J Korean Med Sci.  2011 Jun;26(6):747-752. 10.3346/jkms.2011.26.6.747.

Morphine Attenuates Endothelial Cell Adhesion Molecules Induced by the Supernatant of LPS-Stimulated Colon Cancer Cells

• Min TJ
• Park SH
• Ji YH
• Lee YS
• Kim TW
• Kim JH
• Kim WY
• Park YC

• KMID: 1785959
• DOI: http://doi.org/10.3346/jkms.2011.26.6.747

Abstract

A large reservoir of bacterial lipopolysaccharide (LPS) is available in the colon and this could promote colon cancer metastasis by enhancing tumor cell adhesion, intravasation, and extravasation. Furthermore, adhesion molecules like ICAM-1, VCAM-1, and E-selectin play important roles in the adhesion of tumor cells to endothelium. This study was designed to determine whether morphine can attenuate the expressions of adhesion molecules up-regulated by the supernatant of LPS-stimulated HCT 116 colon cancer cells (LPS-Sup). In this study, we divided to three groups by cell-growth medium of human umbilical vascular endothelial cells (HUVECs): the control group was incubated in growth factor-free endothelial medium, the Sup group was incubated in the supernatant of HCT 116 cells (Sup), and the LPS-Sup group was incubated in LPS-Sup. To observe effect of morphine to the adhesion molecules expressions in the LPS-Sup group, we co-treated morphine with LPS or added it to LPS-Sup. Adhesion molecule expressions on HUVECs in all three groups were measured during incubation period. Consquentially, ICAM-1, VCAM-1, and E-selectin expressions on HUVECs were significantly lower when morphine was co-treated with LPS than not co-treated. Thus, we suggest that morphine affects the expressions of adhesion molecules primarily by attenuating LPS stimuli on tumor cells.

Keyword

Lipopolysaccharides; Morphine; Adhesion Molecule; Supernatant; HUVEC

MeSH Terms

Cell Adhesion Molecules/*metabolism
Cell Line, Tumor
Cell Survival/drug effects
Colonic Neoplasms/*metabolism
E-Selectin/metabolism
Endothelial Cells/drug effects/metabolism
Endothelium, Vascular/cytology
Humans
Intercellular Adhesion Molecule-1/metabolism
Lipopolysaccharides/toxicity
Morphine/*pharmacology
Vascular Cell Adhesion Molecule-1/metabolism

Figure

• 

  Fig. 1 Experimental protocol. The control group; HUVECs were incubated in growth factor-free endothelial medium. The Sup group; HUVECs were incubated in the supernatant of HCT 116 cells. The LPS-Sup; HUVECs were incubated in the supernatant of LPS-stimulated HCT 116 cells.

• 

  Fig. 2 Experimental protocol. Morphine was co-treated in two ways: 1) Morphine + LPS; co-treating morphine with LPS during stimulation of HCT 116 cells or 2) Morphine + LPS-Sup; adding morphine to LPS-Sup prior to incubation of HUVECs. EGM is an endothelial cell growth medium without endothelial growth hormone.

• 

  Fig. 3 Up-regulations of the expressions of the adhesion molecules on the HUVEC cells by LPS-Sup. Graphs show the expressions of ICAM-1, VCAM-1, and E-selectin on HUVECs during a 24-hr of incubation period. ICAM-1, VCAM-1, and E-selectin expressions in the LPS-Sup and Sup groups were compared versus those in the control group. Percentages of positive cells were recorded at 0, 4, 8, 12, and 24 hr of incubation period. Values are expressed as means ± SD. *P < 0.05 vs the control group; †P < 0.05 vs the Sup group.

• 

  Fig. 4 The effect of morphine on the expressions of adhesion molecules on HUVECs. (A) Graphs show the expressions of ICAM-1 on HUVECs incubated in LPS-Sup, Morphine + LPS-Sup and supernatant of Morphine + LPS. (B) Graphs show the expressions of VCAM-1 on HUVECs incubated in LPS-Sup, Morphine + LPS-Sup and supernatant of Morphine + LPS. (C) Graphs show the expressions of E-selectin on HUVECs incubated in LPS-Sup, Morphine + LPS-Sup and supernatant of Morphine + LPS. Values are Aexpressed as means ± SD. (D) Histograms show the characterization of ICAM-1, VCAM-1, and E-selectin expressions on HUVECs at 8-hr of incubation period. Control means unstained value. LPS + 0.1 µM of morphine means the value of when 0.1 µM morphine was co-treated with LPS in the LPS-Sup group. The percentage of positive cells from each group was recorded at 0, 4, 8, 12, and 24 hr of incubation period. In gray box, values are expressed as percentages of gated positive cells. *P < 0.05 versus the LPS-Sup group; †P < 0.05 versus Morphine + LPS-Sup.

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