However, in vitro testing of adaphostin in the NCI 60 cell line panel indi cated that several solid tumor cancer cell lines also dem onstrated considerable sensitivity to adaphostin, indicating there may be a role for adaphostin in treatment of solid tumors. The prostate tumor cell line, PC3 was published as a model to demonstrate signaling cascades involved in adaphostin induced growth inhibition and cell cycle arrest, but this cell line is an order of magni tude more resistant than the lung tumor model NCI H522 to the growth inhibitory effects of the drug in the NCI 60 human tumor cell line screen. An early report showed an anti tumor effect on an orthotopic glioblastoma model U87, in combination with the Flt 1/Fc chimera, and more recent evaluation of adaphostin activity in glioblas toma cell lines identified a high level of HMOX1 induc tion.

HMOX1 is the first and rate limiting step in the degradative pathway of heme, but has also been recog nized as an integral part of a cytoprotective mechanism against oxidative stress. HMOX1 is a target gene of the basic leucine zipper transcription factor, nuclear factor erythroid 2 like 2, Nrf2, a central regulator of cellular oxidative stress response and repre sents an adaptive response that increases cell resistance to oxidative injury. Nrf2 is readily induced in response to ROS through the Nrf2 ARE pathway which transcrip tionally up regulates antioxidant genes in order to protect cells. Nrf2 is regulated through PI3K/AKT pathway, and translocated into the nucleus where it binds to the antioxidant responsive element which results in activation of this enhancer element and initiates the transcription of genes encoding phase II detoxifica tion enzymes.

These enzymes initiate an antioxidant response, which can be beneficial for cancer prevention. However, the Nrf2 ARE pathway has recently been implicated in chemoresistance and the feasibility of Nrf2 inhibition as a strategy for sensitizing cells to chemother apeutics was demonstrated. HMOX1 upregula tion has been identified in the adaphostin response in adherent cell lines, but not in hematopoietic cell line Anacetrapib models, and it appears that adaphostin activates a differ ent oxidative stress response in solid tumor models than in leukemia models.

Thus, we have investigated the mechanism behind HMOX1 induction in the adaphostin sensitive lung tumor cell line NCI H522, and demon strated an enhancement of adaphostin toxicity following inhibition of Nrf2 nuclear translocation with the PI3K inhibitor wortmannin. Methods Drugs and Cell Culture Adaphostin and wortmannin were obtained from the repository of the National Cancer Institutes Developmental Therapeutics Program. Desferrioxamine and N acetyl cysteine were purchased from Sigma. NCI H522, and the leukemia cell lines, were obtained from the NCI 60 Human Tumor Cell Line Screen.