Abstract
Mitochondria play a crucial role in pathways of stress conditions. They can be transported from one cell to another, bringing their features to the cell where they are transported. It has been shown in cancer cells overexpressing multidrug resistance (MDR) that mitochondria express proteins involved in drug resistance such as P-glycoprotein (P-gp), breast cancer resistant protein and multiple resistance protein-1. The MDR phenotype is associated with the constitutive expression of COX-2 and iNOS, whereas celecoxib, a specific inhibitor of COX-2 activity, reverses drug resistance of MDR cells by releasing cytochrome c from mitochondria. It is possible that COX-2 and iNOS are also expressed in mitochondria of cancer cells overexpressing the MDR phenotype. This study involved experiments using the human HCC PLC/PRF/5 cell line with and without MDR phenotype and melanoma A375 cells that do not express the MDR1 phenotype but they do iNOS. Western blot analysis, confocal immunofluorescence and immune electron microscopy showed that iNOS is localized in mitochondria of MDR1-positive cells, whereas COX-2 is not. Low and moderate concentrations of celecoxib modulate the expression of iNOS and P-gp in mitochondria of MDR cancer cells independently from inhibition of COX-2 activity. However, A375 cells that express iNOS also in mitochondria, were not MDR1 positive. In conclusion, iNOS can be localized in mitochondria of HCC cells overexpressing MDR1 phenotype, however this phenomenon appears independent from the MDR1 phenotype occurrence. The presence of iNOS in mitochondria of human HCC cells phenotype probably concurs to a more aggressive behaviour of cancer cells.
Highlights
P-glycoprotein (P-gp), multidrug-resistant protein 1 (MRP1) and breast cancer resistant protein (BCRP) are membrane proteins belonging to the family of multidrug-resistant (MDR) proteins that mediate intrinsic and acquired drug resistance in cancer cells
We have demonstrated that P-gp and BCRP are overexpressed and functionally active in the mitochondrial cristae of an MDR1-positive cancer cell line, and Roundhill et al have shown the presence of MRP1 with efflux activity in human mitochondria [4, 6, 9]
To investigate the presence of COX-2 and inducible nitric oxide synthase (iNOS) in mitochondria, pure fractions of mitochondria were prepared by differential density gradient centrifugation using density media – iodixanol or sucrose, and the fractions of each centrifugation step were analysed by Western blot (Fig. 2A)
Summary
P-glycoprotein (P-gp), multidrug-resistant protein 1 (MRP1) and breast cancer resistant protein (BCRP) are membrane proteins belonging to the family of multidrug-resistant (MDR) proteins that mediate intrinsic and acquired drug resistance in cancer cells. It has been shown that P-gp activity is related to the expression of the inducible isoform of the cyclooxygenase-2 (COX-2) enzyme. The cyclooxygenase pathway plays an important role in the regulation of inflammation and cancer, and COX-2 appears to be important in tumour growth, metastasis process and tumour drug resistance [11, 12]. We have demonstrated that MDR1-positive cancer cells express constitutively COX-2 and that celecoxib, a specific inhibitor of COX-2 activity, can reverse drug resistance to apoptosis of these cells by a P-gp-dependent but COX2-independent mechanism in MDR1-positive cells [15, 16]
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