Characterization of H+-ATPase-dependent activity of multidrug resistance-associated protein in homoharringtonine-resistant human leukemic K562 cells

Abstract

Multidrug resistance (MDR), caused by overexpression of either P-glycoprotein or the multidrug resistance-associated protein (MRP), is characterized by a decreased cellular drug accumulation due to an enhanced drug efflux. Many studies on cells overexpressing MRP and/or Pgp, have shown a concentration of the drug inside cytoplasmic acidic vesicles followed by an exocytotic process. In this study, we examined the effects of 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole or NBD (a H + -ATPase pump inhibitor), buthionine sulphoximine or BSO (an inhibitor of glutathione (GSH) biosynthesis) and verapamil or VPL (a calcium channel blocker) on the subcellular distribution of daunorubicin or DNR in K562 cells overexpressing MRP (K-H30) and Pgp (K-H300) and A549 cells overexpressing spontaneously MRP. Nucleo-cytoplasmic distribution of DNR was carried out using scanning confocal microspectrofluorometry. This technique allows determination of nuclear accumulation of anthracyclines. Our results show that nuclear accumulation of DNR in K-H30 and A549 cells was increased by NBD, BSO and VPL while in K-H300 cells, only VPL was able to increase nuclear accumulation of DNR. Similarly, NBD, BSO and VPL could reverse DNR resistance in K-H30 cells whereas, in K-H300 cells, only VPL increased the sensitivity of these cells. These data suggest a requirement for GSH in MRP-mediated resistance and suggest that even if vesicular sequestration can happen in cells overexpressing MRP and Pgp proteins, probably only the MRP protein is able to extrude the drug through intracellular vesicles and efflux. Finally, NBD and BSO might be a useful agents in facilitating discrimination between Pgp and MRP phenotypes and prognosis in patients.

Synthesis of Antiproliferative Cephalotaxus Esters and Their Evaluation against Several Human Hematopoietic and Solid Tumor Cell Lines: Uncovering Differential Susceptibilities to Multidrug Resistance

“…[98] Previous reports have noted that the activity of homoharringtonine (3), the cephalotaxus ester currently being evaluated in clinical trials, is also compromised in MDR human leukemia cells. [18] Remarkably, the susceptibility of MDR cancer cells to different Cephalotaxus esters has not been systematically probed. Prevention of MDR would significantly improve therapeutic response to this family of chemotherapeutics and extend their use in the clinic.…”

“…These results prompted Phase I clinical evaluation of homoharringtonine (3) in the US in 1981, [16] advancing to more recent phase II studies. [17] While difficulties in production, coupled with its hematologic toxicity and susceptibility to multidrug resistance (MDR), [18] have hindered the development of 3, it is still viewed as a useful drug for the treatment of chronic myeloid leukemia in combination therapy. [17] Cephalotaxine (1) has received considerable and enduring attention in the arena of total synthesis.…”

Hoechst 33342 efflux identifies a subpopulation of cytogenetically normal CD34+CD38− progenitor cells from patients with acute myeloid leukemia

“…Although it is possible that the flow cytometry technique was insufficiently sensitive to detect low levels of Pgp expressionthus leading to false-negative results-it may be more likely that the mechanism of Hoechst dye efflux is more complex than previously thought, perhaps involving ABC transporter molecules sensitive to verapamil inhibition, such as the multidrug resistance protein or breast cancer-related protein or unknown molecules. [37][38][39] Additional studies designed to elucidate the molecular mechanisms involved in Hoechst dye efflux will be necessary to clarify these issues.…”

True

“…72 Other modulators such as genistein, a specific inhibitor of tyrosine kinase, or 7-chloro-4-notrobenz-2-oxa-1,3-diazole (NBD), a vacuolar H+-ATPase, have been reported to modulate MRP1 of a non-specific manner. [73][74][75] However, genistein showed major differences in effects on Rh123 vs DNR transport in the MRP1-mediated MDR cell lines: the accumulation of DNR was increased, whereas the accumulation of Rh123 was decreased by genistein. 73 Recently, van der Kolk et al 40 and ourselves 11 have demonstrated that MRP1 was functional in fresh acute myeloid leukemic blast cells using CF and calcein-AM, respectively.…”