Ecto-5'-nucleotidase (CD73) is an essential enzyme that generates adenosine, an essential molecule for cell growth. CD73 increases significantly in many breast cancers. In this study, α,β-methylene adenosine-5'-diphosphate (APCP), a specific CD73 inhibitor was used to block the hydrolase's activity. Effects of CD73 were examined on human breast cancer cells MDA-MB-231 in culture for proliferation, cell cycle progression, and apoptosis before and after APCP treatment. The in vivo effect of CD73 was examined on MDA-MB-231 tumor xenograft growth in nude mice. Cell growth curve, cell cycle and apoptosis were observed with MTT assays and flow cytometry, respectively. Microvessel density (MVD) and lymph vessel density (LVD) of implanted tumor tissues was analyzed by immunohistochemistry for CD31 and VEGFR-3 staining respectively. Our results showed that APCP inhibited MDA-MB-231 viability in a dose-dependent manner. APCP (12 µM) increased the percentage of G0/G1 phase cells from 49.75 to 59.16% while it decreased S phase and G2/M cells from 24.85 and 18.65% to 21.65 and 12.55%, respectively. The percentages of early and late apoptotic cells were also decreased after APCP treatment. However, APCP treatment did not affect the percentage of normal cells. Xenograft of MDA-MB-231 cells in the APCP treatment group had lower volume and weight than those of control group (2.70±1.14 vs 1.41±0.39 cm3 and 2.7±0.5 vs 1.3±0.2 g), accompanied with less vessel formation with a MVD of 5±1 compared to the control group's 10±2 and an LVD of 4+1 vs 7+2. Our results suggest that CD73 may promote tumor growth and serve as a marker of breast cancer progression.

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