2'[(18)F]-fluoroethylrhodamine B is a promising radiotracer to measure P-glycoprotein function.

In vivo detection of the emergence of P-glycoprotein (Pgp) mediated multidrug resistance in tumors could be beneficial for patients treated with anticancer drugs. PET technique in combination with appropriate radiotracers could be the most convenient method for detection of Pgp function. Rhodamine derivatives are validated fluorescent probes for measurement of mitochondrial membrane potential and also Pgp function. The aim of this study was to investigate whether 2'[(18)F]-fluoroethylrhodamine B ((18)FRB) a halogenated rhodamine derivative previously synthesized for PET assessment of myocardial perfusion preserved its Pgp substrate character. ATPase assay as well as accumulation experiments carried out using Pgp(+) and Pgp(-) human gynecologic (A2780/A2780(AD) and KB-3-1/KB-V1) and a mouse fibroblast cell pairs (NIH 3T3 and NIH 3T3 MDR1) were applied to study the interaction of (18)FRB with Pgp. ATPase assay proved that (18)FRB is a high affinity substrate of Pgp. Pgp(-) cells accumulated the (18)FRB rapidly in accordance with its lipophilic character. Dissipation of the mitochondrial proton gradient by a proton ionophore CCCP decreased the accumulation of rhodamine 123 (R123) and (18)FRB into Pgp(-) cells. Pgp(+) cells exhibited very low R123 and (18)FRB accumulation (around 1-8% of the Pgp(-) cell lines) which was not sensitive to the mitochondrial proton gradient; rather it was increased by the Pgp inhibitor cyclosporine A (CsA). Based on the above data we conclude that (18)FRB is a high affinity Pgp substrate and consequently a potential PET tracer to detect multidrug resistant tumors as well as the function of physiological barriers expressing Pgp.

18FDG, [18F]FLT, [18F]FAZA, and 11C-methionine are suitable tracers for the diagnosis and in vivo follow-up of the efficacy of chemotherapy by miniPET in both multidrug resistant and sensitive human gynecologic tumor xenografts.

Expression of multidrug pumps including P-glycoprotein (MDR1, ABCB1) in the plasma membrane of tumor cells often results in decreased intracellular accumulation of anticancer drugs causing serious impediment to successful chemotherapy. It has been shown earlier that combined treatment with UIC2 anti-Pgp monoclonal antibody (mAb) and cyclosporine A (CSA) is an effective way of blocking Pgp function. In the present work we investigated the suitability of four PET tumor diagnostic radiotracers including 2-[(18)F]fluoro-2-deoxy-D-glucose ((18)FDG), (11)C-methionine, 3'-deoxy-3'-[(18)F]fluorothymidine ((18)F-FLT), and [(18)F]fluoroazomycin-arabinofuranoside ((18)FAZA) for in vivo follow-up of the efficacy of chemotherapy in both Pgp positive (Pgp(+)) and negative (Pgp(-)) human tumor xenograft pairs raised in CB-17 SCID mice. Pgp(+) and Pgp(-) A2780AD/A2780 human ovarian carcinoma and KB-V1/KB-3-1 human epidermoid adenocarcinoma tumor xenografts were used to study the effect of the treatment with an anticancer drug doxorubicin combined with UIC2 and CSA. The combined treatment resulted in a significant decrease of both the tumor size and the accumulation of the tumor diagnostic tracers in the Pgp(+) tumors. Our results demonstrate that (18)FDG, (18)F-FLT, (18)FAZA, and (11)C-methionine are suitable PET tracers for the diagnosis and in vivo follow-up of the efficacy of tumor chemotherapy in both Pgp(+) and Pgp(-) human tumor xenografts by miniPET.

The strong in vivo anti-tumor effect of the UIC2 monoclonal antibody is the combined result of Pgp inhibition and antibody dependent cell-mediated cytotoxicity.

