Yong-Yu Liu

Ceramide glycosylation potentiates cellular multidrug resistance

Ceramide glycosylation, through glucosylceramide synthase (GCS), allows cellular escape from ceramide‐induced programmed cell death. This glycosylation event confers cancer cell resistance to cytotoxic anticancer agents [Liu, Y. Y., Han, T. Y., Giuliano, A. E., and M. C. Cabot. (1999) J. Biol. Chem. 274, 1140–1146]. We previously found that glucosylce‐ramide, the glycosylated form of ceramide, accumulates in adriamycin‐resistant breast carcinoma cells, in vinblastine‐resistant epithelioid carcinoma cells, and in tumor specimens from patients showing poor response to chemotherapy. Here we show that multidrug resistance can be increased over baseline and then totally reversed in human breast cancer cells by GCS gene targeting. In adriamycin‐resistant MCF‐7‐AdrR cells, transfection of GCS upgraded multidrug resistance, whereas transfection of GCS antisense markedly restored cellular sensitivity to anthracyclines, Vinca alkaloids, taxanes, and other anticancer drugs. Sensitivity to the various drugs by GCS antisense transfection increased 7‐ to 240‐fold and was consistent with the resumption of ceramide‐caspase‐apoptotic signaling. GCS targeting had little influence on cellular sensitivity to either 5‐FU or cisplatin, nor did it modify P‐glyco‐protein expression or rhodamine‐123 efflux. GCS anti‐sense transfection did enhance rhodamine‐123 uptake compared with parent MCF‐7‐AdrR cells. This study reveals that GCS is a novel mechanism of multidrug resistance and positions GCS antisense as an innovative force to overcome multidrug resistance in cancer chemotherapy.—Liu, Y.‐Y., Han, T.‐Y., Giuliano, A. E., and Cabot, M. C. Ceramide glycosylation potentiates cellular multidrug resistance. FASEB J. 15, 719‐730 (2001)

Expression of Glucosylceramide Synthase, Converting Ceramide to Glucosylceramide, Confers Adriamycin Resistance in Human Breast Cancer Cells

Multidrug-resistant cancer cells display elevated levels of glucosylceramide (Lavie, Y., Cao, H. T., Volner, A., Lucci, A., Han, T. Y., Geffen, V., Giuliano, A. E., and Cabot, M. C. (1997) J. Biol. Chem. 272, 1682–1687). In this study, we have introduced glucosylceramide synthase (GCS) into wild type MCF-7 breast cancer cells using a retroviral tetracycline-on expression system, and we developed a cell line, MCF-7/GCS. MCF-7/GCS cells expressed an 11-fold higher level of GCS activity compared with the parental cell line. Interestingly, the transfected cells demonstrated strong resistance to adriamycin and to ceramide, whereas both agents were highly cytotoxic to MCF-7 cells. The EC50 values of adriamycin and ceramide were 11-fold (p < 0.0005) and 5-fold (p < 0.005) higher, respectively, in MCF-7/GCS cells compared with MCF-7 cells. Ceramide resistance displayed by MCF-7/GCS cells closely paralleled the activity of expressed GCS with a correlation coefficient of 0.99. In turn, cellular resistance and GCS activity were dependent upon the concentration of the expression mediator doxycycline. Adriamycin resistance in MCF-7/GCS cells was related to the hyperglycosylation of ceramide and was not related to shifts in the levels of either P-glycoprotein or Bcl-2. This work demonstrates that overexpression of GCS, which catalyzes ceramide glycosylation, induces resistance to adriamycin and ceramide in MCF-7 breast cancer cells.

