SDZ PSC 833 the drug resistance modulator activates cellular ceramide formation by a pathway independent of P-glycoprotein

SDZ PSC 833 (PSC 833) is a new multidrug resistance modulator. Recent studies have shown that the principal mechanism of action of PSC 833 is to bind P-glycoprotein (P-gp) and prevent cellular efflux of chemotherapeutic drugs. We previously reported that PSC 833 increases cellular ceramide levels. The present study was conducted to determine whether the impact of PSC 833 on ceramide generation is dependent on P-gp. Work was carried out using the drug-sensitive P-gp-deficient human breast adenocarcinoma cell line, MCF-7, and drug resistant MCF-7/MDR1 clone 10.3 cells (MCF-7/MDR1), which show a stable MDR1 P-gp phenotype. Overexpression of P-gp in MCF-7/MDR1 cells did not increase the levels of glucosylceramide, a characteristic which has been associated with multidrug resistant cells. Treatment of MCF-7 and MCF-7/MDR1 cells with PSC 833 caused similar ceramide elevation, in a dose-responsive manner. At 5.0 microM, PSC 833 increased ceramide levels 4- to 5-fold. The increase in ceramide levels correlated with a decrease in survival in both cell lines. The EC50 (concentration of drug that kills 50% of cells) for PSC 833 in MCF-7 and MCF-7/MDR1 cells was 7.2 +/- 0.6 and 11.0 +/- 1.0 microM, respectively. C6-Ceramide exposure diminished survival of MCF-7 cells; whereas, MCF-7/MDR1 cells were resistant to this short chain ceramide analog. Preincubation of cells with cyclosporine A, which has high affinity for P-gp, did not diminish the levels of ceramide generated upon exposure to PSC 833. These results demonstrate that PSC 833-induced cellular ceramide formation occurs independently of P-gp. As such, these data indicate that reversal of drug resistance by classical P-gp blockers may be modulated by factors unrelated to drug efflux parameters.     

Multidrug resistance modulators and doxorubicin synergize to elevate ceramide levels and elicit apoptosis in drug-resistant cancer cells.

BACKGROUND: To provide insight for the development of more effective clinical agents, the authors attempted to elucidate the mechanisms of action of multidrug resistance (MDR) modulators. Previously, the authors found that MDR modulators blocked the conversion of ceramide to glucosylceramide in MDR cells, thereby enhancing cytotoxicity. Because ceramide is a critical component of the apoptosis signaling cascade, the current study examined the impact of therapy using agents that elicit ceramide formation combined with agents that block ceramide glycosylation. METHODS: Doxorubicin-resistant human breast carcinoma cells (MCF-7-AdrR) were treated with either doxorubicin, tamoxifen, cyclosporine A, or the cyclosporine A analog SDZ PSC 833 (PSC 833) or with combinations thereof, and ceramide and glucosylceramide metabolisms were measured by cell radiolabeling. Cell viability was quantitated spectrophotometrically and apoptosis was evaluated analyzing DNA integrity by gel electrophoresis. RESULTS: Whereas cyclosporine A blocked the generation of glucosylceramide in MCF-7-AdrR cells, a chemical cousin, PSC 833, elicited a 3-fold increase in glucosylceramide and a 5-fold increase in ceramide levels at 24 hours. The PSC 833 response was time-dependent(as early as 30 minutes) and dose-dependent (as low as 0.1 microM). The appearance of ceramide foreran the generation of glucosylceramide. Sphingomyelin levels were not decreased in response to PSC 833; however, Fumonisin B1, a ceramide synthase inhibitor, blocked PSC 833-induced ceramide generation. Adding tamoxifen, which blocks ceramide glycosylation, to the PSC 833 regimen boosted ceramide levels 11-fold over controls and caused DNA fragmentation. A 3-component regimen comprised of tamoxifen, doxorubicin, and PSC 833 increased ceramide levels 26-fold and brought cell viability to zero. CONCLUSIONS: These results demonstrate that MDR modulators can be used separately, in combination, or in conjunction with chemotherapy at clinically relevant concentrations to manipulate cellular ceramide levels and restore sensitivity in the drug resistant setting. As such, this represents a new direction in the treatment of cancer.     