P-glycoprotein (Pgp) extrudes a large variety of chemotherapeutic drugs from the cells, causing multidrug resistance (MDR). The UIC2 monoclonal antibody recognizes human Pgp and inhibits its drug transport activity. However, this inhibition is partial, since UIC2 binds only to 10-40% of cell surface Pgps, while the rest becomes accessible to this antibody only in the presence of certain substrates or modulators (e.g. cyclosporine A (CsA)). The combined addition of UIC2 and 10 times lower concentrations of CsA than what is necessary for Pgp inhibition when the modulator is applied alone, decreased the EC50 of doxorubicin (DOX) in KB-V1 (Pgp+) cells in vitro almost to the level of KB-3-1 (Pgp-) cells. At the same time, UIC2 alone did not affect the EC50 value of DOX significantly. In xenotransplanted severe combined immunodeficient (SCID) mice co-treated with DOX, UIC2 and CsA, the average weight of Pgp+ tumors was only ∼10% of the untreated control and in 52% of these animals we could not detect tumors at all, while DOX treatment alone did not decrease the weight of Pgp+ tumors. These data were confirmed by visualizing the tumors in vivo by positron emission tomography (PET) based on their increased 18FDG accumulation. Unexpectedly, UIC2+DOX treatment also decreased the size of tumors compared to the DOX only treated animals, as opposed to the results of our in vitro cytotoxicity assays, suggesting that immunological factors are also involved in the antitumor effect of in vivo UIC2 treatment. Since UIC2 binding itself did not affect the viability of Pgp expressing cells, but it triggered in vitro cell killing by peripheral blood mononuclear cells (PBMCs), it is concluded that the impressive in vivo anti-tumor effect of the DOX-UIC2-CsA treatment is the combined result of Pgp inhibition and antibody dependent cell-mediated cytotoxicity (ADCC).

¹8FDG a PET tumor diagnostic tracer is not a substrate of the ABC transporter P-glycoprotein.

2-[(18)F]fluoro-2-deoxy-d-glucose ((18)FDG) is a tumor diagnostic radiotracer of great importance in both diagnosing primary and metastatic tumors and in monitoring the efficacy of the treatment. P-glycoprotein (Pgp) is an active transporter that is often expressed in various malignancies either intrinsically or appears later upon disease progression or in response to chemotherapy. Several authors reported that the accumulation of (18)FDG in P-glycoprotein (Pgp) expressing cancer cells (Pgp(+)) and tumors is different from the accumulation of the tracer in Pgp nonexpressing (Pgp(-)) ones, therefore we investigated whether (18)FDG is a substrate or modulator of Pgp pump. Rhodamine 123 (R123) accumulation experiments and ATPase assay were used to detect whether (18)FDG is substrate for Pgp. The accumulation and efflux kinetics of (18)FDG were examined in two different human gynecologic (A2780/A2780AD and KB-3-1/KB-V1) and a mouse fibroblast (3T3 and 3T3MDR1) Pgp(+) and Pgp(-) cancer cell line pairs both in cell suspension and monolayer cultures. We found that (18)FDG and its derivatives did not affect either the R123 accumulation in Pgp(+) cells or the basal and the substrate stimulated ATPase activity of Pgp supporting that they are not substrates or modulators of the pump. Measuring the accumulation and efflux kinetics of (18)FDG in different Pgp(+) and Pgp(-) cell line pairs, we have found that the Pgp(+) cells exhibited significantly higher (p⩽0.01) (18)FDG accumulation and slightly faster (18)FDG efflux kinetics compared to their Pgp(-) counterparts. The above data support the idea that expression of Pgp may increase the energy demand of cells resulting in higher (18)FDG accumulation and faster efflux. We concluded that (18)FDG and its metabolites are not substrates of Pgp.

Tetrodotoxin blocks L-type Ca2+ channels in canine ventricular cardiomyocytes.

