Kenton Howard1, Karen K Lo1, Lihua Ao1, Fabia Gamboni1, Barish H Edil1, Richard Schulick1, Carlton C Barnett2. 1. Department of Surgery, Denver Health Medical Center, University of Colorado, Denver Campus, Denver, Colorado. 2. Department of Surgery, Denver Health Medical Center, University of Colorado, Denver Campus, Denver, Colorado. Electronic address: carlton.barnett@ucdenver.edu.
Abstract
BACKGROUND: Intercellular adhesion molecule-1 (ICAM-1) modulates cell-cell adhesion and is a receptor for cognate ligands on leukocytes. Upregulation of ICAM-1 has been demonstrated in malignant transformation of adenomas and is associated with poor prognosis for many malignancies. ICAM-1 is upregulated on the invasive front of pancreatic metastases and melanomas. These data suggest that the upregulated ICAM-1 expression promotes malignant progression. We hypothesize that the downregulation of ICAM-1 will mitigate tumor progression. METHODS: Mouse colon adenocarcinoma cells (MC38) were evaluated for the expression of ICAM-1 using Western immunoblot analysis. Short hairpin RNA (shRNA) transduction was used to downregulate ICAM-1. Tumor invasion determined via a modified Boyden chamber was used as a surrogate of tumor progression examining MC38 cells, MC38 ICAM-1 knockdowns, and MC38 transduced with vehicle control. The cells were cultured in full media for 24 h and serum-starved for 24 h. A total of 5 × 10(4) cells were plated and allowed to migrate for 24 h using full media with 10% fetal bovine serum as a chemoattractant. Inserts were fixed and stained with crystal violet. Blinded investigators counted the cells using a stereomicroscope. Statistical analysis was performed by analysis of variance with Fischer protected least significant difference and a P value of <0.05 was considered statistically significant. RESULTS: ICAM-1 was constitutively expressed on MC38 cells. Transduction with anti-ICAM-1 shRNA vector downregulated ICAM-1 protein expression by 30% according to the Western blot analysis (P < 0.03) and decreased ICAM-1 messenger RNA expression by 70% according to the reverse transcription-polymerase chain reaction. shRNA knockdown cells had a significant reduction in invasion >45% (P < 0.03). There were no significant differences between the invasion rates of MC38 and MC38 vehicle controls. CONCLUSIONS: Downregulation of ICAM-1 mitigates MC38 invasion. These data suggest that targeted downregulation of tumor ICAM-1 is a potential therapeutic target.
BACKGROUND:Intercellular adhesion molecule-1 (ICAM-1) modulates cell-cell adhesion and is a receptor for cognate ligands on leukocytes. Upregulation of ICAM-1 has been demonstrated in malignant transformation of adenomas and is associated with poor prognosis for many malignancies. ICAM-1 is upregulated on the invasive front of pancreatic metastases and melanomas. These data suggest that the upregulated ICAM-1 expression promotes malignant progression. We hypothesize that the downregulation of ICAM-1 will mitigate tumor progression. METHODS:Mousecolon adenocarcinoma cells (MC38) were evaluated for the expression of ICAM-1 using Western immunoblot analysis. Short hairpin RNA (shRNA) transduction was used to downregulate ICAM-1. Tumor invasion determined via a modified Boyden chamber was used as a surrogate of tumor progression examining MC38 cells, MC38ICAM-1 knockdowns, and MC38 transduced with vehicle control. The cells were cultured in full media for 24 h and serum-starved for 24 h. A total of 5 × 10(4) cells were plated and allowed to migrate for 24 h using full media with 10% fetal bovine serum as a chemoattractant. Inserts were fixed and stained with crystal violet. Blinded investigators counted the cells using a stereomicroscope. Statistical analysis was performed by analysis of variance with Fischer protected least significant difference and a P value of <0.05 was considered statistically significant. RESULTS:ICAM-1 was constitutively expressed on MC38 cells. Transduction with anti-ICAM-1 shRNA vector downregulated ICAM-1 protein expression by 30% according to the Western blot analysis (P < 0.03) and decreased ICAM-1 messenger RNA expression by 70% according to the reverse transcription-polymerase chain reaction. shRNA knockdown cells had a significant reduction in invasion >45% (P < 0.03). There were no significant differences between the invasion rates of MC38 and MC38 vehicle controls. CONCLUSIONS: Downregulation of ICAM-1 mitigates MC38 invasion. These data suggest that targeted downregulation of tumorICAM-1 is a potential therapeutic target.
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