Uncoupling Ceramide Glycosylation by Transfection of Glucosylceramide Synthase Antisense Reverses Adriamycin Resistance

Previous work from our laboratory demonstrated that increased competence to glycosylate ceramide conferred adriamycin resistance in MCF-7 breast cancer cells (Liu, Y. Y., Han, T. Y., Giuliano, A. E., and M. C. Cabot. (1999) J. Biol. Chem. 274, 1140–1146). This was achieved by cellular transfection with glucosylceramide synthase (GCS), the enzyme that converts ceramide to glucosylceramide. With this, we hypothesized that a decrease in cellular ceramide glycosylation would result in heightened drug sensitivity and reverse adriamycin resistance. To down-regulate ceramide glycosylation potential, we transfected adriamycin-resistant breast cancer cells (MCF-7-AdrR) with GCS antisense (asGCS), using a pcDNA 3.1/his A vector and developed a new cell line, MCF-7-AdrR/asGCS. Reverse transcription-polymerase chain reaction assay and Western blot analysis revealed marked decreases in both GCS mRNA and protein in MCF-7-AdrR/asGCS cells compared with the MCF-7-AdrR parental cells. MCF-7-AdrR/asGCS cells exhibited 30% less GCS activity by in vitro enzyme assay (19.7 ± 1.1 versus 27.4 ± 2.3 pmol GC/h/μg protein,p < 0.001) and were 28-fold more sensitive to adriamycin (EC50, 0.44 ± 0.01 versus12.4 ± 0.7 μm, p < 0.0001). GCS antisense transfected cells were also 2.4-fold more sensitive to C6-ceramide compared with parental cells (EC50= 4.0 ± 0.03 versus 9.6 ± 0.5 μm,p < 0.0005). Under adriamycin stress, GCS antisense transfected cells compared with parental cells displayed time- and dose-dependent increases in endogenous ceramide and dramatically higher levels of apoptotic effector, caspase-3. Western blotting showed that adriamycin sensitivity, introduced by asGCS gene transfection, was independent of P-glycoprotein and Bcl-2 expression. In summary, this work shows that transfection of GCS antisense tempers the expression of native GCS and restores cell sensitivity to adriamycin. Therefore, limiting the potential to glycosylate ceramide, which is an apoptotic signal in chemotherapy and radiotherapy, provides a promising approach to combat drug resistance.

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.

A role for ceramide in driving cancer cell resistance to doxorubicin

Advanced cancers acquire resistance to chemotherapy, and this results in treatment failure. The cellular mechanisms of chemotherapy resistance are not well understood. Here, for the first time, we show that ceramide contributes to cellular resistance to doxorubicin through up‐regulating the gene expression of glucosylceramide synthase (GCS). Ceramide, a cellular lipid messenger, modulates doxorubicin‐induced cell death. GCS catalyzes ceramide glycosylation, converting ceramide to glucosylceramide; this process hastens ceramide clearance and limits ceramide‐induced apoptosis. In the present study, we evaluated the role of the GCS gene in doxorubicin resistance using several paired wild‐type and drug‐resistant (doxorubicin‐selected) cancer cell lines, including breast, ovary, cervical, and colon. GCS was overexpressed in all drug‐resistant counterparts, and suppressing GCS overexpression using antisense oligonucleotide restored doxorubicin sensitivity. Characterizing the effect mechanism showed that doxorubicin exposure increased ceramide levels, enhanced GCS expression, and imparted cellular resistance. Exogenous C6‐ceramide and sphingomyelinase treatments mimicked the influence of doxorubicin on GCS, activating the GCS promoter and upregulating GCS gene expression. Fumonisin B1, an inhibitor of ceramide synthesis, significantly suppressed doxorubicin‐up‐regulated GCS expression. Promoter truncation, point mutation, gel‐shift, and protein‐DNA ELISA analysis showed that transcription factor Sp1 was essential for ceramide‐induced GCS up‐regulation. These data indicate that ceramide‐governed GCS gene expression drives cellular resistance to doxorubicin.—Liu, Y.‐Y., Yu, J. Y., Yin, D., Patwardhan, G. A., Gupta, V., Hirabayashi, Y., Holleran, W. M., Giuliano, A. E., Jazwinski, S. M., Gouaze‐Andersson, V., Consoli, D. P., Cabot, M. C. A role for ceramide in driving cancer cell resistance to doxorubicin. FASEB J. 22, 2541–2551 (2008)

N-(4-Hydroxyphenyl)retinamide increases dihydroceramide and synergizes with dimethylsphingosine to enhance cancer cell killing

