Differential expression of sphingolipids in MRP1 overexpressing HT29 cells

We have obtained a novel multidrug resistant cell line, derived from HT29 G(+) human colon carcinoma cells, by selection with gradually increasing concentrations of the anti-mitotic, microtubule-disrupting agent colchicine. This HT29(col) cell line displayed a 25-fold increase in colchicine resistance and exhibited cross-resistance to doxorubicin, VP16, vincristine and taxol. Immunoblotting, combined with RT-PCR showed that the multidrug resistance phenotype was conferred by specific overexpression of the multidrug resistance protein 1. Confocal scanning laser microscopy revealed that multidrug resistance protein 1 specifically localized in the plasma membrane of HT29(col) cells. In a functional assay, using the fluorescent multidrug resistance protein 1 substrate 5-carboxyfluorescein, an increased efflux activity of HT29(col) cells was measured, as compared to the wild-type HT29 G(+) cells. MK571, a specific inhibitor of multidrug resistance protein 1, blocked the 5-carboxyfluorescein efflux, but only partially reversed resistance to colchicine, indicating that additional multidrug resistance mechanisms operate in HT29(col) cells. In conclusion, these results show for the first time overexpression of a functional multidrug resistance protein 1 under colchicine pressure, indicating that colchicine is not a P-glycoprotein-specific substrate. Colchicine-induced overexpression of multidrug resistance protein 1 is accompanied by a changed sphingolipid composition, i.e., enhanced levels of glucosylceramide and galactosylceramide. In addition, ceramide, a lipid messenger molecule involved in apoptosis-related signal transduction processes, was much more abundant in HT29(col) cells, which is indicative of a stress response.     

Therapeutic potential of targeting ceramide/glucosylceramide pathway in cancer

Sphingolipids including ceramides and its derivatives such as ceramide-1-phosphate, glucosylceramide (GlcCer), and sphingosine-1-phosphate are essential structural components of cell membranes. They now recognized as novel bioeffector molecules which control various aspects of cell growth, proliferation, apoptosis, and drug resistance. Ceramide, the central molecule of sphingolipid metabolism, generally mediates anti-proliferative responses such as inhibition of cell growth, induction of apoptosis, and/or modulation of senescence. There are two major classes of sphingolipids. One of them is glycosphingolipids which are synthesized from the hydrophobic molecule, ceramide. GlcCer, generated by glucosylceramide synthase (GCS) that transfers the glucose from UDP-glucose to ceramide, is an important glycosphingolipid metabolic intermediate. GCS regulates the balance between apoptotic ceramide and antiapoptotic GlcCer. Downregulation or inhibition of GCS results in increased apoptosis and decreased drug resistance. The mechanism underlying the drug resistance which develops with increased glucosylceramide expression is associated with P-glycoprotein. In various types of cancers, overexpression of GCS has been observed which renders GCS a good target for the treatment of cancer. This review summarizes our current knowledge on the structure and functions of glucosylceramide synthase and glucosylceramide and on the roles of glucosylceramide synthase in cancer therapy and drug resistance.     

非小细胞肺癌中多药耐药基因的表达及意义

目的:探讨多药耐药基因(MDR1)和多药耐药相关蛋白基因(MRP)在非小细胞肺癌(NSCLC)中的表达、意义及相关关系.方法:采用原位分子杂交对113例NSCLC组织中MDR1和MRP基因mRNA的表达进行检测.结果:MDR1和MRP基因mRNA在NSCLC组织中的阳性表达率分别为51.3%(58/113)、80.5%(91/113),二者与NSCLC肿瘤组织类型、分化程度、淋巴结转移、TNM分期等无关(P＞0.05).MDR1和MRP的协同阳性(MDR1 /MRP )表达率为48.7%(55/113),二者在NSCLC中的表达之间存在明显相关(P＜0.01).结论:MDR1和MRP是NSCLC原发性多药耐药的重要参与因素,二者联合检测对临床NSCLC的化疗具有指导意义.     

