101F6 is a candidate tumor suppressor gene harbored on chromosome 3p21.3, a region with frequent and early allele loss and genetic alterations in many human cancers. We previously showed that enforced expression of wild-type 101F6 by adenoviral vector-mediated gene transfer significantly inhibited tumor cell growth in 3p21.3-deficient non-small cell lung cancer (NSCLC) cells in vitro and in vivo. The molecular mechanism of 101F6-mediated tumor suppression is largely unknown. A computer-aided structural and functional model predicts the 101F6 protein to be a member of the cytochrome b561 protein family that is involved in the regeneration of the antioxidant ascorbate. 101F6 protein is expressed in normal lung bronchial epithelial cells and fibroblasts but is lost in most lung cancers. Treatment with 101F6 nanoparticle-mediated gene transfer in combination with a subpharmacologic dose (200-500 micromol/L) of ascorbate synergistically and selectively inhibited lung cancer cell growth in vitro. Systemic injection of 101F6 nanoparticles plus the i.p. injection of ascorbate synergistically inhibited both tumor formation and growth in human NSCLC H322 orthotopic lung cancer mouse models (P<0.001). Furthermore, exogenous expression of 101F6 enhanced intracellular uptake of ascorbate, leading to an accumulation of cytotoxic H(2)O(2) and a synergistic killing of tumor cells through caspase-independent apoptotic and autophagic pathways. The antitumor synergism showed by the combination treatment with systemic administration of 101F6 nanoparticles and ascorbate on lung cancer offers an attractive therapeutic strategy for future clinical trials in cancer prevention and treatment.