Analysis of Cellular Sub-Fractions in Lung Cancer: A Proteomics Approach
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Objective: Cancer homeostasis may be dependent upon intracellular maintenance of the cell's intrinsic apoptotic pathways. Recent investigations have led to further inquiries as to the role of mitochondria in the regulation of apoptosis. The objective of this study is to determine the feasibility of applying the current techniques of proteomics analysis to the mitochondrial sub-fraction in human lung cancers.
Methods: Human non-small cell lung cancer cell lines were incubated in appropriate media and harvested at sub-confluency. Enriched mitochondrial preparations were obtained after mechanical and chemical cell membrane rupture, followed by differential sucrose gradient separation. Mitochondrial isolation was confirmed using Western blotting for cytochrome c and measurement of cytochrome c oxidase enzymatic activity. Cytosolic fractions were also obtained for comparison. The protein sub-fractions were dispersed via 2-D gel electrophoresis in preparation for mass spectrometric analysis (MALDI), which was used to identify individual proteins.
Results: Qualitative and quantitative comparisons of mitochondrial proteins between cancer cell lines revealed that individual proteins can be readily identified as being unique to one cell line or another. Moreover, a great number of these proteins unique to one cell line teneded to reside in the mitochondrial sub-fraction.
Conclusions: Application of a proteomics approach to analyzing the mitochondrial sub-fraction in lung cancer is feasible and yields intriguing results which may further support a role for mitochondria in the regulation of cellular homeostasis in cancer.
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