Expression and functional activity of the ABC-transporter proteins P-glycoprotein and multidrug-resistance protein 1 in human brain tumor cells and astrocytes

The poor prognosis of glioma patients is partly based on the minor success obtained from chemotherapeutic treatments. Resistance mechanisms at the tumor cell level may be, in addition to the blood–brain barrier, involved in the intrinsic chemo-insensitivity of brain tumors. We investigated the expression of the drug-transporter proteins P-glycoprotein (P-gp) and multidrug-resistance protein 1 (MRP1) in cell lines (N=24) and primary cell cultures (N=36) from neuroectodermal tumors, as well as in brain tumor extracts (N=18) and normal human astrocytes (N=1). We found that a considerable expression of P-gp was relatively rare in glioma cells, in contrast to MRP1, which was constitutively overexpressed in cells derived from astrocytomas as well as glioblastomas. Also, normal astrocytes cultured in vitro expressed high amounts of MRP1 but no detectable P-gp. Meningioma cells frequently co-expressed P-gp and MRP1, while, most of the neuroblastoma cell lines express higher P-gp but lower MRP1 levels as compared to the other tumor types. Both, a drug-exporting and a chemoprotective function of P-gp as well as MRP1 could be demonstrated in selected tumor cells by a significant upregulation of cellular ³H-daunomycin accumulation and daunomycin cytotoxicity via administration of transporter antagonists. Summing up, our data suggest that P-gp contributes to cellular resistance merely in a small subgroup of gliomas, but frequently in neuroblastomas and meningiomas. In contrast, MRP1 is demonstrated to play a constitutive role in the intrinsic chemoresistance of gliomas and their normal cell counterpart.     

Differential metabolism and trafficking of sphingolipids in differentiated versus undifferentiated HT29 cells

Trafficking and metabolism of sphingolipids were examined in undifferentiated (G⁺) and differentiated (G⁺ reversed) HT29 human colon adenocarcinoma cell lines. Metabolic experiments employing a fluorescently labeled sphingolipid precursor, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylceramide (C₆-NBD-ceramide) revealed that both qualitative and quantitative differences exist in sphingolipid synthesis between the 2 cell lines. One of the C₆-NBD-sphingolipids synthesized in G⁺ cells is not found in the G⁺ reversed cells. Furthermore, the ratio of the 2 main products, C₆-NBD-glucosyiceramide and C₆-NBD-sphingomyelin, differs: in G⁺ cells glucosylceramide is by far the main product, whereas G⁺ reversed cells synthesize C₆-NBD-sphingomyelin in slight excess. Once established, these ratios of sphingolipids are quickly restored metabolically when distortion of the ratio is caused by experimental manipulation. This indicates that they represent a true metabolic equilibrium situation of the 2 sphingolipids in these cells, while the distinct ratios are mainly determined by the NBD-lipid pool in the plasma membrane. Preferential synthesis and transfer of glucosylceramide from its site of synthesis to the cell surface do not occur when the plasma membrane pool of glucosylceramide is selectively removed. This suggests that instantaneous replenishment via specific signalling is probably not involved as a mechanism in re-establishing perturbed lipid pools. In conjunction with observations on distinct lipid trafficking pathways of glucosylceramide in G⁺ and G⁺ reversed cells, the present metabolic studies emphasize a relation between the expression of this glycolipid and the state of differentiation of HT29 cells.     

Homocamptothecin-daunorubicin association overcomes multidrug-resistance in breast cancer MCF7 cells

The multidrug-resistance (MDR) status of a novel camptothecin analogue, homocamptothecin (hCPT), was investigated in human colon adenocarcinoma HT29 cells, myelogenous leukemia K562 cells and breast carcinoma MCF7 cells. The cytotoxicity of hCPT was not sensitive to the MDR status in K562 cell lines. However, its cytotoxicity was altered by MRP1, but not Pgp, in naturally MRP1-expressing HT29 cells, and etoposide- and doxorubicin-resistant MCF7/VP and MCF7/DOX cells, respectively. These cells were sensitized to hCPT in presence of MK571, probenecid but not verapamil. These results led to consider hCPT as a substrate for MRP1 and a potential modulator of MRP1 activity. The relationship between the cytotoxic effect of anthracyclines and their nuclear localization had been previously demonstrated. We show that MRP1 mediated the daunorubicin (DNR) efflux in MCF7/VP and MCF7/DOX cells. The combination of sub-toxic doses of hCPT with DNR resulted in the potentiation of DNR activity, well-correlated with an increase in its nuclear accumulation in MCF7/VP cells. Simultaneous pattern was shown to provide higher cytotoxic response than sequential one. In agreement, hCPT increased also the DNR nuclear accumulation in low MRP1-expressing MCF7/DOX cells. However, the enhancement of cytotoxicity in the DNR-hCPT combination was poorly correlated with the nuclear concentration of DNR in MCF7/DOX cells. In addition to the increase in DNR accumulation, the potentiation of DNR activity by hCPT in MCF7/DOX cells implied a synergistic mechanism between both drugs. These data suggest that the present topoisomerase I/II inhibitors combination may be of clinical interest to overcome MDR phenotype in DNR-treated breast cancer patients.     

