Ann Dermatol.  2014 Jun;26(3):363-373.

The Effect of Imiquimod on Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Malignant Melanoma Cell Invasion

Affiliations
  • 1Yeouido Oracle Dermatology and Cosmetic Dermatosurgery Clinic, Seoul, Korea.
  • 2Department of Dermatology, Severance Hospital, Cutaneous Biology Research Institute, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea. kychung@yuhs.ac
  • 3Department of Dermatology, Myongji Hospital, Kwandong University College of Medicine, Goyang, Korea.

Abstract

BACKGROUND
A number of reports have been published regarding the use of imiquimod for the treatment of melanoma in situ and metastatic melanoma. Essential steps in the process of melanoma invasion and metastasis include degradation of basement membranes and remodeling of the extracellular matrix by proteolytic enzymes, including matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs).
OBJECTIVE
To evaluate the antiinvasive effect of imiquimod in human malignant melanoma cell lines, SK-MEL-2 and SK-MEL-24, in vitro, and to investigate imiquimod-induced changes in the expression of MMPs and TIMPs.
METHODS
Invasiveness of melanoma cell lines following imiquimod treatment was evaluated by invasion assays. In order to investigate the mechanism of the anti-invasive effect of imiquimod, mRNA and protein levels of MMP-2, -9, membrane type 1 (MT1)-MMP, TIMP-1, and -2 were assessed by real-time reverse transcription-polymerase chain reaction, gelatin zymography, and western blotting.
RESULTS
Imiquimod treatment decreased in vitro viability of melanoma cells in a concentration-dependent manner. Imiquimod also elicited a concentration-dependent suppression of invasion in both melanoma cell lines. A concentration-dependent decrease in MMP-2 and MT1-MMP protein levels and a concentration-dependent increase in TIMP-1 and -2 protein levels by imiquimod was observed in both melanoma cell lines. However, expression of MMP-9 protein was increased in SK-MEL-2 but decreased in SK-MEL-24 with increasing imiquimod concentrations. Imiquimod elicited alterations in MMPs and TIMPs mRNA levels that parallel the observed changes in protein levels.
CONCLUSION
Imiquimod may elicit an anti-invasive effect on human melanoma cells by regulating MMPs and TIMPs.

Keyword

Imiquimod; Invasion; Melanoma; Matrix metalloproteinase; Tissue inhibitor of metalloproteinase

MeSH Terms

Basement Membrane
Blotting, Western
Cell Line
Extracellular Matrix
Gelatin
Humans
Matrix Metalloproteinase 14
Matrix Metalloproteinases*
Melanoma*
Membranes
Metalloproteases*
Neoplasm Metastasis
Peptide Hydrolases
RNA, Messenger
Tissue Inhibitor of Metalloproteinase-1
Gelatin
Matrix Metalloproteinase 14
Matrix Metalloproteinases
Metalloproteases
Peptide Hydrolases
RNA, Messenger
Tissue Inhibitor of Metalloproteinase-1

Figure

  • Fig. 1 In vitro cell viability of melanoma cell lines SK-MEL-2 (A) and SK-MEL-24 cells (B) following incubation with a range of concentrations of imiquimod. The graphical representation of the dose-effect relationship depicts a concentration-dependent inhibition in the percent viability of cells following imiquimod treatment (IC50 values: 56.32 µg/ml for SK-MEL-2, 62.36 µg/ml for SK-MEL-24).

  • Fig. 2 Invasion assay performed in SK-MEL-2 and SK-MEL-24 cell lines following imiquimod treatment. Suppression of SK-MEL-2 cell migration and invasion following incubation with a range of concentrations of imiquimod (A) 0 µg/ml, (B) 10 µg/ml, and (C) 30 µg/ml imiquimod (A~C: Coomassie stain, ×200). (D) The percentage of invasive SK-MLE-2 melanoma cells was significantly decreased with increasing concentrations of imiquimod (*p<0.05). Suppression of SK-MEL-24 cell migration and invasion following treatment with a range of concentrations of imiquimod (E) 0 µg/ml, (F) 10 µg/ml, and (G) 30 µg/ml imiquimod (E~G: Coomassie stain, ×200). (H) The percentage of invasive SK-MLE-24 melanoma cells was significantly decreased with increasing concentrations of imiquimod (*p<0.05). Con A: concanavalin A.

  • Fig. 3 Expression of matrix metalloproteinase (MMP)-2 and -9 protein and mRNA in SK-MEL-2 and SK-MEL-24 cell lines following incubation with imiquimod. Expression of MMP-2 decreased with increasing concentrations of imiquimod in SK-MEL-2 cells (A) and SK-MEL-24 cells (B). (C) MMP-9 expression in SK-MEL-2 cells was increased by imiquimod treatment in a dose-dependent manner. (D) However, expression of MMP-9 was dose-dependently decreased in SK-MEL-24 cells treated with imiquimod. Conditioned media of the HT1080 cell line was used as a positive control. Relative mRNA expression of MMP-2 in SK-MEL-2 (E) and SK-MLE-24 (F) cells and MMP-9 in SK-MEL-2 (G) and SK-MLE-24 (H) cells, following incubation with a range of concentrations of imiquimod (5, 10, and 30 µg/ml), compared to controls (0 µg/ml). *p<0.05 vs. control.

  • Fig. 4 Expression of membrane type 1 matrix metalloproteinase (MT1-MMP) protein and mRNA in SK-MEL-2 and SK-MEL-24 cell lines following treatment with imiquimod. Expression of MT1-MMP proteins in SK-MEL-2 (A) and SK-MEL-24 (B) cell lines was dose-dependently decreased with increasing concentrations of imiquimod. Relative mRNA expression of MT1-MMP in SK-MEL-2 (C) and SK-MEL-24 (D) cell lines, following treatment with a range of imiquimod concentrations (5, 10, 30 µg/ml), compared to controls (0 µg/ml). Con A: concanavalin A. *p<0.05 vs. control.

  • Fig. 5 Expression of tissue inhibitors of metalloproteinase (TIMP)-1 and -2 protein and mRNA in SK-MEL-2 and SK-MEL-24 cell lines following incubation with imiquimod. Expression of TIMP-1 protein was dose-dependently increased by imiquimod in SK-MEL-2 (A) and SK-MEL-24 cells (B). Expression of TIMP-2 protein was also dose-dependently elevated by imiquimod in SK-MEL-2 (C) and SK-MEL-24 cells (D). Relative mRNA expression of TIMP-1 in SK-MEL-2 (E) and SK-MLE-24 (F) cells and TIMP-2 in SK-MEL-2 (G) and SK-MLE-24 (H) cells, following treatment with a range of concentrations of imiquimod (5, 10, and 30 µg/ml), compared to controls (0 µg/ml). *p<0.05 vs. control.


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