Fenretinide [N-(4-hydroxyphenyl)retinamide (4-HPR)] is cytotoxic in many cancer cell types. Studies have shown that elevation of ceramide species plays a role in 4-HPR cytotoxicity. To determine 4-HPR activity in a multidrug-resistant cancer cell line as well as to study ceramide metabolism, MCF-7/AdrR cells (redesignated NCI/ADR-RES) were treated with 4-HPR and sphingolipids were analyzed. TLC analysis of cells radiolabeled with [3H]palmitic acid showed that 4-HPR elicited a dose-responsive increase in radioactivity migrating in the ceramide region of the chromatogram and a decrease in cell viability. Results from liquid chromatography/electrospray tandem mass spectrometry revealed large elevations in dihydroceramides (N-acylsphinganines), but not desaturated ceramides, and large increases in complex dihydrosphingolipids (dihydrosphingomyelins, monohexosyldihydroceramides), sphinganine, and sphinganine 1-phosphate. To test the hypothesis that elevation of sphinganine participates in the cytotoxicity of 4-HPR, cells were treated with the sphingosine kinase inhibitor d-erythro-N,N-dimethylsphingosine (DMS), with and without 4-HPR. After 24 h, the 4-HPR/DMS combination caused a 9-fold increase in sphinganine that was sustained through +48 hours, decreased sphinganine 1-phosphate, and increased cytotoxicity. Increased dihydrosphingolipids and sphinganine were also found in HL-60 leukemia cells and HT-29 colon cancer cells treated with 4-HPR. The 4-HPR/DMS combination elicited increased apoptosis in all three cell lines. We propose that a mechanism of 4-HPR–induced cytotoxicity involves increases in dihydrosphingolipids, and that the synergy between 4-HPR and DMS is associated with large increases in cellular sphinganine. These studies suggest that enhanced clinical efficacy of 4-HPR may be realized through regimens containing agents that modulate sphingoid base metabolism. [Mol Cancer Ther 2008;7(9):2967–76]

Sphingolipids and expression regulation of genes in cancer.

Sphingolipids including glycosphingolipids have myriad effects on cell functions and affect cancer in aspects of tumorigenesis, metastasis and tumor response to treatments. Bioactive ones like ceramide, sphingosine 1-phosphate and globotriaosylceramide initiate and process cellular signaling to alter cell behaviors immediately responding to oncogenic stress or treatment challenges. Recent studies pinpoint that sphingolipid-mediated gene expression has long and profound impacts on cancer cells, and these play crucial roles in tumor progression and in treatment outcome. More than 10 sphingolipids and glycosphingolipids selectively mediate expressions of approximately 50 genes including c-myc, p21, c-fos, telomerase reverse transcriptase, caspase-9, Bcl-x, cyclooxygenase-2, matrix metalloproteinases, integrins, Oct-4, glucosylceramide synthase and multidrug-resistant gene 1. By diverse functions of these genes, sphingolipids enduringly affect cellular processes of mitosis, apoptosis, migration, stemness of cancer stem cells and cellular resistance to therapies. Mechanistic studies indicate that sphingolipids regulate particular gene expression by modulating phosphorylation and acetylation of proteins that serve as transcription factors (β-catenin, Sp1), repressor of transcription (histone H3), and regulators (SRp30a) in RNA splicing. Disclosing molecular mechanisms by which sphingolipids selectively regulate particular gene expression, instead of other relevant ones, requires understanding of the exact roles of individual lipid instead of a group, the signaling pathways that are implicated in and interaction with proteins or other lipids in details. These studies not only expand our knowledge of sphingolipids, but can also suggest novel targets for cancer treatments.

Ceramide Glycosylation Catalyzed by Glucosylceramide Synthase and Cancer Drug Resistance

