Spontaneous mitochondrial membrane potential change during apoptotic induction by quercetin in K562 and K562/adr cells.

Natural products from plants such as flavonoids are potential drugs to overcome multidrug resistance (MDR) in cancer treatments. However, their modes of action are still unclear. In this study, the effects of quercetin on mitochondrial membrane potential (DeltaPsim) change as well as quercetin's ability to induce apoptosis and inhibit Pgp-mediated efflux of 99mTc-MIBI in K562/adr cells were investigated. Quercetin exhibits cytotoxicity against erythroleukemic cells: IC50 are 11.0 +/- 2.0 micromol/L and 5.0 +/- 0.4 micromol/L for K562 and K562/adr, respectively. Quercetin induces cell death via apoptosis in both K562 and K562/adr cells and does not inhibit Pgp-mediated efflux of 99mTc-MIBI. Quercetin (10 micromol/L, 3 h) and etoposide (100 micromol/L, 24 h) induce similar levels of apoptosis in K562 and K562/adr cells. Quercetin induces an increase followed by a decrease in |DeltaPsim| value depending on its concentration. A decrease in the |DeltaPsim| value is associated with an increase in the percentage of early apoptotic cells. It is clearly shown that quercetin results in a spontaneous DeltaPsim change during apoptotic induction. Therefore, quercetin is potentially an apoptotic-inducing agent, which reacts at the mitochondrial level.

Could 99mTc-MIBI be used to visualize the apoptotic MCF7 human breast cancer cells?

Defects in key components of apoptotic pathways provide a survival advantage to cells and have been implicated as important factors in tumorogenesis. As therapeutic drug-induced apoptosis is a key component in treatment of most cancers, alterations in apoptotic pathways may be critical to drug resistance. The question is: would it be possible to distinguish apoptotic cells and resistant cells with a same radiotracer? In this study, we investigated the ability of sodium phenylacetate (NaPa), a natural cytostatic proapoptotic metabolite, to induce apoptosis in MCF7 human breast cancer cells. Then, we tested the 99mTc-MIBI accumulation in these apoptotic cells. Annexin V-FITC was used to identify apoptotic cells by flow cytometry. Ours results demonstrated that a 72 hr treatment of MCF7 cells with 40 mM NaPa induced apoptosis in 60% of cells. In a parallel way, 99mTc-MIBI accumulation in NaPa treated cells decreased for concentrations higher than 20 mM NaPa. Thus, 99mTc-MIBI accumulation decreased correlatively with the increasing percentage of apoptotic cells obtained by treatment of MCF7 cells with NaPa. These data demonstrate that NaPa induced apoptosis in MCF7 cells and that 99mTc-MIBI is a negative tracer of apoptosis: the more MCF7 cells were engaged in the apoptotic pathway, the more 99mTc-MIBI accumulation decreased in these MCF7 apoptotic cells.

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.

Comparison of the kinetics of active efflux of 99mTc-MIBI in cells with P-glycoprotein-mediated and multidrug-resistance protein-associated multidrug-resistance phenotypes.

The overexpression of two membrane glycoproteins, P-glycoprotein and multidrug-resistance protein (MRP1) is a major cause of resistance to chemotherapeutic agents in the treatment of human cancers. Both proteins confer a similar multidrug-resistant (MDR) phenotype. 99mTc-MIBI, a myocardial imaging agent, which is also useful for the detection of a variety of tumours, has been shown to be a substrate for P-glycoprotein and MRP1. It thus may provide additional information about the P-glycoprotein and MRP1 status of tumour cells. In order to obtain information on the substrate specificity of these proteins, we have studied the transport kinetics of Tc-MIBI in two cell lines, K562/ADR and GLC4/ADR, which overexpress P-glycoprotein and MRP1, respectively. The mean active efflux coefficient ka, which is proportional to the ratio of maximal efflux rate VM to the apparent Michaelis-Menten constant Km, used to characterise the efficiency of the active efflux, was very similar being 1.9 +/- 0.6 x 10(-11) s(-1) x cells x ml and 1.3 +/- 0.5 x 10(-11) s(-1) x cells x ml for drug-resistant K562 and GLC4, respectively. These values are 50-100-times lower than for daunorubicin and other anthracycline derivatives, strongly suggesting that the efficiency of both transporters to pump Tc-MIBI is by far less than that to efflux anthracyclines. Our data show that (a) P-glycoprotein and MRP transporter efficiencies to wash out Tc-MIBI are similar, in spite of a different suspected mechanism of its transport and (b) that both transporters are less efficient to pump Tc-MIBI than to pump anthracyclines (the ka parameter is about 100-times lower for TC-MIBI than for anthracycline).

Isolation of a porcine respiratory, non-enteric coronavirus related to transmissible gastroenteritis.

A porcine respiratory, non-enteric virus which is related to the coronavirus transmissible gastroenteritis virus (TGEV) has been isolated in pigs and in cell culture. The isolate was designated TLM 83. It has become very widespread and enzootic among the swine population in Belgium and in other swine raising countries. It causes an infection of the lungs and appears to spread by aerogenic route. It does not replicate in the enteric tract. The experimental infection in conventional and gnotobiotic pigs in isolation remains subclinical. The infection, either experimental or in the field, results in the formation of antibodies which neutralise the classical enteric TGEV. Based on this relationship, this virus is assumed to be a new TGEV-related porcine respiratory coronavirus or TGEV itself which has totally lost its tropism for the enteric tract.