Accumulation of lactosylceramide and overexpression of a PSC833-resistant P-glycoprotein in multidrug-resistant human sarcoma cells

The selection pressure for resistance to chemotherapy is accompanied by the enhanced expression of ABC proteins and increased cellular glycosphingolipid content. Thus, a possible connection between glycosphingolipid metabolism and ABC proteins in drug resistance has been suggested. In the present study, we established two human multidrug-resistant (MDR) cell lines derived from MESSA sarcoma cells by culturing with increasing concentrations of doxorubicin (DX5 cells) or doxorubicin together with cyclosporin A (GARF cells). Both resistant cell lines overexpressed the MDR1 gene and the wild-type P-glycoprotein at the same level. The cyclosporin derivative PSC833, a potent inhibitor of P-glycoprotein, sensitized DX5 but not GARF cells to the cytotoxic effects of daunorubicin. Moreover, PSC833 increased the nuclear accumulation of daunorubicin and the cellular accumulation of [3H]vinblastine in the DX5 but not in the GARF cells. The cellular incorporation of [3H]-cyclosporin A was lower in DX5 cells compared to MESSA and GARF cells, which incorporated the same level of [3H]-cyclosporin A. Sphingolipid analysis showed that the lactosylceramide level was 2.5- and 5-fold higher in DX5 and GARF cells, respectively, than in MESSA cells. Whereas the pharmacological inhibition of lactosylceramide synthesis was able to reverse only partially the resistance of GARF cells to daunorubicin without significant increase in nuclear accumulation of the drug, the same treatment before the co-treatment with PSC833 and daunorubicin increased the cytotoxic effect of daunorubicin and its nuclear accumulation. These data suggest a possible relationship between lactosylceramide levels and the resistance of P-glycoprotein to modulation by MDR modulators.     

Modulation of resistance to idarubicin by the cyclosporin PSC 833 (valspodar) in multidrug-resistant cells

Idarubicin (IDA) is an anthracycline anticancer drug utilized in the treatment of acute leukemias. There are conflicting data published with regard to the cross-resistance of IDA in multidrug-resistant (MDR) cells expressing P-glycoprotein (P-gp). We evaluated the cytotoxicity and cellular accumulation of IDA in a panel of anthracycline-selected MDR cell lines. Leukemia K562/R7 cells and sarcoma MES-SA/Dx5 cells expressing high levels of the MDR1 (ABCB1) gene were resistant to IDA (42-fold and 150-fold, respectively). In both of these cell lines, resistance to IDA was equivalent to that for doxorubicin, the drug used to select for the MDR variants. The P-gp inhibitor PSC 833 (valspodar) at 2 microM completely restored sensitivity to IDA. IDA accumulation was decreased 12-fold in MES-SA/Dx5 cells vs parental cell line, and drug uptake was restored to control levels by PSC 833. Reduced intracellular IDA was correlated with P-gp content by flow cytometry. Experiments in NIH3T3 murine cells transfected with the human MDR1 gene substantiated the findings of cross-resistance to IDA and reversal of resistance by PSC 833. Our data indicate that IDA is a high-affinity substrate for P-gp.     

Selective susceptibility of transformed T lymphocytes to induction of apoptosis by PSC 833, an inhibitor of P-glycoprotein

P-glycoprotein is a cellular efflux pump. The P-glycoprotein inhibitor PSC 833 causes apoptosis of cancer cells and induces a rise in the intracellular levels of ceramide. Our aims were to determine whether a cause and effect relationship exists between these two actions of PSC 833, and to assess whether the PSC 833-induced apoptosis is restricted to transformed cells. Apoptosis was determined by flow cytometry and radioactive quantitation of DNA fragmentation. PSC 833 induced apoptosis in the human T leukemia cell lines: Molt-4 and Jurkat. Analysis of the apoptosis in Molt-4 and Jurkat cells revealed that PSC 833 induced a rise in the cellular ceramide levels (as measured by the DG kinase assay). PSC 833-induced apoptosis was significantly reduced by specific inhibitors of ceramide de novo synthesis (i.e., fumonisin B1 and L-cycloserine). On the other hand, PSC 833 did not induce apoptosis in normal peripheral blood T cells regardless of whether these cells were quiescent, activated, or proliferating. Our results suggest that PSC 833 induces apoptotic death in human transformed T lymphocytes through an increase in ceramide de novo synthesis. In addition, normal lymphocytes are not susceptible to induction of apoptosis by PSC 833. This difference between normal lymphocytes and leukemia cells presents a potential target for chemotherapy.     

