5-Azacytidine Reduces Growth, Invasiveness, and Clonogenicity of Uveal Melanoma Cells and Decreases the Rate of Metastasis

Abstract

 

Purpose

 

Uveal Melanoma is the most common primary intraocular malignancy in adults. About 50% of cases are complicated by lethal metastases. Epigenetic silencing of tumor suppressor genes is an important factor in tumorigenesis and metastasis of many cancers. Promoter methylation is an epigenetic mechanism that can alter target gene transcription. A number of tumor suppressors have been shown to be methylated in uveal melanoma; such epigenetic modifications may contribute to pathogenicity. We hypothesized that demethylation of uveal melanoma cells may cause a decrease in growth, invasiveness, clonogenicity, and reduce metastatic transformation.

 

Methods

 

We used 5-azacytidine (5-Aza), a drug approved for therapy in myelodysplastic syndrome to demethlyate DNA in uveal melanoma cells. Invasiveness was characterized using transwell migration assays. MTS assays were used to analyze cell growth. Clonogenicity was examined using soft colony agar assays. Metastasis formation was assayed using a murine melanoma intraocular xenograft model which metastasizes hematogenously.

 

Results

 

Treatment of OCM3, 92.1, OCM1, OMM1, Mel285, and Mel290 cells with 1 uM or 2 uM of 5-Aza caused decreased growth compared to DMSO controls at day 7 (p<0.05). Transwell migration assays of OCM3 cells treated with 0.5 uM or 1 uM of 5-Aza demonstrated a decrease in invasion compared to controls (p<0.001). Clonogenicity assays revealed that OCM3, 92.1, and OCM1 cells pretreated with 0.5 uM or 1 uM 5-Aza formed fewer and smaller colonies than controls (p<0.001). In vivo assays of 15 C57Bl6 mice injected with vehicle-treated B16F10 cells and 14 mice injected with 5-Aza-treated cells revealed significantly fewer lung metastases in the latter group.

 

Conclusions

 

Using 5-Aza to demethylate DNA in uveal melanoma cells reduces growth, invasiveness, and clonogenicity in vitro; additionally, treatment with 5-Aza decreases metastasis formation in vivo. These results provide proof of concept for an exciting potential therapy to reduce mortality from this disease. Future work will focus on identifying pathways that mediate these changes.

 

 

A. 5-Aza decreases cell growth in 6 cell lines. B. Transwell migration assays. C. Treatment of OCM3 cells with 5-Aza decreases invasion.

 

 

A. Clonogenicity assays in OCM3, 92.1, and OCM3 lines. B. 5-Aza decreases the number of colonies formed. C. 5-Aza decreases the size of colonies formed.

 

Keywords: 589 melanoma • 624 oncology • 744 tumors