The pH sensitive probe 5-(and-6)-carboxyl seminaphthorhodafluor is a substrate for the multidrug resistance-related protein MRP1

Cellular function is dependent on tight regulation of intracellular pH and numerous reports show cancer cells have abnormal pH values in the cytosol and organelles, such as lysosomes. 5-(and-6)-carboxyl seminaphthorhodafluor (SNARF-1) is a commonly used pH sensitive probe and was used here to determine cytosolic pH of HL-60 leukemia cells and a drug-resistant variant overexpressing multidrug-resistance related protein 1 (MRP1). Resistant cells accumulated significantly less SNARF-1 compared to parental cells but near control levels of probe accumulation were observed by preincubating cells with the specific MRP1 inhibitor MK571. Two new drug-resistant cell lines were generated following exposure to doxorubicin or daunorubicin and these upregulated MRP1 or P-glycoprotein expression, respectively. Experiments in these cells showed that reduced SNARF-1 accumulation was specific to MRP1 overexpression, as cells upregulating P-glycoprotein accumulated control levels of the probe. Confirmation that SNARF-1 is a MRP1 substrate was obtained using K562 and KG1a cells that have been shown to, respectively, constitutively express MRP1 and P-glycoprotein. Together, the data suggest that SNARF-1 is a substrate for MRP1 but not P-glycoprotein, and could therefore be used as a probe to distinguish between expression and activity of these 2 efflux proteins. Finally, we confirm that doxorubicin but not daunorubicin challenged MRP1 overexpressing HL-60 cells have elevated cytosolic pH.     

Inhibition of NADPH oxidase by glucosylceramide confers chemoresistance

The bioactive sphingolipid ceramide induces oxidative stress by disrupting mitochondrial function and stimulating NADPH oxidase (NOX) activity, both implicated in cell death mechanisms. Many anticancer chemotherapeutics (anthracyclines, Vinca alkaloids, paclitaxel, and fenretinide), as well as physiological stimuli such as tumor necrosis factor α (TNFα), stimulate ceramide accumulation and increase oxidative stress in malignant cells. Consequently, ceramide metabolism in malignant cells and, in particular the up-regulation of glucosylceramide synthase (GCS), has gained considerable interest in contributing to chemoresistance. We hypothesized that increases in GCS activity and thus glucosylceramide, the product of GCS activity, represents an important resistance mechanism in glioblastoma. In our study, we determined that increased GCS activity effectively blocked reactive oxygen species formation by NOX. We further showed, in both glioblastoma and neuroblastoma cells that glucosylceramide directly interfered with NOX assembly, hence delineating a direct resistance mechanism. Collectively, our findings indicated that pharmacological or molecular targeting of GCS, using non-toxic nanoliposome delivery systems, successfully augmented NOX activity, and improved the efficacy of known chemotherapeutic agents.     

GCS在胃癌组织中的表达及意义

目的 研究多药耐药蛋白葡萄糖神经酰胺合成酶（GCS）在胃癌组织中的表达。方法 免疫组化法检测63例胃癌组织及30例非癌性胃组织标本中GCS的表达情况,分析其与年龄、性别、组织学类型、分化程度、淋巴结转移的相关性。结果 GCS表达在胃癌组织中高于非癌性胃组织,阳性表达率分别为49.2％和23.3％（P＜0.05）;GCS表达与年龄、性别、分化程度、浸润深度、淋巴结转移均无明显相关性。结论 GCS蛋白在胃癌组织中存在高表达,可能是引起胃癌原发性多药耐药的因素之一。     

Nuclear translocation of MRP1 contributes to multidrug resistance of mucoepidermoid carcinoma