Glucosylceramide synthase blockade down-regulates P-glycoprotein and resensitizes multidrug-resistant breast cancer cells to anticancer drugs

Overexpression of glucosylceramide synthase (GCS), a pivotal enzyme in glycolipid biosynthesis, contributes to cancer cell resistance to chemotherapy. We previously showed that transfection of doxorubicin-resistant MCF-7-AdrR cells with GCS antisense restored cell sensitivity to doxorubicin and greatly enhanced sensitivity to vinblastine and paclitaxel. In that study, doxorubicin promoted generation of ceramide in MCF-7-AdrR/GCS antisense cells; the present study implicates factors in addition to ceramide that augment sensitivity to chemotherapy. Although GCS antisense cells showed enhanced ceramide formation compared with MCF-7-AdrR when challenged with paclitaxel, GCS antisense cells also showed a 10-fold increase in levels of intracellular drug (paclitaxel and vinblastine). In addition, transfected cells had dramatically decreased expression (80%) of P-glycoprotein and a 4-fold decrease in the level of cellular gangliosides. Chemical inhibition of GCS produced the same effects as antisense transfection: exposure of MCF-7-AdrR cells to the GCS inhibitor 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP, 5.0 micromol/L, 4 days) decreased ganglioside levels, restored sensitivity to vinblastine, enhanced vinblastine uptake 3-fold, and diminished expression of MDR1 by 58%, compared with untreated controls. A similar effect was shown in vinblastin-resistant KB-V0.01 cells; after 7 days with PPMP (10 micromol/L), MDR1 expression fell by 84% and P-glycoprotein protein levels decreased by 50%. MCF-7-AdrR cells treated with small interfering RNAs to specifically block GCS also showed a dramatic decrease in MDR1 expression. This work shows that limiting GCS activity down-regulates the expression of MDR1, a phenomenon that may drive the chemosensitization associated with blocking ceramide metabolism. The data suggest that lipids play a role in the expression of multidrug resistance.     

Inhibitory Effects of a Cyclosporin Derivative, SDZ PSC 833, on Transport of Doxorubicin and Vinblastine via Human P-Glycoprotein

The inhibitory effects of SDZ PSC 833 (PSC833), a non-immunosuppressive cyclosporin derivative, on the P-glycoprotein (P-gp)-mediated transport of doxorubicin and vinblastine were compared with those of cyclosporin A (Cs-A). The transcellular transport of the anticancer drugs and PSC833 across a monolayer of LLC-GA5-COL150 cells, which overexpress human P-gp, was measured. Both PSC833 and Cs-A inhibited P-gp-mediated transport of doxorubicin and vinblastine in a concentration-dependent manner and increased the intracellular accumulation of doxorubicin and vinblastine in LLC-GA5-COL150 cells. The values of the 50%-inhibitory concentration (IC₅₀) of PSC833 and Cs-A for doxorubicin transport were 0.29 and 3.66 μ M , respectively, and those for vinblastine transport were 1.06 and 5.10 μ M , respectively. The IC₅₀ of PSC833 for doxorubicin transport was about 4-fold less than that for vinblastine transport, suggesting that the combination of PSC833 and doxorubicin might be effective. PSC833 itself was not transported by P-gp and had higher lipophilicity than Cs-A. These results indicated that the inhibitory effect of PSC833 on P-gp-mediated transport was 5- to 10-fold more potent than that of Cs-A, and this higher inhibitory effect of PSC833 may be related to the absence of PSC833 transport by P-gp and to the higher lipophilicity of PSC833.     