Tetrodotoxin (TTX) is believed to be the most selective inhibitor of voltage-gated fast Na(+) channels in excitable tissues, including nerve, skeletal muscle, and heart, although TTX sensitivity of the latter is lower than the former by at least three orders of magnitude. In the present study, the TTX sensitivity of L-type Ca(2+) current (I (Ca)) was studied in isolated canine ventricular cells using conventional voltage clamp and action potential voltage clamp techniques. TTX was found to block I (Ca) in a reversible manner without altering inactivation kinetics of I (Ca). Fitting results to the Hill equation, an IC(50) value of 55 ± 2 µM was obtained with a Hill coefficient of unity (1.0 ± s0.04). The current was fully abolished by 1 µM nisoldipine, indicating that it was really I (Ca). Under action potential voltage clamp conditions, the TTX-sensitive current displayed the typical fingerprint of I (Ca), which was absent in the presence of nisoldipine. Stick-and-ball models for Cav1.2 and Nav1.5 channel proteins were constructed to explain the differences observed between action of TTX on cardiac I (Ca) and I (Na). This is the first report demonstrating TTX to interact with L-type calcium current in the heart.

Effects of the PKC inhibitors chelerythrine and bisindolylmaleimide I (GF 109203X) on delayed rectifier K+ currents.

Protein kinase C (PKC) inhibitors are useful tools for studying PKC-dependent regulation of ion channels. For this purpose, high PKC specificity is a basic requirement excluding any direct interaction between the PKC inhibitor and the ion channel. In the present study, the effects of two frequently applied PKC inhibitors, chelerythine and bisindolylmaleimide I, were studied on the rapid and slow components of the delayed rectifier K(+) current (I(Kr) and I(Ks)) in canine ventricular cardiomyocytes and on the human ether-à-go-go-related gene (hERG) channels expressed in human embryonic kidney (HEK) cells. The whole cell version of the patch clamp technique was used in all experiments. Chelerythrine and bisindolylmaleimide I (both 1 µM) suppressed I(Kr) in canine ventricular cells. This inhibition developed rapidly, suggesting a direct drug-channel interaction. In HEK cells heterologously expressing hERG channels, chelerythrine and bisindolylmaleimide I blocked hERG current in a concentration-dependent manner, having EC(50) values of 0.11 ± 0.01 and 0.76 ± 0.04 µM, respectively. Both chelerythrine and bisindolylmaleimide I strongly modified gating kinetics of hERG--voltage dependence of activation was shifted towards more negative voltages and activation was accelerated. Deactivation was slowed by bisindolylmaleimide I but not by chelerythrine. I(Ks) was not significantly altered by bisindolylmaleimide I and chelerythrine. No significant effect of 0.1 µM bisindolylmaleimide I or 0.1 µM PMA (PKC activator) was observed on I(Kr) arguing against significant contribution of PKC to regulation of I(Kr). It is concluded that neither chelerythrine nor bisindolylmaleimide I is suitable for selective PKC blockade due to their direct blocking actions on the hERG channel.

Daunorubicin and doxorubicin inhibit the [(11)C]choline accumulation in cancer cells.

We studied how very short (10-40min) incubation with anthracycline derivatives modifies the accumulation of PET tumor-diagnostic radiotracers in cancer cells. The human ovarian A2780 and A2780AD, human B lymphoid JY, human epidermoid KB-3-1 and KB-V-1, and smooth muscle DDT1 MF-2 cells were pre-incubated with daunorubicin and doxorubicin, and the uptake of [(18)F]FDG and [(11)C]choline was measured. Anthracycline treatment decreased remarkably the [(11)C]choline accumulation in a concentration dependent manner, while it did not modify significantly the [(18)F]FDG uptake of the cells.

Functional consequences of Kv1.3 ion channel rearrangement into the immunological synapse.

