Conjugated linoleic acid inhibits peritoneal metastasis in human gastrointestinal cancer cells Hiroki Kuniyasu 1 * , Kazuhiro Yoshida 2 , Takamitsu Sasaki 1 , Tomonori Sasahira 1 , Kiyomu Fujii 1 and Hitoshi Ohmori 1 1 Department of Molecular Pathology, Nara Medical University, Kashihara, Japan 2 Department of Surgical Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University Hiroshima, Japan The effect of conjugated linoleic acid (CLA) on peritoneal metasta- sis was examined by in vitro treatment of cancer cells and mouse peritoneal metastasis models. First, cell growth of MKN28 human gastric cancer cells and Colo320 human colon cancer cells was sup- pressed by CLA in a dose-dependent manner with an increment in apoptosis. CLA significantly inhibited invasion into type IV colla- gen-coated membrane of MKN28 and Colo320 cells (p < 0.05). CLA-induced growth inhibition was recovered by the exposure to antisense S-oligodeoxynucleotide for peroxisome proliferator-acti- vated receptor (PPAR)-c in both cell lines. BALB/c nu-nu mice were inoculated with MKN28 and Colo320 cells into their perito- neal cavity, and administrated with CLA intraperitoneally (weekly, 4 times). CLA treatment did not affect food intake or weight gain of mice. CLA treatment significantly decreased metastatic foci of both cells in the peritoneal cavity (p < 0.005). Survival rate in mice ino- culated with MKN28 or Colo320 cells was significantly recovered by CLA treatment (p 5 0.0025 and 0.0052, respectively). Protein production in MKN28 and Colo320 cells treated with CLA showed a decrease in epidermal growth factor receptor and transforming growth factor-a and an increase in Bax. These findings suggest that CLA inhibits metastasis of human gastric and colon cancer cells. ' 2005 Wiley-Liss, Inc. Key words: peritoneal metastasis; conjugated linoleic acid; PPARg; gastrointestinal cancer Conjugated linoleic acid (CLA) is composed of positional- and stereo-isomers of octadecadienoate (18:2). Chemoprotective prop- erties of CLA are reported in experimental cancer models and in vitro examinations. 1,2 In contrast, linoleic acid (LA) has a carci- nogenic property. LA, an x-6 polyunsaturated fatty acid, is a ster- eoisomer of CLA. Cyclooxygenase (COX)-2 metabolizes LA to prostaglandin (PG) E2, which plays roles in carcinogenesis due to its properties of proinflammation, proproliferatation and immuno- suppression. 3,4 Animal models of colon carcinogenesis revealed the carcinogenic property of LA. 5–8 Differently from LA, CLA is not a precursor of PGE2. CLA activates peroxisome proliferator-activated receptor (PPAR)-g as a ligand. 9,10 PPARg is a nuclear hormone receptor superfamily of ligand-activated transcription factors, which initiate transcription of genes associated with energy homeostasis. 11 PPARg is acti- vated by endogenous secreted prostaglandins and fatty acids; 15- deoxy-d(12,14)-prostaglandin J2 is a strong endogenous ligand of PPARg. PPARg possesses an anti-carcinogenic effect in colon cancer. Synthesized PPARg agonists including troglitazon have been shown to be effective chemopreventive agents in a rat model of carcinogenesis and in AOM-induced colon cancer in mice. 12 Moreover, a decrease in PPARg expression is associated with can- cer metastasis. 13,14 Thus, CLA might act as anti-metastatic agent as well as an anti-carcinogenic agent. In our study, we examined the anti-metastatic effect of CLA on peritoneal dissemination. Peritoneal metastasis of cancer causes poor quality in patients’ lives. 15–17 We tested intraperitoneal administration of CLA in a mouse peritoneal metastasis model, and revealed relevant effects on suppression of metastasis. Material and methods Cell culture A human gastric cancer cell line, MKN28, was kindly provided by Dr. W. Yasui (Hiroshima University Graduate School, Hirosh- ima, Japan). A human colon cancer cell line, Colo320 was pro- vided by American Tissue and Cell Culture. The cells were rou- tinely maintained in RPMI-1640 (Sigma Chemical Co., St. Louis, MO) containing 10% fetal bovine serum (FBS, Sigma Chemical Co.) under the condition of 5% CO 2 in air at 37°C. CLA (Calbio- chem, Darmstadt, Germany) was dissolved with 70% ethanol (50 lg/ll). For negative control, the same amount of 70% ethanol was used. Cell growth The cells were harvested from 80% confluent monolayer cul- tures by a brief trypsinization with 0.1% trypsin and 0.1% EDTA (Sigma Chemical Co.). The cells were seeded at density of 10,000 cells per well of 24-well tissue culture plates and treated under the conditions mentioned in the Results section. These cell number was counted by an autocytometer (Sysmecs, Kobe, Japan) at 48 hr after the treatment. Apoptotic cells were confirmed by Giemsa staining. One thousand cells were observed to measure apoptotic cell percentage. Each experiment was repeated 3 times. Antisense phosphorothioate (S)-oligodeoxynucleotide assay The 18-mer S-oligodeoxynucleotide (ODN) for the antisense sequence from the 1st to the 18th nucleotide of PPARg cDNA coding region were synthesized and purified by reverse-phase high-performance liquid chromatography (Sigma Genosys, Ishi- kari, Japan). The sequence of PPARg was 5 0 -CCT GGG GTA AGG TCG G-3 0 . The sense sequences 18-mer was for negative control. The cells were pretreated with 3lM antisense or sense S- ODN for 6 days, with medium exchanges and additions of anti- sense or sense S-ODN every 2 days. After that, the cells were used for further experiments. Reverse transcriptase-polymerase chain reaction (RT-PCR) PPARg mRNA expression was assessed by RT-PCR with 0.5 lg of total RNA extracted by an RNeasy kit (Qiagen, Hilden, Germany). Primer sets for PPARg were as follows: upper 5 0 -GCC ATC CGC ATC TTT CAG-3 0 and lower 5 0 -CCC CAT CTT TAT TCA TCA-3 0 . PCR products were electrophoresed in a 2% agarose gel and stained with ethidium bromide. b-actin mRNA was also amplified for internal control. The experiment was repeated twice. Animal model BALB/c nu-nu athymic mice were purchased from Japan SLC, Inc. (Shizuoka, Japan). The mice were maintained according to Grant sponsor: Grant-in-Aid for Scientific Research from Mitsui Life Social Welfare Foundation *Correspondence to: 840 Shijo-cho, Kashihara, Japan 634-8521. Fax: 181-0744-25-7308. E-mail: cooninh@zb4.so-net.ne.jp Received 7 March 2005; Accepted after revision 27 May 2005 DOI 10.1002/ijc.21368 Published online 16 August 2005 in Wiley InterScience (www.interscience. wiley.com). Abbreviations: CLA, conjugated linoleic acid; COX, Cyclooxygenase; EGFR, epidermal growth factor receptor; HBSS, Hanks balanced saline solution; LA, linoleic acid; ODN, oligodeoxynucleotide; PBS, phosphate- buffered saline PG, prostaglandin; PPAR, peroxisome proliferator-acti- vated receptor; RT-PCR, reverse transcriptase-polymerase chain reaction; TGF, transforming growth factor. Int. J. Cancer: 118, 571–576 (2006) ' 2005 Wiley-Liss, Inc. Publication of the International Union Against Cancer