Strain differences might contribute to inconsistency of data concerning the anti-carcinogenic activities of some probiotics. Therefore, we study whether Bifidobacterium longum (B.longum) or Lactobacillus gasseri (L.gasseri) protect mouse against 1,2-dimethylhydrazine (DMH)-induced colon tumor. Aberrant crypt foci (ACF) and tumor in colon were induced by i.m. injecting in 6-week-old, ICR male mouse with 20 mg/kg DMH once per week for 10 weeks. Short period (15 weeks) and long period (24 weeks) animal models were designed to evaluate the anti-tumor ability and related pathways of probiotics. The results suggest that mice treated with B. or L. had significantly lower numbers of ACFs and tumors in colon than mice treated with DMH alone. Mitotic index was decreased in mice treated with high or low dose B.longum compared to DMH alone. Apoptosis was slightly increased in mice treated with probiotics. Proliferation rate was decreased in mice treated with high dose B.longum or/and L.gasseri compared to DMH alone. In addition, L.gasseri was found to increase cell viability of RAW 264.7 macrophage by using MTT assay, implicating an enhancement of immunity. Moreover, BrdU incorporation assay revealed an increased DNA synthesis rate in L.-treated RAW 264.7 cells. Cell cycle-related gene expression were analyzed by western blot and the results indicated that Cyclin A and PCNA were up-regulated. Our results indicate a potential anti-tumor activity of B.longum or L.gasseri in colon cancer prevention.

Aattour N., Lemonnier D. Production of interferon induced by Streptococcus thermophilus: role of CD4+ and CD8+ lymphocytes. J Nutri Biochem. 8: 25-31, 1997

Abdelali H., Cassand P., Soussotte V., Daubeze M., Bouley C., Narbonne JF. Effect of dairy products on initiation of precursor lesions of colon cancer in rats. Nutrition and Cancer. 24: 121–132, 1995

Anti M., Marra G., Armelao F. Effect of omega-3 fatty acids on rectal mucosal cell proliferation in subjects at risk for colon cancer. Gastroenterology. 103: 883–891, 1992

Ahotupa M., Saxelin M., Korpela R. Antioxidative properties of Lactobacillus GG. Nutr. Today Suppl. 31: 51S-52S, 1996

Ayebo AD., Angelo IA., Shahani KM. Effect of ingesting Lactobacillus acidophilus milk upon fecal flora and enzyme activity in humans. Milchwissenschaft. 35: 730–733, 1980

Ayebo AD., Shahani KM., Dam R. Antitumor component(s) of yogurt: fractionation. J Dairy Sci. 64(12): 2318-2323, 1981

Balansky R., Gyosheva B., Ganchev G., Mircheva Z., Minkova S., Georgiev G. Inhiitory effects of freeze-dried milk fermented by selected Lactobacillus bulgaricus strains on carcinogenesis induced by 1,2-dimethylhydrazine in rats and by diethylnitrosamine in hamsters. Cancer Letters. 147: 125-137, 1999

Baricault L., Denariaz G., Houri JJ., Bouley C., Sapin C., Trugnan G. Use of HT-29, a cultured human colon cancer cell line, to study the effect of fermented milks on colon cancer cell growth and differentiation. Carcinogenesis. 16: 245–252, 1995

Bird RP., McLellan EA., Bruce WR. Aberrant crypts, putative precancerous lesions, in the study of the role of diet in the aetiology of colon cancer. Cancer Surveys. 8(1): 189-200, 1989

Bingle L., Brown NJ., Lewis CE. The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies. J Pathol. 196(3): 254-65, 2002

Blum S., Delneste Y., Alvarez S., Haller D., Perez PF., Bode Ch., Hammes WP., Pfeifer AMA., Schiffrin EJ. Interactions between commensal bacteria and mucosal immuocompetent cells. Inter. Dairy J. 9: 63-68, 1999

Brandtzaeg P. Current understanding of gastrointestinal immunoregulation and its relation to food allergy. Ann. N.Y. Acad. Sci. 946: 13-45, 2002

