Relationship Between Mitochondrial Alterations And Malignant Phenotypes Of Human Lung Cancer Metastatic Model Cell Lines

Mitochondrial dysfunction is one of the most profound features of cancer cells.Mitochondrial DNA(mtDNA) mutations have been found in a wide range of tumors. We conducted a study on mitochondrial differneces between two homogeneous lung cancer cell lines with differential metastatic capability. Two rho ~ derivatives of PLA801C and PLA801D were generated. Mitochondrial fluorescence was semi-quantitated using laser confocal microscopy while cells were stained with Rh123. Agarose colony formation assays and Transwell invasion assays were carried out to detect the phenotypes of colony formation and invasiveness of the cultured cells respectively. Cell growth assays were determined by MTT method.mtDNA D-loop was amplified and sequenced. Our results showed that the rho ~ cells exhibited stronger capablity of colony formation and invasiveness, and faster growth rates than their respective parental cell lines. PLA801C cells have lower mitochondrial fluorescence density than PLA801D,but there were not significant differences between rho " cells and their respective parental cell lines. Analysis of mtDNA D-loop sequences revealed that a number of point mutations had occurred, including A→G,G→A,T→C and C→T ,and G insertion at np316. In addition, A46G, T16285C and C insertion at np216 were also found in PLA801C mtDNA D-loop, whereas only C insertion at np271 was detected in PLA801D. Sequencing of mtDNA D-loop of rho " cells confirmed that several small fragmentary deletions had occurred. For comparative analysis of expression of mitochondrial proteins of the lung cancermetastatic model cell lines,mitochondria were isolated and purified,mitochondrial proteins were quantitated by Bradford method.2-D electrophoresis was performed,and spots in the gels were trypsinized in-gel.Peptide mass fingerprint and N-terminal sequencing of some spots were identified by MALDI-TOF-MS.Protein data were searched online.The results showed:expression of mitochondrial proteins of both two cell lines were significantly different;Expressions of Annexin A2 and cyclophilin A in PLA801C were significantly higher than that in PLA801D.We conclude that mitochondrial alterations and differential expressions of mitochondrial proteins might contribute to malignant phenotypes of lung cancer.