For real-time PCR, we used commercially available primers selleckchem CHIR99021 designed against human MT1 (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_005958″,”term_id”:”60498974″,”term_text”:”NM_005958″NM_005958, SABiosciences) (45), MT2 (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_005959″,”term_id”:”69122993″,”term_text”:”NM_005959″NM_005959, SABiosciences) (44), and glyceraldehyde-3-phosphate dehydrogenase (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_002046″,”term_id”:”576583510″,”term_text”:”NM_002046″NM_002046, SABiosciences) (37) genes. A ����CT analysis (34) was performed using H69 as the control sample. Data are expressed as relative mRNA levels �� SE (n = 3). In Vivo Studies Male BALB/c 6-wk-old nude (nu/nu) mice (Taconic Farms, Hudson, NY) were kept in a temperature-controlled environment (20�C22��C) with 12:12-h light-dark cycles, fed standard chow ad libitum, and with free access to drinking water.

Mz-ChA-1 cells (5 �� 106) were suspended in 0.2 ml of extracellular matrix gel and injected subcutaneously in the rear flanks of the nude mice (16). After the establishment of the tumor (1 wk with a tumor size of ~5 mm3), mice (n = 4) were treated with vehicle (5% ethanol in water) or melatonin (4 mg/kg body wt, dissolved in 5% aqueous ethanol solution) (51) by intraperitoneal injections three times a week. Tumor variables were measured three times a week by an electronic caliper, and volume was determined as follows: tumor volume (mm3) = 0.5 �� [length (mm) �� width (mm) �� height (mm)]. The measurements started from the first week when the tumor mass was established.

After 43 days of treatment, they were anesthetized with pentobarbital sodium (50 mg/kg body wt ip), and tumor tissues were harvested. Heart, liver, and kidney tissues were collected for evaluation of damage by hematoxylin-eosin staining of paraffin-embedded sections (4- to 5-��m thick). All animal experiments were performed in accordance with a protocol approved by the Scott & White and Texas A&M Health Science Center Institutional Animal Care and Use Committee and conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (publication no. 85�C23, revised 1996). Tumors were allowed to grow until maximum allowable tumor burden was reached, as set forth by the Scott & White and Texas A&M Health Science Center Institutional Animal Care and Use Committee tumor burden policy.

Tumor tissues were fixed for 4 h in 10% buffered formalin and embedded in low-temperature fusion paraffin. Subsequently, sections (4�C5 ��m) were stained 1) Anacetrapib with hematoxylin-eosin for evaluation of necrosis (16); and 2) for proliferating cell nuclear antigen (PCNA) (a marker of proliferation) (16), cleaved caspase-3 (for measurement of apoptosis) (4), AANAT, ASMT, melatonin, MT1, and MT2 by immunohistochemistry (16).