Formation of immunological synapse (IS), the interface between T cells and antigen presenting cells, is a crucial step in T cell activation. This conjugation formation results in the rearrangement and segregation of a set of membrane bound and cytosolic proteins, including that of the T cell receptor, into membrane domains. It was showed earlier that Kv1.3, the dominant voltage-gated potassium channel of T cells redistributes into the IS on interaction with its specific APC. In the present experiments we investigated the functional consequences of the translocation of Kv1.3 channels into the IS formed between mouse helper T (T(h)2) and B cells. Biophysical characteristics of whole-cell Kv1.3 current in standalone cells (c) or ones in IS (IS) were determined using voltage-clamp configuration of standard whole-cell patch-clamp technique. Patch-clamp recordings showed that the activation of Kv1.3 current slowed (tau(a,IS)=2.36+/-0.13 ms (n=7); tau(a,c)=1.36+/-0.06 ms (n=18)) whereas the inactivation rate increased (tau(i,IS)=263+/-29 ms (n=7); tau(i,c)=365+/-27 ms (n=17)) in cells being in IS compared to the standalone cells. The equilibrium distribution between the open and the closed states of Kv1.3 (voltage-dependence of steady-state activation) was shifted toward the depolarizing potentials in T cells engaged into IS (V(1/2,IS)=-20.9+/-2 mV (n=7), V(1/2,c)=-26.4+/-1.5 mV (n=12)). Thus, segregation of Kv1.3 channels into the IS modifies the gating properties of the channels. Application of protein kinase (PK) inhibitors (PKC: GF109203X, PKA: H89, p56Lck: damnacanthal) demonstrated that increase in the inactivation rate can be explained by the dephosphorylation of the channel protein. However, the slower activation kinetics of Kv1.3 in IS is likely to be the consequence of the redistribution of the channels into distinct membrane domains.

Functional implications of membrane modification with semenogelins for inhibition of sperm motility in humans.

Semenogelin I and II (Sgs) are the major component of human semen coagulum. The protein is rapidly cleaved after ejaculation by a prostate-specific antigen, resulting in liquefaction of the semen coagulum and the progressive release of motile spermatozoa. Sgs inhibit human sperm motility; however, there is currently no information on its effect on the sperm membrane. This study investigated the role of Sgs on human sperm motility through regulation of membrane potential and membrane permeability. Fresh semen samples were obtained from normozoospermic volunteers, and studies were conducted using motile cells selected using the swim-up method. Sgs changed the characteristics of sperm motion from circular to straightforward as evaluated by a computer-assisted motility analyzer, and all parameters were decreased more than 2.5 mg/mL. The results demonstrate that Sgs treatment immediately hyperpolarized the membrane potential of swim-up-selected sperm, changed the membrane structure, and time-dependently increased membrane permeability, as determined through flow cytometric analysis. The biphasic effects of Sgs were time- and dose-dependent and partially reversible. In addition, a monoclonal antibody against Sgs showed positive binding to cell membrane proteins in fixed cells, observed with confocal fluorescence microscopy. These results demonstrate that Sgs modifies the membrane structure, indirectly inhibiting motility, and provides suggestions for a therapy for male infertility through selection of a functional sperm population using Sgs.

Involvement of membrane channels in autoimmune disorders.

Ion channels are ubiquitous transmembrane proteins that are involved in a wide variety of cellular functions by selectively controlling the passage of ions across the plasma membrane. Among these functions many immune processes, including those in autoimmune reactions, also rely on the operation of ion channels, but the roles of ion channels can be very diverse. Here the participation of ion channels in three different roles in autoimmune processes is discussed: 1. ion channels in effector immune cells attacking other tissues causing autoimmune diseases, such as multiple sclerosis; 2. ion channels as direct targets of the immune system whereby loss of channel function leads to disease, as in myasthenia gravis; 3. ion channels whose function is modulated in the target cells by an apoptotic signal transduction cascade, such as the Fas/Fas ligand pathway. The numerous tasks that ion channels perform in autoimmune disorders and the wealth of information that has been gathered about them in recent years together provide a good basis for the design and production of drugs that may be effectively used in the therapy of these diseases.

Nutrition and immune system: certain fatty acids differently modify membrane composition and consequently kinetics of KV1.3 channels of human peripheral lymphocytes.