Multidrug resistance-related protein 1 (MRP1 or ABCC1), a membrane-bound energy-dependent efflux transporter, is overexpressed in several kinds of multidrug-resistant cell lines and related to multidrug-resistance (MDR) of various cancers. In this study, we investigated whether MRP1 was involved in the chemoresistance of mucoepidermoid carcinoma (MEC). We demonstrated that down-regulation of MRP1 in MC3/5FU, a drug-resistant MEC cell line, by RNA interference increased the drug sensitivity of the cells to 5-fluorouracil, doxorubicin, pharmorubicin, bleomycin-A5, cis-platinum and taxol. However, no significant quantitative difference of MRP1 mRNA and protein expression was found between MC3/5FU cells and its parental cell line (MC3) as determined by RT-PCR and Western blot. Interestingly, MRP1 was translocated from the cytoplasmic membrane of the MC3 cells to the nuclei of MC3/5FU cells as revealed by indirect immunofluorescence staining. Furthermore, MRP1 down-regulation mainly decreased the nuclear expression of MRP1 rather than the cytoplasmic membrane expression. Our results suggested that MRP1 was involved in the chemoresistance of MEC and MRP1 may confer drug-resistance by a mechanism associated with its nuclear translocation.     

Resistance to the antiproliferative effect induced by a short-chain ceramide is associated with an increase of glucosylceramide synthase,P-glycoprotein,and multidrug-resistance gene-1 in cervical cancer cells

Purpose

Ceramide is glycosylated to glucosylceramide or lactosylceramide, and this glycosylation is a novel multidrug-resistance (MDR) mechanism. In this work, a short-chain ceramide (C6), lactosylceramide (LacCer), and an inhibitor of ceramide glycosylation (d-threo-1-phenyl-2-decanoylamino-3-1-propanol, PDMP) were evaluated on the proliferation of cervical cancer cells. The participation of glucosylceramide synthase (GCS), P-glycoprotein (P-gp), and multidrug-resistance gene-1 (MDR-1) in the resistance to the antiproliferative effect induced by C6 was also evaluated.

Methods

Cell proliferation was determined by crystal violet staining. GCS and MDR-1 mRNA expression was evaluated by real-time RT-PCR assay. GCS and P-gp protein expressions, as well as Rhodamine 123 uptake, which is a functional test for P-gp efflux activity, were determined by flow cytometry.

Results

C6 inhibited proliferation of CaLo and CasKi cells with an IC₅₀ of 2.5 μM; however, 50 % proliferation of ViBo cells was inhibited with 10 μM. LacCer increased the proliferation of all cells. When cells were treated with PDMP plus C6, no additional effect on antiproliferation induced by C6 was observed in CaLo and CasKi cells; however, proliferation diminished in comparison with C6 alone in ViBo cells. C6 increased GCS and MDR-1 expression in all cells, as well as P-gp expression in CasKi cells.

Conclusions

Cells that have more capacity to glycosylate ceramide and express a higher level of GCS, MDR-1, and P-gp, are more resistant to the antiproliferative effect induced by C6.     

GCS在人乳腺癌细胞多药耐药中的作用及与P-gP的关系

目的探讨葡萄糖神经酰胺合成酶（GCS）在人乳腺癌细胞多药耐药中的作用及其与P-糖蛋白（P-gP）的关系。方法采用MTT法检测多柔比星（阿霉素）对人乳腺癌耐药细胞株MCF-7／Adr和敏感株MCF-7的抑制率和IC50。以GCS抑制剂D，L-threo-1-phenyl-2-decanoyl—amino-3-morpholino-1-propanol（PDMP）预处理MCF-7／Adr后检测抑制率和IC50。运用流式细胞术（FCM）检测人MCF-7及MCF-7／Adr中GCS、P-gp的表达，以PDMP预处理细胞后检测GCS、P-gP的表达。FCM法检测细胞中ADM的荧光强度。结果MCF-7／Adr对MCF-7的耐药倍数为22．7倍，PDMP作用后阿霉素对MCF-7／Adr的抑制率升高，IC50下降（P〈0．05）。MCF-7／Adr中GCS和P-gp的表达均高于MCF-7，PDMP使MCF-7／Adr中GCS表达下降（P〈0．05），对P—gp表达无明显影响（P〉0．05）。FCM检测显示PDMP可使阿霉素在MCF-7内潴留增多。结论GCS在MCF-7／Adr多药耐药中起重要作用，PDMP能影响P-gP功能，GCS与P-gP有-定关系。     

A very early induction of major vault protein accompanied by increased drug resistance in U-937 cells.

