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Giant Lysosomes as a Chemotherapy Resistance Mechanism in Hepatocellular Carcinoma Cells.

Colombo F, Trombetta E, Cetrangolo P, Maggioni M, Razini P, De Santis F, Torrente Y, Prati D, Torresani E, Porretti L - PLoS ONE (2014)

Bottom Line: ABC expression analyses showed that the main ABC protein harboured by all of the cell lines was PGP, whose expression was not limited to the cell membrane but was also found on lysosomes.The findings of this study demonstrate the involvement of PGP-positive lysosomes in drug sequestration and MDR in HCC cell lines.The possibility of modulating this mechanism using PGP inhibitors could lead to the development of new targeted strategies to enhance HCC treatment.

View Article: PubMed Central - PubMed

Affiliation: Clinical Chemistry and Microbiology Laboratory, Flow Cytometry and Experimental Hepatology Service, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.

ABSTRACT
Despite continuous improvements in therapeutic protocols, cancer-related mortality is still one of the main problems facing public health. The main cause of treatment failure is multi-drug resistance (MDR: simultaneous insensitivity to different anti-cancer agents), the underlying molecular and biological mechanisms of which include the activity of ATP binding cassette (ABC) proteins and drug compartmentalisation in cell organelles. We investigated the expression of the main ABC proteins and the role of cytoplasmic vacuoles in the MDR of six hepatocellular carcinoma (HCC) cell lines, and confirmed the accumulation of the yellow anti-cancer drug sunitinib in giant (four lines) and small cytoplasmic vacuoles of lysosomal origin (two lines). ABC expression analyses showed that the main ABC protein harboured by all of the cell lines was PGP, whose expression was not limited to the cell membrane but was also found on lysosomes. MTT assays showed that the cell lines with giant lysosomes were more resistant to sorafenib treatment than those with small lysosomes (p<0.01), and that verapamil incubation can revert this resistance, especially if it is administered after drug pre-incubation. The findings of this study demonstrate the involvement of PGP-positive lysosomes in drug sequestration and MDR in HCC cell lines. The possibility of modulating this mechanism using PGP inhibitors could lead to the development of new targeted strategies to enhance HCC treatment.

No MeSH data available.


Related in: MedlinePlus

Cytoplasmic vesicle localisation.Fluorescence microscopy images confirming intra-cellular vesicle localisation. (A) Hcc-1, (B) HepG2, (C) Hep3B, and (D) SNU475 cells. Sunitinib autofluorescence (green), CD29 staining (red) and Hoechst 33342 nuclear staining (blue). Original magnification 40x.
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pone-0114787-g002: Cytoplasmic vesicle localisation.Fluorescence microscopy images confirming intra-cellular vesicle localisation. (A) Hcc-1, (B) HepG2, (C) Hep3B, and (D) SNU475 cells. Sunitinib autofluorescence (green), CD29 staining (red) and Hoechst 33342 nuclear staining (blue). Original magnification 40x.

Mentions: Fig. 2 clearly shows the intra-cellular localisation of green vesicles (sunitinib autofluorescence) after staining the cytoplasmic membranes of HCC cells with β1 integrin. No Oil-red-O staining of the cytoplasmic vesicles in any of the cell lines was observed (Fig. 3), thus ruling out the possibility that they were mere lipid droplets. Furthermore, cell pre-incubation with NH4Cl did not allow sunitinib accumulation in cell vesicles, while sunitinib-loaded vesicles released the drug after NH4Cl addition in culture medium, thus suggesting a lysosomal origin of cell vesicles (S1 Figure). Moreover, we also observed an increased number of lysosomes in Hcc-1, HepG2, PLC/PRF/5 and HuH7 after NH4Cl incubation.


Giant Lysosomes as a Chemotherapy Resistance Mechanism in Hepatocellular Carcinoma Cells.

Colombo F, Trombetta E, Cetrangolo P, Maggioni M, Razini P, De Santis F, Torrente Y, Prati D, Torresani E, Porretti L - PLoS ONE (2014)

Cytoplasmic vesicle localisation.Fluorescence microscopy images confirming intra-cellular vesicle localisation. (A) Hcc-1, (B) HepG2, (C) Hep3B, and (D) SNU475 cells. Sunitinib autofluorescence (green), CD29 staining (red) and Hoechst 33342 nuclear staining (blue). Original magnification 40x.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4262459&req=5

pone-0114787-g002: Cytoplasmic vesicle localisation.Fluorescence microscopy images confirming intra-cellular vesicle localisation. (A) Hcc-1, (B) HepG2, (C) Hep3B, and (D) SNU475 cells. Sunitinib autofluorescence (green), CD29 staining (red) and Hoechst 33342 nuclear staining (blue). Original magnification 40x.
Mentions: Fig. 2 clearly shows the intra-cellular localisation of green vesicles (sunitinib autofluorescence) after staining the cytoplasmic membranes of HCC cells with β1 integrin. No Oil-red-O staining of the cytoplasmic vesicles in any of the cell lines was observed (Fig. 3), thus ruling out the possibility that they were mere lipid droplets. Furthermore, cell pre-incubation with NH4Cl did not allow sunitinib accumulation in cell vesicles, while sunitinib-loaded vesicles released the drug after NH4Cl addition in culture medium, thus suggesting a lysosomal origin of cell vesicles (S1 Figure). Moreover, we also observed an increased number of lysosomes in Hcc-1, HepG2, PLC/PRF/5 and HuH7 after NH4Cl incubation.

Bottom Line: ABC expression analyses showed that the main ABC protein harboured by all of the cell lines was PGP, whose expression was not limited to the cell membrane but was also found on lysosomes.The findings of this study demonstrate the involvement of PGP-positive lysosomes in drug sequestration and MDR in HCC cell lines.The possibility of modulating this mechanism using PGP inhibitors could lead to the development of new targeted strategies to enhance HCC treatment.

View Article: PubMed Central - PubMed

Affiliation: Clinical Chemistry and Microbiology Laboratory, Flow Cytometry and Experimental Hepatology Service, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.

ABSTRACT
Despite continuous improvements in therapeutic protocols, cancer-related mortality is still one of the main problems facing public health. The main cause of treatment failure is multi-drug resistance (MDR: simultaneous insensitivity to different anti-cancer agents), the underlying molecular and biological mechanisms of which include the activity of ATP binding cassette (ABC) proteins and drug compartmentalisation in cell organelles. We investigated the expression of the main ABC proteins and the role of cytoplasmic vacuoles in the MDR of six hepatocellular carcinoma (HCC) cell lines, and confirmed the accumulation of the yellow anti-cancer drug sunitinib in giant (four lines) and small cytoplasmic vacuoles of lysosomal origin (two lines). ABC expression analyses showed that the main ABC protein harboured by all of the cell lines was PGP, whose expression was not limited to the cell membrane but was also found on lysosomes. MTT assays showed that the cell lines with giant lysosomes were more resistant to sorafenib treatment than those with small lysosomes (p<0.01), and that verapamil incubation can revert this resistance, especially if it is administered after drug pre-incubation. The findings of this study demonstrate the involvement of PGP-positive lysosomes in drug sequestration and MDR in HCC cell lines. The possibility of modulating this mechanism using PGP inhibitors could lead to the development of new targeted strategies to enhance HCC treatment.

No MeSH data available.


Related in: MedlinePlus