Bolognani F., Rumney CJ. Effect of lactobacilli, bifidobacteria and inulin on the formation of aberrant crypt foci in rats. Eur Nutr. 40: 293-300, 2001

Carter JW., Lancaster HK., Hardman WE., Cameron IL. Distribution of intestine-associated lymphoid tissue, aberrant crypt foci and tumors in the large bowel of 1,2-dimethylhydrazine-treated mice. Cancer Res. 54: 4304–4307, 1994

Caderni G., Giannini A., Lancioni L., Luceri C., Biggeri A., Dolara P. Characterisation of aberrant crypt foci in carcinogen-treated rats: association with intestinal carcinogenesis. Brit J Cancer. 71: 763–769, 1995

Celis JE., Celis A. Cell cycle-dependent variations in the distribution of the nuclear protein cyclin proliferating cell nuclear antigen in cultured cells: subdivision of S phase. Proc Natl Acad Sci. 82: 3262-3266, 1985

Celis JE., Madsen P. Increased nuclear cyclin/PCNA antigen staining of non S phase transformed human amnion cells engaged in nucleotide excision DNA repair. FEBS Lett. 209: 277-283, 1986

Challa A., Rao DR., Chawan CB., Shackelford L. Bifidobacterium longum and lactulose suppress azoxymethaneinduced colonic aberrant crypt foci in rats. Carcinogenesis. 18: 517–521, 1997

Chang WWL. Histogenesis of colon cancer in experimental animals. Scand. J. Gastroenterol. 19: 27–43, 1984

Chang WC., Chapkin RS., Lupton JR. Predictive value of proliferation, differentiation and apoptosis as intermediate markers for colon tumorigenesis. Carcinogenesis. 18: 721–730, 1997

Chen T., Isomaki P., Rimpilainen M., Toivanen P. Human cytokine responses induced by gram-positive cell walls of normal intestinal microbiota. Clin Exp Immunol. 118: 261-267, 1999

Crissman HA., Oishi N., Habbersett R. Detection of BrdUrd-labeled cells by differential fluorescence analysis of DNA fluorochromes: pulse-chase and continuous labeling methods. Methods Cell Biol. 41:341-349, 1994

Davies MJ., Bowey EA., Adlercreutz H., Rowland IR., Rumsby PC. Effects of soy or rye supplementation of high fat diets on colon tumour formation in azoxymethane-treated rats. Carcinogenesis. 20: 927–931, 1999

Dziarski R. Demonstration of peptidoglycan-binding sites on lymphocytes and macrophages by photoaffinity cross-linking. J Biol Chem. 266: 4713-4718, 1991

Fairey AS., Courneya KS., Field CJ., Mackey JR. Physical exercise and immune system function in cancer survivors: a comprehensive review and future directions. Cancer. 94(2): 539-551, 2002

Ermak TH., Dougherty EP., Bhagat HR., Kabok Z., Pappo J. Uptake and transport of copolymer biodegradable microspheres by rabbit Peyer's patch M cells. Cell & Tissue Research. 279(2): 433-436, 1995

Fearon ER., Vogelstein B. A genetic model for colorectal tumorigenesis. Cell. 61: 759-767, 1990

Fernandes CF., Shahani KM. Anticarcinogenic and immunological properties of dietary lactobacilli. J. Food Prot. 53: 704-707, 1990

Friend BA., Shahani KM. Antitumor properties of lactobacilli and dairy products fermented by lactobacilli. J. Food Prot. 47: 717-720, 1984

Fodde R., Smits R., Clevers H. APC, signal transduction and genetic instability in colorectal cancer. Nat Rev Cancer. 10(1): 55-67, 2001

Fosslien E. Molecular pathology of cyclooxygenase-2 in neoplasia. Annals of Clinical & Laboratory Science. 30(1): 3-21, 2000

Gill H. S. Stimulation of the immune system by lactic cultures. Int. Dairy J. 8: 535-544, 1998
Gill HS., Rutherfurd KJ., Cross ML., Gopal PK. Enhancement of immunity in the elderly by dietary supplementation with the probiotic Bifidobacterium lactis HN019. Am J Clin Nutr Dec. 74(6): 833-839, 2001

