Inhibition of Glucosylceramide Synthase Does Not Reverse Drug Resistance in Cancer Cells

Abstract

The multidrug-resistant cancer cell lines NCI/AdR RES and MES-SA/DX-5 have higher glycolipid levels and higher P-glycoprotein expression than the chemosensitive cell lines MCF7-wt and MES-SA. Inhibiting glycolipid biosynthesis by blocking glucosylceramide synthase has been proposed to reverse drug resistance in MDR cells by causing an increased accumulation of proapoptotic ceramide during treatment of cells with cytotoxic drugs. We treated both multidrug-resistant cell lines with the glucosylceramide synthase inhibitors PDMP (D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol), C 9 DGJ (N-nonyl-deoxygalactonojirimycin) or C 4 DGJ (N-butyl-deoxygalactonojirimycin). PDMP achieved a significant reversal of drug resistance in agreement with previous reports. However, the Nalkylated iminosugars C 9 DGJ and C 4 DGJ, which are more selective glucosylceramide synthase inhibitors than PDMP, failed to cause any reversal of drug resistance despite depleting glycolipids to the same extent as PDMP. Our results suggest that (a) inhibition of glucosylceramide synthase does not reverse multidrug resistance and (b) the chemosensitization achieved by PDMP cannot be caused by inhibition of glucosylceramide synthase alone.A major limitation in chemotherapy for cancer is multidrug resistance (MDR), 1 an innate or acquired phenotype, which allows cancer cells to resist a broad spectrum of chemotherapeutic drugs. One of the most extensively studied and clinically relevant mechanisms of drug resistance is the overexpression by cancer cells of the transmembrane multidrug transporter P-glycoprotein (MDR1, ABCB1, EC 3.6.3.44) (1). However, a drug resistance phenotype comprises many, often interacting, mechanisms of resistance (2). These include increased DNA repair, altered target sensitivity, decreased apoptotic response and numerous aberrant signal transduction pathways. Elevated levels of glycolipids have been correlated with multidrug resistance in cancer (3, 4). Manipulation of glycolipid levels in some MDR cancer cells was able to reverse drug resistance (5-12). This led to the hypothesis that elevated glucosylceramide synthase (GCS, EC 2.4.1.80) activity is a novel form of multidrug resistance and that inhibition of GCS is a promising therapeutic strategy for combating multidrug resistance. The biochemical basis is that an elevated GCS activity prevents the accumulation of ceramide, which is thought to precede, and trigger, apoptosis in response to some cytotoxic drugs (13-16). Therefore, inhibition of GCS activity will promote the accumulation of pro-apoptotic ceramide and enhance cell death in response to cytotoxic agents (17-21).This hypothesis rests largely on three lines of evidence. The first is that multidrug resistance correlates with elevated glycolipid levels (4), which is unfortunately hindered by the small number of samples analyzed and the fact that variability of glycolipid levels in cancers remains unknown.Second, genetic manipulation of the GCS enzyme in MCF7 cells can affect their sensitivity to various cytot...

The chemosensitizing activity of inhibitors of glucosylceramide synthase is mediated primarily through modulation of P-gp function

“…In general, the assumption has been that the sensitizing effect was due to inhibition of GCS, thereby resulting in increased ceramide levels and induction of apoptosis. However, recent studies suggest that the chemosensitizing effect of PDMP and its analogues may not be related to GCS inhibition (25)(26)(27). Iminosugar-based GCS inhibitors which are from a different chemical class than the ceramide-based inhibitor PDMP were observed to be ineffective in chemosensitizing multidrug-resistant tumor cells (25).…”

PDMP sensitizes neuroblastoma to paclitaxel by inducing aberrant cell cycle progression leading to hyperploidy

“…These compounds, which seem to be more specific and less toxic, did not sensitize multidrug-resistant tumor cells to cytostatics (9,12). NorrisCervetto et al (9) concluded from their studies that the effect of PDMP on drug resistance could not be explained by inhibition of GCS alone. However, there are no studies providing a mechanism unrelated to GCS inhibition by which PDMP could chemosensitize multidrug-resistant tumor cells.…”

“…10,11). It was observed that iminosugars, which are potent inhibitors of GCS, do not chemosensitize multidrug-resistant tumor cells to cytostatics (9,12). The notion thus arises that chemosensitization achieved by PDMP may not involve GCS but instead is caused by an alternative mechanism.…”

“…Recently, however, the involvement of GCS in drug sensitization by PDMP has been questioned (ref. 9; for reviews on this issue, see refs. 10,11).…”

Apoptotic modification of glycosphingolipids of human granulocytes

“…Високоефективну рідинну хроматографію проводили за методикою, опи-саною у [21][22][23]. Зразки для ВЕРХ аналізу готували таким чином: клітини відми-вали двічі у ЗФР, осад ресуспендували у 100 мкл ЗФР, додавали 200 мкл дейоні зованої води і проводили три цикли заморожування/розтавання.…”