fenretinide and Retinoblastoma

Retinoblastoma arises from a subset of developing retinal cells lacking the RB-1 gene product pRB, which have lost the ability to respond to apoptotic signals. A better understanding of retinoblastoma biological response to therapeutic agents with low toxicity could improve the development of novel approaches for treatment and prevention of the disease. Naturally occurring retinoids inhibit growth and induce differentiation of Y79 human retinoblastoma cells in vitro. The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) has been shown to induce apoptosis and/or necrosis of tumor cells of neuroectodermal origin. We examined the sensitivity of Y79 retinoblastoma cells to 4HPR in vitro, and in a xenograft model of tumor growth in nude mice in vivo. 4HPR treatment in the range 2.5 to 10 microM induced a loss of Y79 cell viability, as determined by crystal violet, trypan blue exclusion, and long-term clonogenic assays, and impairment of mitochondrial function detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Reactive oxygen species were elevated in 4HPR-treated cells and antioxidants rescued cell viability, indicating that 4HPR-induced cell death was mediated by oxidative stress. 4HPR inhibited growth of Y79 xenografts in vivo in both chemoprevention and intervention settings. Tumor growth inhibition by 4HPR was also associated with significant inhibition of angiogenesis in vivo. These findings could have an important translational value for chemoprevention or early intervention in the treatment of retinoblastoma.

Topics: Animals; Antineoplastic Agents; Cell Death; Cell Survival; Disease Models, Animal; Fenretinide; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms, Experimental; Neovascularization, Pathologic; Reactive Oxygen Species; Retinoblastoma; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR or fenretinide) is toxic to myeloid leukemia and cervical carcinoma cell lines, probably in part due to its ability to increase levels of reactive oxygen species (ROS). We have studied the effects of 4-HPR on neuroblastoma cell lines. Since neuroblastomas commonly relapse in bone marrow, a hypoxic tissue compartment, and many chemotherapeutic agents are antagonized by hypoxia, our purpose was to study in these cell lines several factors influencing 4-HPR-induced cytotoxicity, including induced levels of ROS, effects of physiologic hypoxia and antioxidants, levels of ceramide, and the mechanism of cell death.. ROS generation was measured by carboxydichlorofluorescein diacetate fluoresence. Ceramide was quantified by radiolabeling and thin-layer chromatography. Immunoblotting was used to assess p53 protein levels. Apoptosis (programmed cell death) and necrosis were analyzed by nuclear morphology and internucleosomal DNA fragmentation patterns. Cytotoxicity was measured by a fluorescence-based assay employing digital imaging microscopy in the presence or absence of the pancaspase enzyme inhibitor BOC-d-fmk. Statistical tests were two-sided.. In addition to increasing ROS, 4-HPR (2.5-10 microM) statistically significantly increased the level of intracellular ceramide (up to approximately 10-fold; P<.001) in a dose-dependent manner in two neuroblastoma cell lines, one of which is highly resistant to alkylating agents and to etoposide. Cell death induced by 4-HPR was reduced but not abrogated by hypoxia in the presence or absence of an antioxidant, N-acetyl-L-cysteine. Expression of p53 protein was not affected by 4-HPR. Furthermore, the pan-caspase enzyme inhibitor BOC-d-fmk prevented apoptosis, but not necrosis, and only partially decreased cytotoxicity induced by 4-HPR, indicating that 4-HPR induced both apoptosis and necrosis in neuroblastoma cells.. 4-HPR may form the basis for a novel, p53-independent chemotherapy that operates through increased intracellular levels of ceramide and that retains cytotoxicity under reduced oxygen conditions.

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Caspase Inhibitors; Ceramides; DNA Fragmentation; DNA, Neoplasm; Drug Interactions; Fenretinide; Free Radical Scavengers; Gene Expression Regulation, Neoplastic; Humans; Hypoxia; Necrosis; Reactive Oxygen Species; Retinoblastoma; Tumor Cells, Cultured; Tumor Suppressor Protein p53