Goldin BR., Gorbach SL. The effect of milk and lactobacillus feeding on human intestinal bacterial enzyme activity. American Journal of Clinical Nutrition. 39: 756–761, 1984b

Havennar R., Husis JHJ. The lactic acid bacteria. Wood B. J. B. 99: 151-170, 1992

Hyun SP., Ji HR., Yeong LH., Jung HYP. Dietary conjugated linoleic acid (CLA) induces apoptosis of colonic mucosa in 1,2-dimethy1hydrazine-treated rats: a possible mechanism of the anticarcinogenic effect by CLA British Journal of Nutrition. 86: 549-555, 2001

Kaziu H., Sasaki M., Nakajima H., Suzuki Y. Effect of antioxidative lactic acid bacteria on rats fed a diet deficient vitamin E.J. Dairy Sci. 76: 2493-2499, 1993

Jacoby RF., Hohman C., Marshall DJ., Frick TJ., Schlack S., Broda M., Smutko J., Elliott RW. Genetic analysis of colon cancer susceptibility in mice. Genomics. 22: 381–387, 1994

Kelman Z. PCNA: structure, functions and interactions. Oncogene. 14: 629-640, 1997

Key TJ., Allen NE., Spencer EA., Travis RC. The effect of diet on risk of cancer. Lancet. 360: 861-868, 2002

Kim JM., Araki S., Kim DJ., Park CB., Takasuka N., Baba-Toriyama H., Ota T., Nir Z., Khachik F., Shimidzu N., Tanaka Y., Osawa T., Uraji T., Murakoshi M., Nishino H., Tsuda H. Chemopreventive effects of carotenoids and curcumins on mouse colon carcinogenesis after 1,2-dimethylhydrazine initiation Carcinogenesis. 19: 81-85, 1998

King K., Cidlowski J. Cell cycle and apoptosis: Common pathways to life and death. J Cell Biochem. 58: 160-175, 1995

Kulkarni N., Reddy BS. Inhibitory effect of Bifidobacterium longum cultures on the azoxymethane-induced aberrant crypt foci formation and fecal bacterial β-glucuronidase. Proceedings of the Society for Experimental Biology and Medicine. 207: 278–283, 1994

Lankaputhra WE., Shah NP. Antimutagenic properties of probiotic bacteria and of organic acids. Mutat Res. 397(2): 169-182, 1998

Lipkin M., Uehara K., Winawer S., Sanchez A., Bauer C., Phillips R., Lynch HT., Blattner WA., Fraumeni JFJr. Seventh-Day Adventist vegetarians have a quiescent proliferative activity in colonic mucosa. Cancer Letters. 26(2): 139-144, 1985

Lidbeck A., Geltner-Allinger U., Orrhage KM., Ottava L., Brismar B., Gustafsson JA., Rafter JJ., Nord CE. Impact of Lactobacillus acidophilus supplements on the faecal microflora and soluble faecal bile acids in colon cancer patients. Microbial Ecology in Health & Disease. 4: 81–88, 1991

Lipkin M., Reddy B., Newmark H., Lamprecht SA. Dietary factors in human colorectal cancer. Annu Rev Nutr. 19: 545-586, 1999

Liu S., Bishop WR., Liu M. Differential effects of cell cycle regulatory protein p21(WAF1/Cip1) on apoptosis and sensitivity to cancer chemotherapy. Drug Resist Updat. 6(4): 183-195, 2003

Markowitz SD., Dawson DM., Willis J. Willson JKV. Focus on colon cancer. Cancer Cell. 1: 233-236, 2002

Mathews MB., Bornstein RM., Franza BR., Garrels JI. Identify of the proliferating cell nuclear antigen and cyclin. Nature. 309: 374-376, 1984

Marin ML., Lee JH., Murtha J., Ustunol Z., Pestka JJ. Differential cytokine production in clonal macrophage and T-cell lines cultured with bifidobacteria. J Dairy Sci. 80: 2713-2720, 1997

