The tumor-suppressor gene cyclin-dependent kinase inhibitor 1B (P27) is downregulated in gastric cancer cells mainly through proteolytic degradation mediated by the SKP-Cullin1-F-Box (SCF) complex. But the correlation between its downregulation and gastric cancer prognosis still remains indefinite. MLN4924, an anti-tumor agent, which suppresses the SCF complex by inhibiting Cullin1 neddylation, emerges as a promising tool to elucidate its functions in gastric cancer cells. In this study, MLN4924 induced significant growth inhibition of gastric cancer cells in a dose-dependent manner, along with the simultaneous accumulation of P27 and cell cycle abnormalities such as G2/M arrest. Importantly, we found that P27 silencing in MLN4924-treated cells resulted in an enhancement of growth inhibition both in vitro and in vivo. Mechanism analysis revealed the antagonism effects of antioxidants to this excess apoptosis, suggesting reactive oxygen species (ROS) overproduction especially in the mitochondria was the principal cause of the augmentation. Moreover, the robust ROS attacked the mitochondria to initiate collapse of the mitochondrial membrane permeability and the exportation of apoptosis-inducing factor (AIF), IAP-binding mitochondrial protein (SMAC/DIABLO) and cytochrome c. Finally, we also found that P27 knockdown affected the expression profile of several critical BH3 family members to amplify the mitochondrial dysfunction and apoptosis. In summary, our findings unveiled a protective role of P27 by maintaining mitochondrial membrane permeability in MLN4924-treated gastric cancer cells, and therefore highlighted the potential combination of MLN4924 with P27 inhibition to improve its therapeutic efficacy.

ABSTRACT. This study was primarily undertaken to test the hypothesis that mitochondrial DNA (mtDNA) mutations may be associated with aplastic anemia (AA). We analyzed mtDNA sequences from 15 patients with AA. The samples were obtained from bone marrow, and patients' oral epithelial cells were collected for normal tissue comparison. Total DNA was amplified by PCR after extraction, and these segments were then sent for sequencing. The results were compared with those of oral epithelial tissues as well as mtDNA sequences in the revised Cambridge Reference Sequence (rCRS) database. We detected 61 heteroplasmic mutations in 11 genes, including those encoding NADH dehydrogenase (ND)1-2 and 4-6, tRNA glutamic acid (TRNE), ribosomal RNA (RNR) 1 and 2, cytochrome c oxidase (COX1), cytochrome b (CYTB), and tRNA glycine (TRNG); mutation rates were particularly high in ND2 2131©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 11 (3): 2130-2137 (2012) Mitochondrial DNA mutation in aplastic anemia (34.4%) and ND4 (21.3%) in the patients' mtDNA genomes. The products of these genes are involved in oxidation in the respiratory chain, and a large number of homoplasmic mutations were found. Interestingly, these 162 polymorphisms were mostly in the D-loop DNA structure (54.3%), in which numerous mutations associated with leukemia and myelodysplastic syndromes are found. We conclude that functional impairment of the mitochondrial respiratory chain induced by mutation may be an important reason for hematopoietic failure in AA patients.