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. 2015 Jun;19(6):1410-7.
doi: 10.1111/jcmm.12528. Epub 2015 Feb 18.

Mitochondria of a human multidrug-resistant hepatocellular carcinoma cell line constitutively express inducible nitric oxide synthase in the inner membrane

Ornella Fantappiè  1 , Chiara Sassoli  2 , Alessia Tani  2 , Daniele Nosi  2 , Serena Marchetti  3 , Lucia Formigli  2 , Roberto Mazzanti  1
Affiliations

Mitochondria of a human multidrug-resistant hepatocellular carcinoma cell line constitutively express inducible nitric oxide synthase in the inner membrane

Ornella Fantappiè et al. J Cell Mol Med. 2015 Jun.

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.

Keywords: COX-2; MDR; coxib; iNOS; mitochondrion.

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Figures

Figure 1
Figure 1
Expression of P-gp, COX-2, BCRP and iNOS in P5 and MDR1-positive P1(0.5) cells. (A) Western blot analysis of P-gp, COX-2, BCRP and iNOS expression in total cell lysate of P5 and P1(0.5) cells at basal conditions. β-actin was used as a protein-loading control. (B) P-gp, COX-2, BCRP and iNOS protein expression in different fractions of P1(0.5) cells obtained during the preparation of mitochondria by differential centrifugation following purification on an iodixanol or sucrose gradient as described in the Materials and methods section. WCS: whole cell lysate; LSP: low speed pellets; CM: crude mitochondria; MFI: mitochondrial fraction iodixanol; MFS: mitochondrial fraction sucrose. AIF and HSP-60 were used as mitochondrial marker proteins. One representative experiment out of three performed is shown.
Figure 2
Figure 2
Confocal immunofluorescence analysis of mitochondrial localization of iNOS in P5 and MDR1-positive P1(0.5) cells. Representative confocal immunofluorescence images of (A) P5 and (C) MDR1-positive P1(0.5) cells grown on glass coverslips, incubated with 100 nmol/l of MitoTracker red CMXRos to label mitochondria (red), fixed and immunostained for iNOS expression (green). Note the colocalization between the two fluorescent signals (yellow) indicating the mitochondrial localization of the iNOS. (B and D) Qualitative assessment of the colocalized points (white) obtained by using Image J colocalization Plugin Software (NIH). The images are representative of at least three separate experiments with similar results.
Figure 3
Figure 3
Immunoelectron cytochemistry analysis of iNOS protein in MDR1-positive P1(0.5) cells. Representative transmission electron microscopy image of Epon-812-embedded samples of MDR1-positive P1(0.5) cells processed for immunogold labelling of anti-iNOS antibody. The photograph shows a mitochondrion with the cristae decorated with iNOS-gold particles (arrows). The image represents at least three separate experiments with similar results.
Figure 4
Figure 4
Effect of celecoxib on the expression of P-gp, BCRP and iNOS in mitochondria of MDR1-positive P1(0.5) cells. Mitochondria of P1(0.5) cells were purified by using the fraction methodology based on iodixanol separation as described in the Materials and methods section. HSP-60 were used as a protein-loading control. One experiment representative of the three performed is shown.
Figure 5
Figure 5
Analysis of iNOS expression in MDR1-positive P1(0.5) cells cultured at basal condition (0) and after treatment with 10 or 50 μmol/l celecoxib for 6 hrs. (A) Western blot analysis of iNOS in total cell lysate. β-actin was used as a protein-loading control. (B–G) Representative confocal immunofluorescence images of MDR1-positive P1(0.5) cells grown on glass coverslips incubated with 100 nmol/l of MitoTracker red CMXRos to label mitochondria (red) fixed and immunostained for iNOS expression (green). The colocalization between the two fluorescent signals (yellow) indicates the mitochondrial localization of the iNOS. In C, E, G, qualitative assessment of the colocalized points (white) between the two fluorescent signals obtained by using Image J colocalization Plugin Software (NIH) is shown. Note that the treatment with 10 μM/l celecoxib (D and E) reduced iNOS expression as compared with the untreated cells (B and C), whereas the treatment with 50 μM/l celecoxib (F and G) did not modify its expression. The localization of iNOS at mitochondrial level did not vary upon celecoxib treatment (C, E and G). The images are representative of at least three separate experiments with similar results.
Figure 6
Figure 6
Analysis of expression of iNOS in A375 melanoma cells. (A) iNOS protein expression in different fractions of A375 cells obtained during the preparation of mitochondria by differential centrifugation following purification on an iodoxanol or sucrose gradient as described in the Materials and methods section. WCS: whole cell lysate; LSP: low speed pellets; CM: crude mitochondria; MFI: mitochondrial fraction iodixanol; MFS: mitochondrial fraction sucrose. AIF and HSP-60 were used as mitochondrial marker proteins. One representative experiment out of three performed is shown. (B) Representative confocal immunofluorescence images of A375 melanoma cells grown on glass coverslips, incubated with 100 nmol/l of MitoTracker red CMXRos to label mitochondria (red), fixed and immunostained for iNOS expression (green). Note the colocalization between the two fluorescent signals (yellow) indicating the mitochondrial localization of the iNOS and the qualitative assessment of the colocalized points (white) obtained by using Image J colocalization Plugin Software (NIH). The images are representative of at least three separate experiments with similar results.
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