Abstract:

Objective To explore the effects of HINT1 on the proliferation and apoptosis of human melanoma cell line A375. Methods Three cell clones were used in the experiment, including A375 cells that were previously transfected with eukaryotic expressing vector pcDNA.3.1/myc-His（-）A-HINT1 and highly expressed HINT1 （HINT1-A375）, A375 cells transfected with pcDNA.3.1/myc-His（-）A empty vector （neo-A375） and untransfected A375 cells. MTT assay, flow cytometry and terminal deoxynucleotidyl transferase （TdT）-mediated dUTP nick end labeling （TUNEL） were performed to detect the proliferation, cell cycle and apoptosis of the three kinds of cells, respectively. The relative activity of Caspase 3, 8 and 9 was measured by spectrophotometry, and the protein expression of Bcl-2, Bax, Cytochrome C and p53 by Western blot. Results Compared with neo-A375 and untransfected A375 cells, HINT1-A375 cells grew more slowly （P < 0.05） with an increase in G1-phase population （73.17% ± 3.99%, F = 25.65, P < 0.05）, a decrease in S-phase population（16.75% ± 1.62%, F = 75.48, P < 0.01）, and a pronounced late apoptosis （23.57% ± 9.58%, F = 11.71, P < 0.01）. A significant increase was also observed in the percentage of apoptotic cells （12% ± 1%, F = 358.02, P < 0.01） as well as in the relative activity of Caspase 9 （0.45 ± 0.03, F = 135.62, P < 0.01） and Caspase 3 （0.46 ± 0.04, F = 90.28，P < 0.01） in HINT1-A375 cells compared with the other two kinds of cells. Western blot showed upregulated expressions of Bax, Cytochrome C and p53 but downregulated expression of Bcl-2 in HINT1-A375 cells. Conclusions The overexpression of HINT1 could inhibit the proliferation and promote the apoptosis of A375 cells with cell cycle arrest in G1 phase, hinting that HINT1 may be a tumor suppressor gene in human melanoma.

Key words: HINT1, Melanoma, Proliferation, Apoptosis