Potassium (K(+)) channels of human peripheral lymphocytes play a considerable role in the signalling processes required for immune responses. Modification of the fatty acid composition of the membrane influences the functions of various membrane enzymes and ion channels. We set out to establish how the incorporation of fatty acids with different carbon chain lengths and degrees of unsaturation into the cell membrane influences the function of K(V)1.3 channels of lymphocytes, thereby potentially modifying the immune responses of the cells. The incorporation of the fatty acids into the cell membrane was monitored by gas chromatography. Whole-cell patch-clamp experiments demonstrated that the polyunsaturated linoleic acid, arachidonic acid and docosahexaenoic acid all decreased the activation and inactivation time constants of the K(V)1.3 channels, but did not affect the voltage-dependence of steady-state activation and steady-state inactivation of the channels. Treatment with the saturated palmitic acid, stearic acid and the monounsaturated oleic acid did not result in significant changes in the biophysical parameters of K(V)1.3 gating studied. We conclude that the incorporation of fatty acids unsaturated to different degrees into the cell membrane of lymphocytes influenced the rate of gating transitions but not the equilibrium distribution of the channels between different states. This effect depended on the degree of unsaturation and the chain length of the fatty acids: no effects of saturated and monounsaturated fatty acids (16:0, 18:0 and 18:1) were observed whereas treatment with polyunsaturated fatty acids (18:2, 20:4 and 22:6) resulted in significant changes in the channel kinetics.

Complete inhibition of P-glycoprotein by simultaneous treatment with a distinct class of modulators and the UIC2 monoclonal antibody.

P-glycoprotein (Pgp) is one of the active efflux pumps that are able to extrude a large variety of chemotherapeutic drugs from the cells, causing multidrug resistance. The conformation-sensitive UIC2 monoclonal antibody potentially inhibits Pgp-mediated substrate transport. However, this inhibition is usually partial, and its extent is variable because UIC2 binds only to 10 to 40% Pgp present in the cell membrane. The rest of the Pgp molecules become recognized by this antibody only in the presence of certain substrates or modulators, including vinblastine, cyclosporine A (CsA), and SDZ PSC 833 (valspodar). Simultaneous application of any of these modulators and UIC2, followed by the removal of the modulator, results in a completely restored steady-state accumulation of various Pgp substrates (calcein-AM, daunorubicin, and 99mTc-hexakis-2-methoxybutylisonitrile), indicating near 100% inhibition of pump activity. Remarkably, the inhibitory binding of the antibody is brought about by coincubation with concentrations of CsA or SDZ PSC 833 approximately 20 times lower than what is necessary for Pgp inhibition when the modulators are applied alone. The feasibility of such a combinative treatment for in vivo multidrug resistance reversal was substantiated by the dramatic increase of daunorubicin accumulation in xenotransplanted Pgp+ tumors in response to a combined treatment with UIC2 and CsA, both administered at doses ineffective when applied alone. These observations establish the combined application of a class of modulators used at low concentrations and of the UIC2 antibody as a novel, specific, and effective way of blocking Pgp function in vivo.

Na+/Ca2+ exchanger inhibitors modify the accumulation of tumor-diagnostic PET tracers in cancer cells.

AIM: To establish the effects of Na(+)/Ca(2+) exchanger (NCX) blockers on 2-[(18)F]fluoro-2-deoxy-D-glucose ((18)FDG) and (11)C-choline accumulation in different cancer cells. METHODS: The tumor cells were incubated with NCX inhibitors, and the uptakes of (18)FDG and (11)C-choline were measured. Flow cytometric measurements of intracellular Ca(2+) and Na(+) concentrations were carried out. The presence of the NCX antigen in the cancer cells was proved by Western blotting, flow cytometry and confocal laser scanning microscopy. RESULTS: The NCX is expressed at a noteworthy level in the cytosol and on the cytoplasmic membrane of the examined cells. Incubation of the cells with three chemically unrelated NCX blockers (bepridil, KB-R7943 or 3',4'-dichlorobenzamil hydrochloride) resulted in an increase in the intracellular Ca(2+) concentration, with a simultaneous decrease in the intracellular Na(+) concentration. The treatment with the NCX inhibitors increased the energy consumption of the tumor cells by 50-100%. Thapsigargin abolished the NCX-induced (18)FDG accumulation in the cells. The NCX blockers applied decreased the (11)C-choline accumulation of all the investigated cancer cells by 60-80% relative to the control. CONCLUSION: A possible masking effect of NCX medication must be taken into consideration during the diagnostic interpretation of PET scans.

