4B). We demonstrated a decrease in CCA proliferation (by PCNA) and an increase in CCA selleckchem apoptosis (by cleaved caspase-3) in tumor sections from melatonin-treated mice compared with vehicle-treated mice (Fig. 4B). By immunohistochemistry in tumor sections, we found an increase in the expression of AANAT, ASMT, and melatonin, and a decrease in MT1/MT2 expression in nude mice treated with melatonin compared with controls (Fig. 4B). For semiquantitative data, see Table 2. Fig. 4. After 34 days of melatonin administration, there was a significant decrease in tumor size in nude mice treated with melatonin compared with mice treated with vehicle. Representative pictures of the tumors from vehicle- and melatonin-treated mice and a … Table 1.

Measurement of liver and body weight, and liver to body weight ratio, in nude mice treated with vehicle or melatonin in vivo Table 2. Semiquantitative data for the expression of necrosis, PCNA, cleaved caspase-3, melatonin, AANAT, ASMT, MT1, and MT2 in nude mice treated with vehicle or melatonin Effect of Overexpression of AANAT on the Proliferation, Apoptosis, and MT1/MT2 Expression of Mz-ChA-1 Cells Since 1) CCA lines proliferate at higher rate because they express lower amount of AANAT and secrete less melatonin compared with nonmalignant cholangiocytes, and 2) melatonin inhibits Mz-ChA-1 growth in athymic mice by upregulation of AANAT, we tested the hypothesis that in vitro overexpression of AANAT in Mz-ChA-1 reduces (similar to what is shown in vivo in nude mice) the proliferation and MT1/MT2 expression and increases apoptosis in these cells.

Stable transfection of AANAT cDNA into Mz-ChA-1 cells increased the mRNA expression of AANAT in these cells compared with Mz-ChA-1 cells transfected with the control vector (Fig. 5A). In Mz-ChA-1 cells (overexpressing AANAT), there was 1) a reduction in CCA proliferation (Fig. 5, B and C); 2) an increase in apoptosis (Fig. 5D); and 3) a decrease in the expression of MT1/MT2 (Fig. 5E) compared with Mz-ChA-1 cells transfected with the control vector (Fig. 5, B�CE). Fig. 5. A: AANAT AV-951 were stably overexpressed in Mz-ChA-1 cells, overexpression that was confirmed by real-time PCR. Values are means �� SE of 3 evaluations. Stable transfection of AANAT cDNA into Mz-ChA-1 cells increased the mRNA and protein expression of … DISCUSSION In this study, we found that melatonin synthesis is down regulated in CCA, and that melatonin inhibits CCA growth by enhanced biliary expression of AANAT, the key enzyme regulating melatonin synthesis (17). The data introduce the novel concept that decreased melatonin synthesis in CCA suppresses melatonin antiproliferative actions via an autocrine loop.