J Korean Med Sci.  2009 Jun;24(3):474-480. 10.3346/jkms.2009.24.3.474.

EC-18, a Synthetic Monoacetyldiacylglyceride, Inhibits Hematogenous Metastasis of KIGB-5 Biliary Cancer Cell in Hamster Model

• Kim MH
• Chang HM
• Kim TW
• Lee SK
• Park JS
• Kim YH
• Lee TY
• Jang SJ
• Suh CW
• Lee TS
• Kim SH
• Lee SG

• KMID: 1779166
• DOI: http://doi.org/10.3346/jkms.2009.24.3.474

Abstract

EC-18 (monoacetyldiacylglyceride) stimulates T cell production of IL-2, IL-4, IL-12, IFN-gamma, and GM-CSF in vitro. To study the effects of these cytokines stimulated by EC-18 on cancer cells, we applied hamster biliary cancer model, a difficult cancer to treat. Cancer (KIGB-5) cells were given intravenously to produce hematogenous metastatic lung lesions which were treated with EC-18 at 10, 25, and 50 mg/kg/day respectively. The fourth group was untreated control. At 4th, 8th, and 12th week the lungs were examined. EC-18 treated groups showed only a few microscopic lung lesions and no evidence of metastatic lesion with highest dose whereas widespread gross lung lesions were observed in untreated control. To investigate whether the anti-tumor effect of EC-18 is associated with suppression of tumor cell Toll-like receptor 4 (TLR-4) expression in addition to stimulation of the immune cells, KIGB-5 cells were exposed to LPS with or without EC-18. TLR-4 mRNA and protein expression, measured by reverse transcriptase PCR (RT-PCR), real-time quantitative PCR and western blot analysis, showed suppression of TLR-4 expression in KIGB-5 cells treated with EC-18 compared with control. In conclusion, EC-18 has a significant anti-tumor effect in this experimental model of biliary cancer suggesting potential for clinical application to this difficult cancer.

Keyword

EC-18; Anti-Tumor Effect; TLR-4; Biliary Cancer

MeSH Terms

Animals
Antineoplastic Agents/*therapeutic use
Biliary Tract Neoplasms/*drug therapy/pathology
Cricetinae
Cytokines/metabolism
Female
Glycerides/*therapeutic use
Lung/pathology
Neoplasm Metastasis
T-Lymphocytes/immunology/metabolism
Toll-Like Receptor 4/genetics/metabolism
Tumor Cells, Cultured

Figure

• 

  Fig. 1 IL-2 secretion by 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (EC-18) treated T-cells. Brown spots indicate IL-2 producing cells. Data are expressed as mean±S.E.M. *P<0.05 compared with the control group.

• 

  Fig. 2 Cytokine secretion by EC-18 treated T cells compared with Anti-CD3+Anti-CD28 treated control (IL-2, IL-4, IL-12[p70], GM-CSF, IFN-γ and TNF-α). *P<0.05, †P<0.005 compared with the control group.

• 

  Fig. 3 Effect of EC-18 on [Ca2+]i of mouse lymphocyte after Ionomycin exposure. Change in [Ca2+]i was compared with EC-18 treated group and control group for a period of 0-120 sec after exposure of Ca2+ mobilizing agent (5 mM Ionomyicin).

• 

  Fig. 4 Expression of TLR-4 in KIGB-5 cells by RT-PCR. EC-18 inhibits TLR-4 expression by RT-PCR in KIGB-5 cells. Line 1: Control: KIGB-5 were exposed medium alone, 2: LPS: KIGB-5 were exposed LPS (10 µg/mL), 3: EC-18: KIGB-5 were exposed EC-18 (1 µg/mL), 4: LPS + EC-18: KIGB-5 were exposed LPS and EC-18.

• 

  Fig. 5 Expression of TLR-4 in KIGB-5 cells by western blot analysis. EC-18 (1 µg/mL) treated cells reduced the level of TLR-4 protein when compared with untreated control and LPS (10 µg/mL) treated cells. Lane 1, Control: KIGB-5 were exposed medium alone; 2, LPS: KIGB-5 were exposed LPS (10 µg/mL); 3, EC-18: KIGB-5 were exposed EC-18 (1 µg/mL); 4, LPS+EC-18: KIGB-5 were exposed LPS and EC-18. Band densities were quantified by means of a Bio-Rad Versa Doc Imaging System. *P<0.05 compared with the control group (n=3).

• 

  Fig. 6 Microscopic findings (H&E, ×100) of the lungs at 8 week after injection of KIGB-5 cells. (A) Control hamsters, (B) hamsters treated with EC-18 10 mg/kg/day, (C) EC-18 25 mg/kg/day, (D) EC-18 50 mg/kg/day. Control group showed multiple metastatic lesions. Hamster groups treated with EC-18 10, 25 mg/kg/day in respect showed metastatic lesions, except EC-18 50 mg/kg/day treated group which showed no evidence of the lesion.

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  Fig. 7 Gross pathological and microscopic findings (H&E, ×100) of the lungs at 12 weeks after injection of KIGB-5 cells. (A, a) Control hamsters, (B, b) hamsters treated with EC-18 10 mg/kg/day, (C, c) EC-18 25 mg/kg/day, (D, d) EC-18 50 mg/kg/day. Control group showed multiple metastatic lesions whereas, EC-18 10, 25, and 50 mg/kg/day treated group showed no evidence of the lesion.

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