Combined treatment of Bcl-2 antisense oligodeoxynucleotides (G3139), p-glycoprotein inhibitor (PSC833), and sterically stabilized liposomal doxorubicin suppresses growth of drug-resistant growth of drug-resistant breast cancer in severely combined immunodeficient mice

We studied the possibility of increasing sensitization of drug-resistant MDA435/LCC6 multidrug-resistant (MDR) human breast cancer cells to doxorubicin (DOX) by increasing cellular drug retention with P-glycoprotein (P-gp) inhibitor PSC833 in combination with induction of cell death through down-regulation of Bcl-2 protein using Bcl-2 antisense (G3139). In in vitro cytotoxicity assays, the combination of G3139 with DOX exhibited 40% increased cytotoxicity in both wild-type (WT) and MDR cells. PSC833 increased the cytotoxicity of DOX and Taxol with complete and partial reversal of the resistance of MDR cells to DOX and Taxol, respectively. The presence of G3139 did not increase the cytotoxicity of PSC833 combined with DOX or Taxol in both cell lines. In vivo studies with WT and MDR cell lines transplanted into severely combined immunodeficient mice demonstrated that G3139 (5 mg/kg) was able to suppress the growth of both WT and MDR tumors to an equivalent extent. PSC833 (100 mg/kg) partially restored the sensitivity of resistant tumors to DOX, and the combination of G3139 and PSC833 with liposomal DOX showed maximum growth suppression of MDR tumors compared with individual treatments. The improved efficacy of this treatment was attributed to Bcl-2 antisense-induced apoptosis, combined with cellular retention of DOX in tumor cells via P-gp blockade.     

Effect of PSC 833 on the cytotoxicity and pharmacodynamics of mitoxantrone in multidrug-resistant K562 cells

We examined the effect of PSC 833, a nonimmunosuppressive cyclosporin analogue, on the cytotoxicity, accumulation and retention of an anthraquinone antileukemia drug mitoxantrone (MIT). This was done in P-glycoprotein (PGP)-overexpressing multidrug-resistant K562/D1-9 cells and compared with the effect of cyclosporin A (CsA). We also compared MIT with the effect of PSC 833 on the cytotoxicity of daunorubicin (DNR) and doxorubicin (DOX). While PSC 833 and CsA had no effect on the cytotoxicity, accumulation and retention of MIT in the parent K562 cells, PSC 833 and CsA restored accumulation and retention of MIT in K562/D1-9 cells dose-dependently. Consequently, there was increased sensitivity of K562/D1-9 cells to MIT. The reversing activity of PSC 833 on the cytotoxicity of MIT was stronger than that of CsA, and was almost the same as the reversing activity of PSC 833 on the cytotoxicity of DNR and DOX. The resistance index of MIT decreased from 43.9-fold to 2.8-fold by 0.4 microM PSC 833, which is a clinically achievable plasma concentration. These results suggest that the combination of PSC 833 with MIT could be a promising treatment in reversing PGP-mediated MDR in leukemia patients.     

A Phase I study of infusional vinblastine in combination with the P-glycoprotein antagonist PSC 833 (valspodar).

BACKGROUND: PSC 833 is a second-generation P-glycoprotein (Pgp) antagonist developed to reverse multidrug resistance (MDR). The authors conducted a Phase I study of orally administered PSC 833 in combination with vinblastine administered as a 5-day continuous infusion. METHODS: Seventy-nine patients with advanced malignant disease were enrolled in the trial and treated with escalating doses of PSC 833. Pharmacokinetic interactions between PSC 833 and vinblastine were anticipated. Accordingly, when dose limiting toxicities were observed, the dose of vinblastine was reduced as PSC 833 was escalated. Three schedules and two formulations of PSC 833 were used in the study. RESULTS: The maximum tolerated doses of PSC 833 were 12.5 mg/kg orally every 12 hours for 8 days for the liquid formulation in combination with 0.9 mg/m(2) per day vinblastine as a continuous intravenous infusion (CIV) for 5 days; and 4 mg/kg orally every 6 hours for 8 days for the microemulsion formulation in combination with 0.6 mg/m(2) per day vinblastine CIV for 5 days. The principal toxicities for PSC 833 were ataxia and paresthesias and for the combination, constipation, fever. and neutropenia. Increased oral bioavailability and increased peak and trough concentrations were observed with the microemulsion formulation. Significant interpatient variability in pharmacokinetic parameters was observed. Ten patients studied at the MTD for PSC 833 (4 mg/kg orally every 6 hours for 8 days) had inhibition of rhodamine efflux from CD56 positive peripheral lymphocytes as a surrogate for Pgp antagonism. Among 43 evaluable patients with clear cell carcinoma of the kidney, 3 patients had complete responses, and 1 patient had a partial response. CONCLUSIONS: PSC 833 in combination with vinblastine can be administered safely to patients provided the vinblastine dose is adjusted for pharmacokinetic interactions. The high interpatient variability is a significant confounding factor. Surrogate studies with CD56 positive cells suggest that Pgp inhibition in the clinical setting is achievable. Improved methods for predicting pharmacokinetic interactions should improve future studies.     