Masaru F., Takehiko F., Koichiro T., Toru M., Hiroko Y., Takako S., Masakazu F., Osami N. The tumor-preventing effect of a mixture of several lactic acid bacteria on 1,2-dimethylhydrazine-induced colon carcinogenesis in mice. Oncology Reports. 8: 1073-1078, 2001

McLennan EA., Bird RP. Aberrant crypts: potential preneoplastic lesions in the murine colon. Cancer Res. 48: 6187–6192, 1988

Miyachi K., Fritzler MJ., Tan EM. Autoantibody to a nuclear antigen in proliferating cells. J Immunol. 121(6): 2228-2234, 1978

Moen CJA., Groot PC., Hart AAM., Snoek M., Demant P. Fine mapping of colon tumor susceptibility (Scc) genes in the mouse, different from the genes known to be somatically mutated in colon cancer. Proc. Natl Acad. Sci. 93: 1082–1086, 1996

Morata de Ambrosini V., Gonzalez S. de Ruiz Holgado AP., Oliver G. Study of the morphology of the cell walls of some strains of lactic acid bacteria and related species. Journal of Food Protection. 61(5): 557-562, 1998
Newmark HL., Lipkin M., Maheswari N. Colonic hyperplasia and hyperproliferation induced by a nutritional stress diet with four components of western style diet. J.N.C.I. 82: 491–496, 1990

Orrhage K., Sillerstrom E., Gustafsson JA., Nord CE., Rafter J. Binding of mutagenic heterocyclic amines by intestinal and lactic acid bacteria. Mutat Res. 311(2): 239-48, 1994

Pan Q., Hamilton SR., Hyland J., Boitnott JK. Effects of carcinogen dosage on experimental colonic carcinogenesis by azoxymethane: an ultrastructural study of grossly normal colonic mucosa. Journal of the National Cancer Institute. 74(3): 689-698, 1985

Park HS., Goodlad RA., Wright NA. The incidence of aberrant crypt foci and colonic carcinoma in dimethylhydrazine-treated rats varies in a site-specific manner and depends on tumor histology. Cancer Res. 57: 4507–4510, 1997

Perdigon G., Alvarez S., Demacias MEN., Margni RA., Oliver G., Deruiz A. Lactobacilli administered orally induce release of an enzymes from peritoneal macrophages in mice. Milchwissenschaft. 41: 244-347, 1986a

Perdigon G., Demacias MEN., Alvarez S., Oliver G., Holgdo AAP. Effect of a mixture of Lactobacillus acidophilus administered orally on the immune system in mice. J. Food Prot. 49: 986-989, 1986b

Perdigon G., Nader de Macias ME., Alvarez S., Oliver G., Pesce de Ruiz Holgado AA. Enhancement of immune response in mice fed with Streptococcus thermophilus and Lactobacillus acidophilus. J Dairy Sci. 70(5): 919-926, 1987

Perdigon G., Alvarez S., Pesce de Ruiz Holgado A. Immunoadjuvant activity of oral Lactobacillus casei: influence of dose on the secretory immune response and protective capacity in intestinal infections. J Dairy Res. 58(4): 485-496, 1991

Perdigon G., Rachid M., De Budeguer MV., Valdez JC. Effect of yogurt feeding on the small and large intestine associated lymphoid cells in mice.J Dairy Res. 61(4):553-62, 1994

Perdigon G., Maldonado GC., Valdez JC., Medici M. Interaction of lactic bacteria ith the gut immune system. Eur J Cliwn Nutr. 56: 21s-26s, 2002

Peta E., Jackson PJ., O’Connor, Donald P., Cooper, GP., Margison, Andrew C., Povey Associations between tissue-specific DNA alkylation, DNA repair and cell proliferation in the colon and colon tumour yield in mice treated with 1,2-dimethylhydrazine Carcinogenesis. 24: 527 – 533, 2003

Pool-Zobel BL., Neudecker C., Domizlaff I., Ji S., Schillinger U., Rumney C., Moretti M., Vilarini I., Scassellati-Sforzolini R., Rowland I. Lactobacillus- and Bifidobacterium-mediated antigenotoxicity in the colon of rats. Nutrition and Cancer. 26: 365–380, 1996

