Characterization of Acidic Vesicles in Multidrug-resistant and Sensitive Cancer Cells by Acridine Orange Staining and Confocal Microspectrofluorometry

Abstract

SUMMARYTo study the pH gradient status through membranes of acidic vesicles, either in sensitive or in multidrug-resistant living cancer cells, we monitored the fluorescenceemission spectra of acridine orange. Successive stainings with a pH-sensitive dye and AO showed that low-pH organelles were stained red by AO. In these compartments, high AO concentrations are driven by the pH gradient through membrane vesicles. The resulting rise in the dye's oligomeric/monomeric ratio induced an increase in the red/green (655-nm/530-nm) emission intensity ratio. Therefore, the accumulation of AO in acidic organelles was appraised by determination of the contribution of the red emission intensity (R%) in each emission spectrum, using laser scanning confocal microspectrofluorometry. In vesicles of multidrug-resistant K562-R cells, R% is significantly higher (72 Ϯ 10%) than the value (48 Ϯ 8%) from K562-sensitive cells ( p Ͻ 0.001). This result is interpreted as a more important accumulation of AO in acidic cytoplasmic structures of resistant cells, which induces a shift from AO monomers (green emission) to self-associated structures (red emission). Equilibration of the pH gradient through acidic organelles was performed by addition of weak bases and carboxylic ionophores. Ammonium chloride (0.1 mM), methylamine (0.1 mM), monensine (10 M), or nigericine (0.3 M) all suppressed the initial difference of local AO accumulation between both cell lines. These agents decreased the red emission intensity for the resistant cell line but not for the sensitive one. The same effects were induced by 50 M verapamil, a pleiotropic drug-resistance modulator. Our data allow the hypothesis of a higher pH gradient through membranes of acidic organelles, which would be a potential mechanism of multidrug resistance via the sequestration of weak bases inside these organelles.

Na+/H+ exchanger activity is increased in doxorubicin‐resistant human colon cancer cells and its modulation modifies the sensitivity of the cells to doxorubicin

“…16 In addition, vacuolar type H þ -ATPase may be expressed at the plasma membrane of certain tumor cells, and the ability of its inhibitors bafilomycin A1 and concanamycin A to revert MDR in vitro has suggested that this pump could be responsible for the different vesicular acidification and sequestration of anthracyclines in breast and leukaemic resistant cells. [18][19][20][21] Our group has already demonstrated that the inhibition of NHE activity with the amiloride analogue EIPA may increase doxorubicin accumulation and improve the antiproliferative effect of the drug in a resistant human colon cell line. 10 In the present work, we provide further and more specific evidences in support of a link between NHE activity and MDR.…”

Photodynamic Inactivation with Acridine Orange on a Multidrug‐resistant Mouse Osteosarcoma Cell Line

“…----demonstrated that there was no difference in AO accumulation between sensitive and multidrug-resistant K562 cells, but resistant cells had significantly higher red fluorescence than sensitive cells. 22) Therefore, it is of interest to clarify the effect of PDT with AO on the MDR mouse osteosarcoma cell line, MOS/ADR1, which expresses P-glycoprotein.…”

Considerations for point-of-care diagnostics: evaluation of acridine orange staining and postprocessing methods for a three-part leukocyte differential test

“…33,35 These local variations in intracellular pH are dependent on the presence of intracellular lysosomes, vesicles, and other components specific to the role that each leukocyte population plays; this enables differentiation and classification of each cell population based on its colorimetric features following AO staining. 33,36 In Fig. 1, granulocytes represent the most acidic cell population containing many acidic vesicles resulting in the greatest red emission.…”