Na+/Ca2+ exchanger modulates the flagellar wave pattern for the regulation of motility activation and chemotaxis in the ascidian spermatozoa.

Ion channels and ion exchangers are known to be important participants in various aspects of sperm physiology, e.g. motility activation, chemotaxis, the maintenance of motility and the acrosome reaction in the sperm. We report here on a role of the K+ -independent Na+/Ca2+ exchanger (NCX) on ascidian sperm. Reverse-transcriptase PCR reveals that the NCX is expressed in the testis while immunoblotting and immunolocalization demonstrate that the NCX exists on the sperm in the ascidian Ciona savignyi and C. intestinalis. A potent blocker of the NCX, KB-R7943 was found to block sperm-activating and -attracting factor (SAAF)-induced motility activation, sperm motility and sperm chemotaxis. We further analyzed the effects of this blocker on motility parameters such as the flagellar waveform, curvature, beat frequency, amplitude and wavelength of the sperm flagella. Inhibition of the NCX caused two distinct effects: a low concentration of KB-R7943 induced symmetric bending, whereas a high concentration of KB-R7943 resulted in asymmetric flagellar bending. These findings suggest that the NCX plays important roles in the regulation of SAAF-induced sperm chemotaxis, motility activation and motility maintenance in the ascidian. This study provides new information toward an understanding of Ca2+ transport systems in sperm motility and chemotaxis.

Role of the Na+/Ca2+ exchanger in calcium homeostasis and human sperm motility regulation.

A number of cell functions, such as flagellar beating, swimming velocity, acrosome reaction, etc., are triggered by a Ca2+ influx across the cell membrane. For appropriate physiological functions, the motile human sperm maintains the intracellular free calcium concentration ([Ca2+]i) at a submicromolar level. The objective of this study was to determine the role of the Na+/Ca2+ exchanger (NCX) in the maintenance of [Ca2+]i in human spermatozoa. Spermatozoa maintained in extracellular medium containing>or=1 microM Ca2+ exhibited motility similar to that of the control. In addition to several calcium transport mechanisms described earlier, we provide evidence that the NCX plays a crucial role in the maintenance of [Ca2+]i. Three chemically unrelated inhibitors of the NCX (bepridil, DCB (3',4'-dichlorobenzamil hydrochloride), and KB-R7943) all blocked human sperm motility in a dose and incubation time dependent manner. The IC50 values for bepridil, DCB, and KB-R7943 were 16.2, 9.8, and 5.3 microM, respectively. The treatment with the above-mentioned blockers resulted in an elevated [Ca2+]i and a decreased [Na+]i. The store-operated calcium channel (SOCC) inhibitor SKF 96365 also blocked the sperm motility (IC50=2.44 microM). The presence of the NCX antigen in the human spermatozoa was proven by flow cytometry, confocal laser scanning microscopy, and immunoblotting techniques. Calcium homeostasis of human spermatozoa is maintained by several transport proteins among which the SOCC and the NCX may play a major role.

Ion channels in T cells: from molecular pharmacology to therapy.

Ion channels of a variety of cell types, such as cardiac and smooth muscle cells and neurons, serve as targets for many drugs used in therapy. T cells also express an assortment of ion channels that are in the focus of intensive research, as they may provide efficient ways to specifically manipulate T cell function and, consequently, immune responses. T cell activation relies on the operation of voltage-gated and Ca2+-activated potassium channels and Ca2+ release-activated Ca2+ channels. Many peptide toxin and small molecule blockers of these channels are known, but inhibitors of even higher affinity and selectivity would be needed for safe and effective clinical use. The recent discovery that the expression pattern of potassium channels in T cells is subset specific emphasizes the potential that these proteins have in immunomodulation. Compounds that could suppress T cells involved in autoimmunity without affecting T cells in normal immune responses would be of enormous value. In this paper the basic properties of these channels and compounds known to influence their operation are reviewed.