Prelich G., Stillman B. Coordinated leading and lagging strand synthesis during SV40 DNA replication in vitro requires PCNA. Cell 53: 117-126, 1998

Reddy BS., Rivenson A. Inhibitory effect of Bifidobacterium longum on colon, mammary, and liver carcinogenesis induced by 2-amino-3-methylimidazo [4,5-f] quinoline, a food mutagen. Cancer Res. 53(17): 3914-3918, 1993

Reddy GV., Friend BA., Shahani KM., Farmer RE. Antitumor activity of yogurt components. J. Food Prot. 46: 8-11, 1983

Rowland IR., Grasso P. Degradation of N-nitrosamines by intestinal bacteria. Applied Microbiology. 29: 7–12, 1975
Rowland IR., Rumney CJ., Coutts JT., Lievense LC. Effect of Bifidobacterium longum and inulin on gut bacterial metabol ismand carcinogen-induced aberrant crypt foci in rats. Carcinogenesis. 19: 281–285, 1998

Savage DC. Mechanisms by which indigenous microorganisms colonize gastrointestinal epithelial surfaces. Prog Food Nutr Sci. 7(3-4): 65-74, 1983

Takahashi T., Oka T., Iwana H., Kuwata T., Yamamoto Y. Immune response of mice to orally administered lactic acid bacteria. Biosci Biotechnol Biochem. 57: 1557-1560, 1993

Shahani KM., Ayebo AD. Role of dietary lactobacilli in gastrointestinal microecology. American Journal of Clinical Nutrition. 33: 2448–2457, 1980

Sherr CJ., Roberts JM. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 13(12): 1501-1512, 1999

Tejada-Simon MV., Lee JH., Ustunol Z., Pestka JJ. Ingestion of yogurt containing Lactobacillus acidophilus and Bifidobacterium spp. to potentiate immunoglobulin A responses to cholera toxin in mice. J. Dairy Sci. 82: 649-660, 1999

Thorup I., Meyer O., Kristiansen E. Influence of a dietary fiber on development of dimethylhydrazine-induced aberrant crypt foci and colon tumor incidence in wistar rats. Nutr Cancer. 21: 177–182, 1994

Thurnherr N., Deschner EE., Stonehill EH., Lipkin M. Induction of adenocarcinoma of the colon in mice by weekly injections of 1,2-dimethylhydrazine. Cancer Research. 33: 940-945, 1973

Tsurimoto T. PCNA, a multifunctional ring on DNA. Biochim Biophys Acta. 1443: 23-39, 1998

Underdown BJ., Schiff JM. Immunoglobulin A: Strategic defense initiative at the mucosal surface. Annu Rev Immunol. 4: 389-417, 1986

Valdez JC., Rachid M., Bru E., Perdigon G. The effect of yoghurt on the cytotoxic and phagocytic activity of macrophages in tumor-bearing mice. Food Agrc Immunol. 9: 299-308, 1997

Van WT., Stassen APM., Moen CJA., Hart AAM., Valk MA., Demant P. Gene interaction and single gene effects in colon tumour susceptibility in mice. Nat. Genet. 14: 468–470, 1996

Van WT., Ruivenkamp CA., Stassen AP., Moen CJ., Demant P. Four new colon cancer susceptibility loci, Scc6 to Scc9 in the mouse. Cancer Res. 59: 4216–4218, 1999

Watson WH., Cai J., Jones DP. Diet and apoptosis. Annu Rev Nutr. 20: 485-505, 2000

Yoo BH., Lee BH., Kim JS., Kim NJ., Kim SH., Ryu KW. Effects of Shikunshito-Kamiho on Fecal Enzymes and Formation of Aberrant Crypt Foci Induced by 1,2-Dimethylhydrazine Biol. Pharm. Bull. 24(6): 638-642, 2001

Zheng L. Lee WH. The retinoblastoma gene: a prototypic and multifunctional tumor suppressor. Exp Cell Res. 264(1): 2-18, 2001

Zhang XB., Ohta Y. Binding of mutagens by fractions of the cell wall skeleton of lactic acid bacteria on mutagens. J. Dairy Sci. 74: 1477-1481, 1991