U-937 human leukemia cells were selected for resistance to doxorubicin in the presence or absence of a specific drug modulator that inhibits the activity of P-glycoprotein (Pgp), encoded by the multidrug-resistance gene (MDR1). Parental cells expressed low basal levels of the multidrug-resistance-associated gene (MRP1) and major vault protein (MVP) mRNAs and no MDR1 mRNA. Two doxorubicin-resistant cell lines were selected. Both drug-resistant cell lines upregulated the MVP mRNA level 1.5-fold within 1 cell passage. The MVP mRNA level continued to increase over time as the doxorubicin selection pressure was increased. MVP protein levels generally paralleled the mRNA levels. The 2 high molecular weight vault protein mRNAs were always expressed at constitutive levels. Fully formed vault particles consisting of the MVP, the 2 high molecular weight proteins and the vault RNA assembled and accumulated to increased levels in drug-selected cells. MVP induction is therefore the rate-limiting step for vault particle formation in U-937 cells. By passage 25 and thereafter, the selected cells were resistant to doxorubicin, etoposide, mitoxantrone and 5-fluorouracil by a pathway that was independent of MDR1, MRP1, MRP2 and breast cancer resistance protein. In summary, U-937 doxorubicin-selected cells are programmed to rapidly upregulate MVP mRNA levels, to accumulate vault particles and to become multidrug resistant.     

Keap1 mutations and Nrf2 pathway activation in epithelial ovarian cancer

Resistance to platinum-based chemotherapy develops in the majority of patients with epithelial ovarian cancer (EOC). Platinum compounds form electrophilic intermediates that mediate DNA cross-linking and induce double-strand DNA breaks. Because the cellular response to electrophilic xenobiotics is partly mediated by Keap1-Nrf2 pathway, we evaluated the presence of Kelch-like ECH-associated protein 1 (Keap1) mutations and NF-E2-related factor 2 (Nrf2) pathway activation in EOC and correlated these with platinum resistance and clinical outcome. Nrf2 immunohistochemistry revealed nuclear localization (a surrogate of pathway activation) in over half of EOC patient specimens examined, with more common occurrence in the clear cell EOC subtype. Quantitative real-time PCR revealed that Nrf2 target genes were upregulated in tumors with nuclear positivity for Nrf2. Microarray analysis also showed upregulation of Nrf2 target genes in clear cell EOCs compared with other EOC subtypes. In addition, Keap1 sequence analysis revealed genetic mutations in 29% of clear cell samples and 8% of nonclear cell tumors. RNAi-mediated knockdown of Keap1 was associated with Nrf2 pathway activation and resistance to carboplatin in vitro. Importantly, patients with evidence of Nrf2 pathway activation had fewer complete clinical responses to platinum-based therapy, were enriched for platinum resistance, and had shorter median overall survival compared with those who did not show evidence of Nrf2 pathway activation. Our findings identify Keap1 mutations in EOC and they suggest a previously unrecognized role for the Keap1-Nrf2 pathway in mediating chemotherapeutic responses in this disease.     

Nitric oxide reverts the resistance to doxorubicin in human colon cancer cells by inhibiting the drug efflux

Multidrug resistance (MDR) is a phenomenon by which cancer cells evade the cytotoxic effects of chemotherapeutic agents. It may occur through different mechanisms, but it often correlates with the overexpression of integral membrane transporters, such as P-glycoprotein (Pgp) and MDR-associated proteins (MRPs), with resulting decrease of drug accumulation and cellular death. Doxorubicin is a substrate of Pgp; it has been suggested that its ability to induce synthesis of nitric oxide (NO) could explain, at least in part, its cytotoxic effects. Culturing the human epithelial colon cell line HT29 in the presence of doxorubicin, we obtained a doxorubicin-resistant (HT29-dx) cell population: these cells accumulated less intracellular doxorubicin, were less sensitive to the cytotoxic effects of doxorubicin and cisplatin, overexpressed Pgp and MRP3, and exhibited a lower NO production (both under basal conditions and after doxorubicin stimulation). The resistance to doxorubicin could be reversed when HT29-dx cells were incubated with inducers of NO synthesis (cytokines mix, atorvastatin). Some NO donors increased the drug accumulation in HT29-dx cells in a guarosine-3':5'-cyclic monophosphate-independent way; this effect was associated with a marked reduction of doxorubicin efflux rate in HT29 and HT29-dx cells, and tyrosine nitration in the MRP3 protein. Our results suggest that onset of MDR and impairment of NO synthesis are related; this finding could point to a new strategy to reverse doxorubicin resistance in human cancer.     

