Melatonin (N-acetyl-5-methoxytryptamine), the major research use secretory hormone of the pineal gland (42), is also secreted from several extrapineal tissues, such as gastrointestinal tract and liver (21). Melatonin is synthesized from serotonin by two enzymes, serotonin N-acetyltransferase or arylalkylamine N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT) (23). There are three melatonin membrane receptor subtypes, MT1, MT2, and MT3 (found only in nonmammals), through which melatonin exerts its multiple effects (59). Melatonin receptors are present in the central nervous system, as well as peripheral tissues, including small intestine and hepatocytes (36, 40). There is growing evidence that cholangiocytes secrete a number of neuroendocrine factors (e.g.

, dopamine and serotonin) regulating the neoplastic growth of cholangiocytes by autocrine mechanisms (1, 10). A number of studies have shown that melatonin secretion is impaired in patients suffering from breast cancer, endometrial cancer, and colorectal cancer (49, 53, 58), suggesting that reduced melatonin levels are linked to higher incidence of several types of tumors. However, no data exist regarding the role of melatonin as an autocrine regulator of CCA growth. In the liver, melatonin protects against cholestatic oxidative stress in rats during cholestasis (13) and reduces the regeneration and stimulated the apoptosis of hepatocytes in hepatectomized rats (26). In support of the specificity of the present study, we have shown that melatonin inhibits biliary hyperplasia in cholestatic rats (43).

Melatonin is also chemopreventive against O. viverrini-induced CCA by reducing oxidative and nitrosative DNA damage in rabbits (32). On the basis of this background, we hypothesize that 1) in CCA, there is a decrease in the expression of AANAT and ASMT and the secretion of melatonin, changes that induce an increase in CCA growth by an autocrine mechanism; and 2) increases in the biliary expression of melatonin (by in vivo administration of melatonin or in vitro overexpression of AANAT in CCA cells) decreases CCA growth. MATERIALS AND METHODS Reagents were purchased from Sigma (St. Louis, MO), unless differently indicated. The antibodies against AANAT (FL-207), ASMT (C-20), MEL-1A-R (V-15), and MEL-1B-R (T-18) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), unless differently indicated.

The rabbit polyclonal antibody against melatonin (ab35137) was purchased AV-951 from Abcam (Cambridge, MA). The ELISA kits for detecting melatonin levels in nonmalignant and CCA cell lines, and serum and bile from controls and CCA patients, were purchased from GenWay Biotech. In Vitro Studies Cell lines. We used six human CCA cell lines (Mz-ChA-1, HuH-28, TFK-1, CCLP1, SG231, and HUCC-T1) with different biliary origin. Mz-ChA-1 cells (from human gallbladder) (28) were a gift from Dr. G. Fitz (University of Texas Southwestern Medical Center, Dallas, TX).