Biphasic accumulation kinetics of [99mTc]-hexakis-2-methoxyisobutyl isonitrile in tumour cells and its modulation by lipophilic P-glycoprotein ligands.

AIM: To study the accumulation and washout kinetics of [99mTc]-hexakis-2-methoxyisobutyl isonitrile (99mTc-MIBI) in MDR positive and MDR negative tumour cells and how this is modified by lipophilic P-glycoprotein ligands. METHODS: The tumour cells were incubated in the presence and absence of the ligands and the uptakes of 99mTc-MIBI, rhodamine 123 and 2-[18F]fluoro-2-deoxy-D-glucose (18FDG) were measured. RESULTS: The accumulation of 99mTc-MIBI in the tumour cells followed biphasic kinetics. Verapamil and cyclosporin A increased the membrane fluidity and significantly enhanced the 99mTc-MIBI uptake of the MDR negative cells, while the rhodamine 123 uptake was not affected. Verapamil significantly increased the uptake of rhodamine 123 and 18FDG but did not modify that of 99mTc-MIBI in the MDR positive cells. Cyclosporin A significantly increased the 18FDG uptake of the MDR positive and negative tumour cells; these effects were ouabain-sensitive. Depolarization of the cytoplasmic membrane, acidification of the extracellular medium and the administration of CCCP decreased the accumulation of 99mTc-MIBI and rhodamine 123 uptake in the tumour cells. CONCLUSIONS: Lipophilic P-glycoprotein ligands modified the biphasic accumulation kinetics of the 99mTc-MIBI uptakes of MDR negative and positive tumour cells in different and complex ways and could therefore mask the P-glycoprotein pump-dependent changes in tracer accumulation.

Effects of miltefosine on membrane permeability and accumulation of [99mTc]-hexakis-2-methoxyisobutyl isonitrile, 2-[18F]fluoro-2-deoxy-D-glucose, daunorubucin and rhodamine123 in multidrug-resistant and sensitive cells.

Miltefosine is a phospholipid analog that exhibits antineoplastic activity against breast cancer metastases, but its mechanism of action remains uncertain. The aim of this study was to investigate the transport mechanism for the removal of miltefosine and [99mTc]-hexakis-2-methoxyisobutyl isonitrile (99mTc-MIBI) from multidrug-resistant cells. The P-glycoprotein pump function, cell viability, and 99mTc-MIBI and 2-[18F]fluoro-2-deoxy-D-glucose (18FDG) uptakes were measured in NIH 3T3 (3T3) and NIH 3T3MDR1 G185 (3T3MDR1) mouse fibroblasts and human lymphoid B JY cells. Miltefosine treatment increased the permeability and fluidity of these tumor cells in a concentration-dependent manner. The multidrug-sensitive cells were 3-4 times more sensitive to miltefosine than the multidrug-resistant ones. The extent of 99mTc-MIBI accumulation in the P-glycoprotein-expressing cells increased in the presence of miltefosine, whereas the rhodamine123 and daunorubicin uptakes of the cells did not change significantly. In the 3T3MDR1 cells verapamil reinstated the rhodamine123 and daunorubicin accumulation, but not the 99mTc-MIBI uptake. Cyclosporin A reinstated the uptakes of 99mTc-MIBI, daunorubicin and rhodamine123 by the 3T3MDR1 cells. In a concentration-dependent manner miltefosine decreased the extents of 99mTc-MIBI, rhodamine123, daunorubicin and 18FDG accumulation in the JY and 3T3 cells. Our findings indicate a common transport mechanism for 99mTc-MIBI and miltefosine, which is distinct from that for rhodamine123 and daunorubicin in MDR cells.