Double impact on p‐glycoprotein by statins enhances doxorubicin cytotoxicity in human neuroblastoma cells

Abstract

The development of multidrug resistance (MDR) is a major problem during cancer treatment. Drug efflux via ATP-binding cassette (ABC) transporters is the main mechanism responsible for resistance to chemotherapeutics. We have recently observed that statins enhance susceptibility to doxorubicin-induced apoptosis in human rhabdomyosarcoma cells, which is now also observed in human SH-SY5Y neuroblastoma cells. We have therefore investigated the ABC transporter activity to confirm a possible inhibition by statins in SH-SY5Y cells. Indeed, simvastatin directly inhibited dye transport at equimolar concentrations of the ABC transporter inhibitor, verapamil. Making use of the fluorescence behavior of doxorubicin the accumulation of anthracycline was monitored in real-time confocal microscopy. The intracellular doxorubicin accumulation was immediately enhanced by statins in SH-SY5Y cells and also in a MYCN-amplified neuroblastoma cell line STA-NB-10. The heavily glycosylated P-glycoprotein (ABCB1, P-gp) transporter appeared as a 180-and 140-kDa species. Atorvastatin and simvastatin reduced the 180-kDa form of P-gp, but not verapamil. Thereby the fully glycosylated species is shifted to the core glycosylated species (140 kDa), which was only seen at statin exposure times longer than 24 hr. The functional importance of glycosylation of the transporter was highlighted by exogenous application of N-glycosidase F, which was sufficient to enhance doxorubicin accumulation. Hence, these novel findings of statins' dual impact on P-gp have clinical implications. The enhanced intracellular accumulation of chemotherapeutics or other ABC transporter substrates in the presence of statins may represent a novel concept to overcome MDR in cancer therapy and improve drug safety.Neuroblastoma is the most common solid tumor in early childhood. About 55% of patients older than 1 year already have metastases at the time of diagnosis and therefore have a poor survival rate despite intensive therapy. 1 In $20% of the neuroblastoma a MYCN oncogene amplification occurs, which is the most unfavorable prognostic factor. 2,3 The MYCN amplification is the best established clinical and biological marker, which allows determination of the prognosis of the tumor, with numerous reports confirming the association with rapid tumor progression, advanced clinical stage and poor outcome. 3,4 Studies have shown that the expression of the multidrug resistance-associated protein 1 (MRP1, ABCC1) in neuroblastoma also correlates with poor clinical outcome and prognosis. 5,6 Recently, a prospective study has provided further evidence that high levels of MRP1 expression are strongly associated with MYCN oncogene amplification and predictive of poor outcome. 7 ATP-binding cassette (ABC) transporters, also termed multidrug resistance (MDR) proteins, play an important role in the efflux of many drugs. 8,9 Moreover, these ABC transporters are overexpressed in a wide range of tumors, for example, colon, kidney, adrenocortical or hepatocellular carcinomas, and thereby conf...

Establishment and characterization of a bladder cancer cell line with enhanced doxorubicin resistance by mevalonate pathway activation

“…Interestingly, the cooperation of statins with doxorubicin may be partly based on an off-target effect on multidrug efflux pumps, as reported e.g. for neuroblastoma [35]orrhabdomyosarcoma [36] cells. With regard to urothelial bladder carcinoma cells, this is, to our knowledge, the first report showing a clear therapeutic cooperativity of a statin with a conventional anticancer drug.…”

Functional roles of glycogene and N‐glycan in multidrug resistance of human breast cancer cells

“…Additionally, for deglycosylation of membrane protein, intact MCF/ADR cells were incubated with 25 units of PNGase for 0, 8, 16, 24 hours, washed, and subsequently treated as described for drug sensitivity and in vivo antitumor activity assays (18). The cell survival rate was 87%, 85%, 85%, and 81% by trypan blue dye exclusion assay, respectively.…”

The ABC of glycosylation

“…This phenotype is not due to mutations in the putative N-glycosylation sites of these transporters, but is instead related to the reduced expression of two glycosyltransferases, GNPTG (which accounts for the first step in the biosynthesis of N-glycans) and MGAT5 (which catalyses the addition of mannose to proteins). In keeping with this, a protective role has been ascribed to the glycosylation of ABCB1 (also known as P-glycoprotein), which can be shut down by statins 13 , thereby suggesting new therapeutic options. Therefore, we propose that glycosylation is a relevant regulatory mechanism of ABC transporter expression and should be considered so that we can better understand the contribution of transporters to both drug resistance and tumour initiation and progression.…”