Glucosylceramide synthase (GCS), converting ceramide to glucosylceramide, catalyzes the first reaction of ceramide glycosylation in sphingolipid metabolism. This glycosylation by GCS is a critical step regulating the modulation of cellular activities by controlling ceramide and glycosphingolipids (GSLs). An increase of ceramide in response to stresses, such as chemotherapy, drives cells to proliferation arrest and apoptosis or autophagy; however, ceramide glycosylation promptly eliminates ceramide and consequently, these induced processes, thus protecting cancer cells. Further, persistently enhanced ceramide glycosylation can increase GSLs, participating in selecting cancer cells to drug resistance. GCS is overexpressed in diverse drug-resistant cancer cells and in tumors of breast, colon, and leukemia that display poor response to chemotherapy. As ceramide glycosylation by GCS is a rate-limiting step in GSL synthesis, inhibition of GCS sensitizes cancer cells to anticancer drugs and eradicates cancer stem cells. Mechanistic studies indicate that uncoupling ceramide glycosylation can modulate gene expression, decreasing MDR1 through the cSrc/β-catenin pathway and restoring p53 expression via RNA splicing. These studies not only expand our knowledge in understanding how ceramide glycosylation affects cancer cells but also provide novel therapeutic approaches for targeting refractory tumors.

Tamoxifen induces selective membrane association of protein kinase C epsilon in MCF-7 human breast cancer cells

Tamoxifen, a synthetic antiestrogen, is known for its antitumoral action in vivo; however, it is well accepted that many tamoxifen effects are elicited via estrogen receptor‐independent routes. Previously, we reported that tamoxifen induces PKC translocation in fibroblasts. In the present study, we investigated the influence of tamoxifen, and several triphenylethylene derivatives, on protein kinase C (PKC) in MCF‐7 human breast cancer cells. As measured by Western blot analysis, tamoxifen elicited isozyme‐specific membrane association of PKC‐ϵ, which was time‐dependent (as early as 5 min post‐treatment) and dose‐dependent (5.0–20 μM). Tamoxifen did not influence translocation of α, β, γ, δ or ζ PKC isoforms. Structure‐activity relationship studies demonstrated chemical requirements for PKC‐ϵ translocation, with tamoxifen, 3‐OH‐tamoxifen and clomiphene being active. Compounds without the basic amino side chain, such as triphenylethylene, or minus a phenyl group, such as N,N‐dimethyl‐2‐[(4‐phenylmethyl)phenoxy]ethanamine, were not active. In vitro cell growth assays showed a correlation between agent‐induced PKC‐ϵ translocation and inhibition of cell growth. Exposure of cells to clomiphene resulted in apoptosis. Since PKC‐ϵ has been associated with cell differentiation and cellular growth‐related processes, the antiproliferative influence of tamoxifen on MCF‐7 cells may be related to the interaction with PKC‐ϵ. Int. J. Cancer 77:928–932, 1998.© 1998 Wiley‐Liss, Inc.

Olive Phenolics as c-Met Inhibitors: (-)-Oleocanthal Attenuates Cell Proliferation, Invasiveness, and Tumor Growth in Breast Cancer Models

Dysregulation of the Hepatocyte growth factor (HGF)/c-Met signaling axis upregulates diverse tumor cell functions, including cell proliferation, survival, scattering and motility, epithelial-to-mesenchymal transition (EMT), angiogenesis, invasion, and metastasis. (-)-Oleocanthal is a naturally occurring secoiridoid from extra-virgin olive oil, which showed antiproliferative and antimigratory activity against different cancer cell lines. The aim of this study was to characterize the intracellular mechanisms involved in mediating the anticancer effects of (-)-oleocanthal treatment and the potential involvement of c-Met receptor signaling components in breast cancer. Results showed that (-)-oleocanthal inhibits the growth of human breast cancer cell lines MDA-MB-231, MCF-7 and BT-474 while similar treatment doses were found to have no effect on normal human MCF10A cell growth. In addition, (-)-oleocanthal treatment caused a dose-dependent inhibition of HGF-induced cell migration, invasion and G1/S cell cycle progression in breast cancer cell lines. Moreover, (-)-oleocanthal treatment effects were found to be mediated via inhibition of HGF-induced c-Met activation and its downstream mitogenic signaling pathways. This growth inhibitory effect is associated with blockade of EMT and reduction in cellular motility. Further results from in vivo studies showed that (-)-oleocanthal treatment suppressed tumor cell growth in an orthotopic model of breast cancer in athymic nude mice. Collectively, the findings of this study suggest that (-)-oleocanthal is a promising dietary supplement lead with potential for therapeutic use to control malignancies with aberrant c-Met activity.