Cytotoxic effect of the cyclosporin PSC 833 in multidrug-resistant leukaemia cells with increased expression of P-glycoprotein.

Multidrug resistance (MDR) to anti-cancer agents is frequently associated with overexpression of the drug efflux transporter P-glycoprotein (Pgp) in cancer cells, ensuing drug expulsion and maintenance of tolerable intracellular levels of certain cytotoxic drugs. Pgp may also be present in normal tissue, providing protection against toxic substances, but the physiological role of Pgp is not fully understood. Recently, it was shown that Pgp also takes part in the transport of certain growth-regulating cytokines (Drach et al, 1996; Raghu et al, 1996). Therefore, we studied the effect of the highly potent Pgp inhibitor PSC 833 on proliferation of three pairs of MDR and parental human cell lines (HB8065 hepatoma cells, KG1a and K562 leukaemia cells). The MDR phenotypes were characterized by Pgp overexpression, which was demonstrated by flow cytometry using the anti-Pgp antibody MRK16. Electronic cell counting of 72-96 h cultures revealed a dose-dependent antiproliferative effect of PSC 833 in the resistant KG1a/200 and K562/150 cells. The half-maximal growth inhibitory concentrations (GI50) were 0.2 microM and 0.7 microM respectively. Exposure to PSC 833 induced cell death by apoptosis in both cell types, as revealed by flow cytometry and detection of 3''-hydroxy ends of DNA (the result of DNA fragmentation associated with apoptosis), by terminal transferase-mediated dUTP-biotin nick end-labelling (TUNEL). Similar effects were not found in the hepatoma cell lines or the parental leukaemia lines. These results demonstrated a discriminating cytotoxicity of PSC 833 in two human leukaemia MDR variants, representing a possible therapeutic indication which warrants consideration during the ongoing clinical evaluation of this drug.     

Comparative Study on Reversal Efficacy of SDZ PSC 833, Cyclosporin a and Verapamil on Multidrug Resistance in vitro and in vivo

A non-immunosuppressive cyclosporin, SDZ PSC 833 (PSC833), shows a reversal effect on multidrug resistance (MDR) by functional modulation of MDR1 gene product, P-glycoprotein. The objective of the present study was to compare the reversal efficacy of three multidrug resistance modulators, PSC833, cyclosporin A (CsA) and verapamil (Vp). PSC833 has approximately 3-10-fold greater potency than CsA and Vp with respect to the restoring effect on reduced accumulation of doxorubicin (ADM) and vincristine (VCR) in ADM-resistant K562 myelogenous leukemia cells (K562/ADM) in vitro and also on the sensitivity of K562/ADM to ADM and VCR in in vitro growth inhibition. The in vivo efficacy of a combination of modifiers (PSC833 and CsA: 50 mg/kg, Vp 100 mg/kg administered p.o. 4 h before the administration of anticancer drugs) with anticancer drugs (ADM 2.5 mg/kg i.p., Q4D days 1, 5 and 9, VCR 0.05 mg/kg i.p., QD days 1-5) was tested in ADM-resistant P388-bearing mice. PSC833 significantly enhanced the increase in life span by more than 80%, whereas CsA and Vp enhanced by less than 50%. This reversal potency, which exceeded that of CsA and Vp, was confirmed by therapeutic experiments using colon adenocarcinoma 26-bearing mice. These results demonstrated that PSC833 has signficant potency to reverse MDR in vitro and in vivo, suggesting that PSC833 is a good candidate for reversing multidrug resistance in clinical situations.     

