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Expression of three major resistance genes MDR1, MRP1 and LRP was investigated in small cell lung cancer, non-small cell lung cancer and metastasis. Single biopsies of bronchoscopy from 73 patients were performed to investigate expression of these three resistance genes by reverse transcriptase-polymerase chain reaction. Relations between gene expression and patient age, smoking status, histology, and chemotherapy were evaluated. A more frequent expression of MDR1 (77 versus 66%), MRP1 (91 versus 72%) and LRP (77 versus 63%) genes was detected in the malignant biopsies than in the non-malignant, respectively. In the metastasis biopsies, expression of these genes was markedly increased. No significant difference was observed between specimens before and after chemotherapy. Biopsies from progressing cancer showed higher MDR1, MRP1 and LRP gene expression. In conclusion, these data reveal a major role of MRP1 in intrinsic resistance and the high gene expression of MDR1 and MRP1 in relapsed diseases.

A selective analysis of adsorbed mitoxantrone (MTX) was performed by surface-enhanced Raman scattering (SERS) at the range of cellular membrane. Disruption of the membrane fluidity was carried out to appraise changes in membrane adsorption of MTX and drug uptake in sensitive (HCT-116 S) and resistant BCRP/MXR (HCT-116 R) cells. Based on spectral MTX modifications, micro-SERS spectroscopy discriminated clearly drug adsorption phenomena on plasma membrane from drug in solution. A 3-fold higher SERS intensity of MTX for HCT-116 R was observed concluding to a higher drug adsorption on resistant membrane. The increase of membrane fluidity with benzyl alcohol (BA) or chloroform (CF) resulted in a 3-fold decrease of MTX adsorption on HCT-116 R, exclusively. BA and CF improved intracellular accumulation of MTX (e.g., 823 and 191 pmol MTX/10(6) HCT-116 R incubated with or without BA). At 4 degrees C, drug accumulation measurements showed a decrease of MTX permeability in resistant membrane (42 pmol MTX/10(6) cells), restored with fluidizers (e.g., 342 pmol MTX/10(6) cells with BA). Fluorescence confocal microscopy involved an exclusive MTX emission around the plasma membrane of resistant cells whereas fluidizers increased the intracellular uptake of MTX in both cell lines at the same time with less drug emission around the plasma membrane. Changes of the membrane structure of resistant cells should modify both drug adsorption and membrane permeation.

We used confocal microspectrofluorometry to investigate intracellular distribution of pirarubicin or THP-DOX in parental K562, CEM, and LR73 tumor cells and their corresponding multidrug-resistant (MDR) strains. Each spectrum of a recorded image was considered as a combination of cell autofluorescence and fluorescence of the drug. In the cytoplasm of parental K562, CEM, and LR73 cells, THP-DOX fluorescence emission profile was similar to that of free drug in aqueous buffer. The (I550nm/I600nm) ratio was 0. 50 +/- 0.1. However, in the cytoplasm of resistant cells the 550-nm band profile was modified. The I550nm/I600nm ratio was 0.85 +/- 0.2 in MDR K562 cells, which is significantly different from the ratio in sensitive cells (p<0.01). This appeared first to correspond to accumulation and self-oligomerization of THP-DOX in cytoplasmic organelles of MDR cells. Transfection of LR73 cells with the mdr1 gene conferred this characteristic on the resistant LR73R cells. Bodipy-ceramide, a trans-Golgi probe, was co-localized with the typical fluorescence emission peak at 550 nm observed in the cytoplasm of MDR cells. This organelle has been shown to be more acidic in MDR cells. Moreover, this specific pattern was similar to that observed when anthracycline is complexed with sphingomyelin. The typical fluorescence emission peak at 550 nm decreased in MDR cells incubated simultaneously in the presence of the drug and quinine, verapamil, or S9788. Growth inhibitory effect and nuclear accumulation of THP-DOX data obtained on LR73R and LR73D cell lines showed that only during reversion of resistance by verapamil and S9788 was an increase of nuclear THP-DOX accumulation observed. Our data suggest that characteristics of molecular environment, such as higher pH gradient or lipid structures, would be potential mechanisms of multidrug-resistance via the sequestration of anthracyclines.

To study the pH gradient status through membranes of acidic vesicles, either in sensitive or in multidrug-resistant living cancer cells, we monitored the fluorescence-emission 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 +/- 10%) than the value (48 +/- 8%) from K562-sensitive cells (p < 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 microM), or nigericine (0.3 microM) 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 microM 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.