no studies have addressed the effect of mTOR inhibitors on ovarian cancer cells that have acquired resistance following the contact with platinum agents. More over, since most tumor specimens Decitabine Antimetabolites inhibitor and tumor derived cell lines utilized in these investigations have now been ovarian SACs, the position of mTOR in CCC remains largely as yet not known. It has been noted that lack of PTEN expression is widespread in CCC of the ovary. In addition it is reported that ovarian endometriosis, from which CCC is considered to occur, is seen as a hyperactivation of the AKT mTOR pathway. CCC may be a good candidate for therapy with a mTOR inhibitor, as it is well known that loss of PTEN expression and consequent activation of AKT signaling end in hypersensitivity to mTOR inhibition. In the present investigation, we examined the activation status of mTOR both in early stage and high level stage CCC, and we decided whether RAD001 has anti-neoplastic effectiveness in both in vitro and in vivo models of CCC. More over, we examined the function of AKT/mTOR signaling in the acquired resistance to cisplatin in CCC cells. Methods and materials Reagents/Antibodies Gene expression RAD001 was obtained from Novartis Pharma AG. ECL Western blotting detection reagents were from Perkin Elmer. Antibodies recognizing phospho p70S6K, p70S6K, mTOR, phospho mTOR, AKT, phospho AKT, PARP, LC3B and B actin were obtained from Cell Signaling Technology. The Cell Titer 96 well growth assay kit was obtained from Promega. Cisplatin was obtained from Sigma. Drug Preparation RAD001 was produced at a day later in a microemulsion vehicle. RAD001 was prepared according to the company s standards. Therefore, for animal reports, RAD001 was diluted to the correct focus in double distilled water right before administration by gavage. For in vitro studies, RAD001 was prepared in DMSO before addition to cell cultures. Clinical trials All surgical specimens were collected Cediranib solubility and archived in accordance with standards permitted by the institutional review boards of the parent institutions. Proper informed consent was obtained from each patient. The tumors involved 46 SACs and 52 CCCs. Based on criteria of the International Federation of Obstetrics and Gynecology criteria, 22 SACs were stage I 24 and II tumors were stage III IV tumors. Among CCCs, 27 were stage I II tumors and 25 were stage III IV tumors. Immunohistochemistry Cyst samples were fixed in 10 percent neutral buffered formalin overnight and then embedded in paraffin. In all individuals, the diagnosis was predicated on a light microscopy examination using mainstream hematoxylin and eosin stain. Ovarian cancer muscle microarrays composed of two cores from each tumor sample were prepared by the Tumor Bank Facility at Fox Chase Cancer Center, as described previously.