Noninvasive molecular imaging of hypoxia in human xenografts: comparing hypoxia-induced gene expression with endogenous and exogenous hypoxia markers

Tumor hypoxia is important in the development and treatment of human cancers. We have developed a novel xenograft model for studying and imaging of hypoxia-induced gene expression. A hypoxia-inducible dual reporter herpes simplex virus type 1 thymidine kinase and enhanced green fluorescence protein (HSV1-TKeGFP), under the control of hypoxia response element (9HRE), was stably transfected into human colorectal HT29 cancer cells. Selected clones were further enriched by repeated live cell sorting gated for hypoxia-induced eGFP expression. Fluorescent microscopy, fluorescence-activated cell sorting, and radioactive substrate trapping assays showed strong hypoxia-induced expression of eGFP and HSV1-tk enzyme in the HT29-9HRE cells in vitro. Sequential micropositron emission tomography (PET) imaging of tumor-bearing animals, using the hypoxic cell tracer (18)F-FMISO and the reporter substrate (124)I-FIAU, yielded similar tumor hypoxia images for the HT29-9HRE xenograft but not in the parental HT29 tumor. Using autoradiography and IHC, detailed spatial distributions in tumor sections were obtained and compared for the following hypoxia-associated biomarkers in the HT29-9HRE xenograft: (124)I-FIAU, (18)F-FMISO, Hoechst (perfusion), lectin-TRITC (functional blood vessels), eGFP, pimonidazole, EF5, and CA9. Intratumoral distributions of (124)I-FIAU and (18)F-FMISO were similar, and eGFP, pimonidazole, EF5, and CA9 colocalized in the same areas but not in well-perfused regions that were positive for Hoechst and lectin-TRITC. In enabling the detection of hypoxia-induced molecular events and mapping their distribution in vivo with serial noninvasive positron emission tomography imaging, and multiple variable analysis with immunohistochemistry and fluorescence microscopy, this human xenograft model provides a valuable tool for studying tumor hypoxia and in validating existing and future exogenous markers for tumor hypoxia.     

Expression of P-glycoprotein in HeLa cells confers resistance to ceramide cytotoxicity

The role of glucosylceramide synthase (GCS) in regulating ceramide-induced apoptosis has been widely studied. The purpose of this investigation was to evaluate the role of P-glycoprotein (P-gp) in regulating ceramide cytotoxicity by using C6-ceramide. To accomplish this, we employed HeLa cells with conditional expression of the multidrug resistance gene 1/P-gp. HeLa cells expressing P-gp (P-gp/on cells) challenged with [14C]C6-ceramide (6 μM), synthesized 4.5-fold the amount of C6-glucosylceramide (GC) compared to HeLa cells with suppressed expression of P-gp (P-gp/off cells), whereas the generated levels of C6-sphingomyelin were almost equal (33 and 29% of intracellular 14C, respectively). Tamoxifen, a P-gp antagonist, decreased the C6-GC levels from 3.5-1.0% in the P-gp/off and from 17-2.8% of the total lipid 14C levels in the P-gp/on cells. Tamoxifen did not inhibit cell-free C6-GC synthesis in the P-gp/off or P-gp/on homogenates. However, a specific GCS inhibitor, ethylenedioxy-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (ethylenedioxy-P4), blocked synthesis by 90%. In the cytotoxicity assays, the P-gp/off cells were sensitive to C6-ceramide and the P-gp/on cells were resistant. Resistance to C6-ceramide in the P-gp/on cells was reversed by tamoxifen but not by ethylenedioxy-P4. Experiments in another cervical cancer model showed that multidrug-resistant P-gp-rich KB-V1 cells synthesized 3-fold more C6-GC from C6-ceramide than the parental, P-gp-poor KB-3-1 cells, and whereas tamoxifen had no effect on the C6-GC synthesis in the KB-3-1 cells, it inhibited synthesis by 70% in the KB-V1 cells. This study demonstrates that P-gp potentiates C6-ceramide glycosylation and if antagonized augments C6-ceramide sensitivity, both features previously ascribed to GCS. We propose that P-gp can be an effective target for enhancing short-chain ceramide cytotoxicity in the treatment of drug-resistant cancer.