Alteration of the daunorubicin-triggered sphingomyelin-ceramide pathway and apoptosis in MDR cells: influence of drug transport abnormalities

We have previously shown that in myeloid leukemic cells, daunorubicin (DNR) induces apoptosis via the activation of the sphingomyelin-ceramide pathway. We have now investigated sphingomyelin (SM) hydrolysis, ceramide generation, and apoptosis in vincristine-selected multidrug resistant (MDR) HL-60 cells (HL-60/Vinc), compared with their parental counterparts. We show that DNR triggers the SM cycle (stimulation of neutral sphingomyelinase, SM hydrolysis, and ceramide generation) and apoptosis in both parental and MDR cells, when used at isotoxic doses (ie., 1 and 100 microM for HL-60 and HL-60/Vinc, respectively). However, in MDR cells treated with either 10 microM DNR or 1 microM DNR in association with the P-glycoprotein (P-gp) blocker verapamil (treatment conditions which yield an intracellular DNR concentration similar to that achieved with 1 microM in the parental cells), we were unable to detect SM hydrolysis, ceramide generation and apoptosis. This implies that inhibition of the DNR-induced SM cycle in MDR cells is not directly related to P-gp. We have also investigated the influence of intracellular drug localization on the DNR-induced SM-cycle in HL-60/Vinc cells. In these cells, DNR at 10 microM is mainly localized in cytoplasmic vesicles, while the drug is diffusely distributed when used at 100 microM. A diffuse distribution pattern was also observed when MDR cells were treated with 1 microM DNR in association with the cyclosporine derivative PSC-833, but not with verapamil. In parallel, PSC-833, but not verapamil, restored the induction of the SM cycle and the apoptotic potential of DNR, and markedly increased drug cytotoxicity in MDR cells. Our results suggest that altered intracellular drug transport plays an important role in limiting ceramide generation and cell death in MDR cells.     

Potentiation of the reversal activity of SDZ PSC833 on multi-drug resistance by an anti-p-glycoprotein monoclonal antibody MRK-16

SDZ PSC833 (PSC833), an analogue of cyclosporines, is one of the most potent modulators of multi-drug resistance (MDR). We previously reported that MRK-16, an anti-P-glycoprotein MAb, enhanced MDR reversal activity of cyclosporin A (CsA) through inhibition of P-glycoprotein-mediated CsA transport. We have examined here whether MRK-16 can enhance MDR reversal activity of PSC833. We found that MRK-16 potentiated the MDR reversal activity of PSC833, and of CsA, in MDR sublines of human myelocytic leukemia K562 and human ovarian cancer A2780 cells. Like MRK-16 combined with CsA, MRK-16 enhanced the effect of a sub-optimum dose of PSC833 on vincristine accumulation in MDR cells. However, MRK-16 could not increase cellular accumulation of PSC833 in MDR tumor cells, yet it could increase cellular accumulation of CsA. P-glycoprotein could not transport PSC833 but could transport CsA. Our results indicate that MRK-16 potentiates the MDR reversal activity of both PSC833 and CsA, yet also suggest that the molecular mechanism of the potentiation differs between the two substances. © 1996 Wiley-Liss, Inc.     

Resistance to microtubule-targeted cytotoxins in a K562 leukemia cell variant associated with altered tubulin expression and polymerization

A vinblastine resistant cell line, KCVB2, was established by co-selecting the parental erythroleukemic cell line K562 with step-wise increased concentrations of vinblastine (Velban) in the presence of the cyclosporin D analogue PSC 833 (2 microM), a potent modulator of the multidrug resistance phenotype. KCVB2 cells are 8-fold resistant to the selecting agent, vinblastine, but also exhibit significant resistance to other vinca alkaloids, including 14-fold resistance to vinorelbine, as well as 6-fold cross-resistance to paclitaxel. Doubling time and morphology were similar to the parental K562 cells. Rt-PCR analysis revealed no alterations in the expression of the mdr1 and MRP genes. Intracellular vinblastine accumulation was unchanged. Disruption of the mitotic spindles and multiple mitotic asters occurred in both cell lines but required higher concentrations of vinblastine in KCVB2 cells than in K562 cells. Significant differences were observed in the tubulin content of KCVB2 cells: reduction of total tubulin content, increased polymerized fraction of total tubulin, and overexpression of class III beta-tubulin which is expressed at very low levels in the parental K562 cells. K562 cells transfected with murine class III beta-tubulin did not display the resistance pattern observed in KCVB2 cells. Revertants of KCVB2 manifested reversion to parental drug sensitivity, an increase in total tubulin level, and a decrease in polymerized tubulin. In conclusion, the KCVB2 cell line displays a novel mechanism of resistance to both depolymerizing and stabilizing microtubule-targeted cytotoxins which does not involve altered cellular drug accumulation, but corresponds to alterations in the total tubulin content and polymerization status, and may involve an effect on microtubule dynamics.     

