Involvement of glutathione in loss of technetium-99m-MIBI accumulation related to membrane MDR protein expression in tumor cells.

UNLABELLED: It was reported recently that 99mTc-hexakis-2-methoxyisobutyl isonitrile (MIBI) uptake is drastically reduced in cancer cells that express the multidrug resistance (MDR) product, Pgp 170 kDa (Pgp), suggesting that 99mTc-MIBI is a transport substrate for this transmembrane glycoprotein. In our study, we explored if another pump, a multidrug resistance-associated protein (MRP), could affect 99mTc-MIBI uptake. In addition, we studied the involvement of intracellular glutathione (GSH) as a modulator of 99mTc-MIBI uptake by both Pgp and MRP proteins. METHODS: MDR1 and MRP gene expression in seven human tumor cell lines was determined on a transcriptional level by reverse transcriptase polymerase chain reaction and on a protein level using immunocytochemistry. Technetium-99m-MIBI uptake was quantified by measuring radioactivity retained in the cells incubated at 37 degrees C in the presence or absence of buthionine sulfoximine (BSO), which depletes cellular GSH. The cellular GSH content was determined with Ellman's reagent. RESULTS: Cell lines were classified according to their phenotypic characteristics: 1/MRP-/Pgp-: breast cancer cells (MCF7), lung carcinoma cells (H69S) and mouth epidermoid tumor cells (KB 3.1), 2/MRP-/Pgp+: MCF7 mdr+, KBA.1; and 3/MRP+/Pgp-: small-cell lung carcinoma (H69 AR and A 549). Technetium-99m-MIBI uptake was significantly lower in cells expressing MRP as well as Pgp compared to MRP/Pgp cells. Depletion of GSH by BSO resulted in an increase of 99mTc-MIBI uptake in multidrug resistant cells overexpressing MRP but not expressing Pgp. CONCLUSION: Technetium-99m-MIBI is extruded by both Pgp and MRP efflux pumps. However, MRP action is indirect and involves intracellular GSH for a presumed interaction with the 99mTc-MIBI before its effLux. Technetium-99m-MIBI seems to be a good candidate for a noninvasive marker to diagnose MDR1 related to Pgp and MRP expression in tumors of different origin.

Uptake of liposome-encapsulated 99Tcm-MIBI by sensitive and multidrug-resistant tumour cell lines.

It is well established that accumulation of 99Tcm-sestamibi (99Tcm-MIBI) is much higher in sensitive than multidrug-resistant tumour cells expressing the permeability glycoprotein 170 (Pgp 170) as well as a multidrug-resistance related protein (MRP). Thus 99Tcm-MIBI is a good candidate for diagnosing the multidrug-resistance phenotype by in vivo imaging. However, the blood clearance of 99Tcm-MIBI is too rapid to achieve optimal accumulation in tumours and uptake in the liver, spleen, heart and muscle is too high for it to be an excellent in vivo tumour tracer. One way of prolonging the bioavailability of 99Tcm-MIBI is to use liposomes which do not affect its accumulation in tumour cells. We explored this possibility in vitro using two sensitive and five resistant cell lines, two of them expressing Pgp 170 and three others over-expressing MRP. 99Tcm-MIBI was incorporated into liposomes prepared by thin film hydration with phosphate-buffered saline using distearoyl phosphatidyl choline, distearoyl phosphatidyl ethanolamine and cholesterol in a ratio of 1.85:0.15:1.00. Liposome diameter was 97.9 +/- 4.5 nm as determined by dynamic light scattering. 99Tcm-MIBI uptake was quantified by measuring radioactivity retained in the cells incubated at 37 degrees C with liposome-encapsulated 99Tcm-MIBI or with free radiotracer in the presence of empty liposomes. In both experimental cases, 99Tcm-MIBI accumulation was similar to that obtained in the presence of free 99Tcm-MIBI only: it was much higher in sensitive than in resistant Pgp 170-positive and MRP-positive cells. Encapsulation in liposomes does not alter the potency of 99Tcm-MIBI to distinguish the sensitive and resistant tumour cells. Our results suggest that future studies should assess the usefulness of the encapsulated form of 99Tcm-MIBI for in vivo imaging of tumours.