Selection of non-P-glycoprotein mediated high-level etoposide resistant cell lines by adriamycin with P-gp inhibitors

In murine erythroleukemia (MEL) A20 cells (grown in 20 ng/ml adriamycin), mutation(s) producing 10-fold adriamycin (doxorubicin) resistance emerged via an unknown mechanism. Exposure of A20 cells to further stepwise increasing concentrations of ADR in combination with MDR modulators (PSC833 and verapamil) aimed to amplify the undetermined A20 mechanism while controlling P-glycoprotein (P-gp) overexpression. The growth of the derived cell lines A30P, A40P and A60P (grown in 30, 40 and 60 ng/ml ADR with PSC833 and verapamil) was initially slow, but eventually reached near WT rates. The new cell lines A30P and A40P were only 1.3- and 1.6-fold more resistant to adriamycin than PC4 A20. Resistance to vincristine was unchanged, but resistance to etoposide (VP-16) was 3.7-fold higher in A40P than A20 (itself 97-fold higher than wild-type). Expression of mdr3 and mrp mRNA tested by RT-PCR showed no increase. Daunorubicin and etoposide accumulation was not different among the cell lines, and no changes were detected in the number of daunorubicin fluorescent lysosomes. In comparison to WT, reduced topoisomerase IIalpha (EC 5.99.1.3) activity (20%) and protein expression (80%) was similar to the parental A20 cells. No mutations in the coding sequence of topoisomerase IIalpha could be located to account for the high etoposide resistance levels. The inhibitor combination of verapamil and PSC833 prevented the emergence of transporter mediated MDR, but not ADR selection of cell lines highly resistant to etoposide.     

Potential role of acid ceramidase in conversion of cytostatic to cytotoxic end-point in pancreatic cancer cells

Purpose

Acid ceramidase (AC) occupies an important place in the control of cancer cell proliferation. We tested the influence of AC inhibition on the effects of PSC 833, a P-glycoprotein antagonist with potent ceramide-generating capacity, to determine whether AC could be a therapeutic target in pancreatic cancer.

Methods

Ceramide metabolism was followed using ³H-palmitate, and molecular species were determined by mass spectroscopy. Apoptosis was measured by DNA fragmentation, autophagy by acridine orange staining, and cell cycle was assessed by flow cytometry and RB phosphorylation. AC was measured in intact cells using fluorescent substrate.

Results

Exposure of human PANC-1 or MIA-PaCa-2 cells to PSC 833 promoted increases in de novo (dihydro)ceramides, (dihydro)glucosylceramides, and (dihydro)sphingomyelins, demarking ceramide generation and robust metabolism. Despite the multifold increases in (dihydro)ceramide levels, cells were refractory to PSC 833. However, PSC 833 produced a dose-dependent decrease in DNA synthesis and dose- and time-dependent decreases in RB phosphorylation, consistent with cell cycle arrest as demonstrated at G1. Cytostatic effects of PSC 833 were converted to cytotoxic end-point by acid ceramidase inhibition. Cytotoxicity was accompanied by formation of acridine orange-stained acidic vesicles and an increase in LC3 expression, indicative of autophagic response. Cell death was not reversed by preexposure to myriocin, which blocks PSC 833-induced ceramide generation.

Conclusion

Although the role of ceramide in end-point cytotoxicity is unclear, our results suggest that acid ceramidase is a viable target in pancreatic cancer. We propose that AC inhibition will be effective in concert with other anticancer therapies.