Oligonucleotides blocking glucosylceramide synthase expression selectively reverse drug resistance in cancer cells

High glucosylceramide synthase (GCS) activity is one factor contributing to multidrug resistance (MDR) in breast cancer. Enforced GCS overexpression has been shown to disrupt ceramide-induced apoptosis and to confer resistance to doxorubicin. To examine whether GCS is a target for cancer therapy, we have designed and tested the effects of antisense oligodeoxyribonucleotides (ODNs) to GCS on gene expression and chemosensitivity in multidrug-resistant cancer cells. Here, we demonstrate that antisense GCS (asGCS) ODN-7 blocked cellular GCS expression and selectively increased the cytotoxicity of anticancer agents. Pretreatment with asGCS ODN-7 increased doxorubicin sensitivity by 17-fold in MCF-7-AdrR (doxorubicin-resistant) breast cancer cells and by 10-fold in A2780-AD (doxorubicin-resistant) ovarian cancer cells. In MCF-7 drug-sensitive breast cancer cells, asGCS ODN-7 only increased doxorubicin sensitivity by 3-fold, and it did not influence doxorubicin cytotoxicity in normal human mammary epithelial cells. asGCS ODN-7 was shown to be more efficient in reversing drug resistance than either the GCS chemical inhibitor d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol or the P-glycoprotein blocking agents verapamil and cyclosporin A. Experiments defining drug transport and lipid metabolism parameters showed that asGCS ODN-7 overcomes drug resistance mainly by enhancing drug uptake and ceramide-induced apoptosis. This study demonstrates that a 20-mer asGCS oligonucleotide effectively reverses MDR in human cancer cells.     

Glycosylation of ceramide potentiates cellular resistance to tumor necrosis factor-alpha-induced apoptosis.

Ceramide, as a second messenger, initiates one of the major signal transduction pathways in tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis. Glucosylceramide synthase (GCS) catalyzes glycosylation of ceramide and produces glucosylceramide. By introduction of the GCS gene, cytotoxic resistance to TNF-alpha has been conferred in human breast cancer cells. MCF-7/GCS-transfected cells expressed 4.1-fold higher levels of GCS activity and exhibited a 15-fold (P < 0.0005) greater EC(50) for TNF-alpha, compared with the parental MCF-7 cell line. DNA fragmentation and DNA synthesis studies showed that TNF-alpha had little influence on the induction of apoptosis or on growth arrest in MCF-7/GCS cells, compared to MCF-7 cells. These studies reveal that TNF-alpha resistance in MCF-7/GCS cells is closely related to ceramide hyperglycosylation, a hallmark of this transfected cell line, and resistance was not aligned with changes in TNF receptor 1 expression. This work demonstrates that GCS, which catalyzes ceramide glycosylation, potentiates cytotoxic resistance to TNF-alpha.     

Ascorbate promotes the cellular accumulation of doxorubicin and reverses the multidrug resistance in breast cancer cells by inducing ROS-dependent ATP depletion

Chemotherapy is the most effective strategy for the treatment of metastatic breast cancer. However, P-glycoprotein (P-gp)-mediated multidrug resistance severely limits the efficacy of chemotherapy and is a major cause of the failure during chemotherapeutic treatment. In this study, we investigated the anticancer effects of combining chemotherapeutic drugs with ascorbate (AA) in human breast cancer cells. We found that combined administration of AA can improve the sensitivity of both MCF-7 and doxorubicin (Dox)-resistant MCF-7/Adr cells to Dox in vitro and in vivo by a reactive oxygen species (ROS)-dependent mechanism. Further studies proved that AA can promote the accumulation of Dox in MCF-7/Adr cells when combined with doxorubicin. AA had no effect on the expression of P-gp at the mRNA and protein levels, but could decrease its activity as demonstrated by an obvious inhibition of efflux of P-gp substrate Rh 123. AA reduced ATP levels in both MCF-7 and MCF-7/Adr cells, and pretreating AA-stimulating cells with catalase completely rescued ATP levels. With ATP reduction, we observed an increased cellular calcium and the appearance of vacuoles and micropores on the cell surface, indicating the increased cell membrane permeability in AA-treated MCF-7/Adr cells. The above results suggest that AA could promote the cellular accumulation of doxorubicin by inducing ROS-dependent ATP depletion. Clinically, a combination of AA with doxorubicin would be a novel strategy for reversal of the multidrug resistance in human breast cancer cells during chemotherapy.     

Glycosylation of Ceramide Potentiates Cellular Resistance to Tumor Necrosis Factor-α-Induced Apoptosis

Ceramide, as a second messenger, initiates one of the major signal transduction pathways in tumor necrosis factor-α (TNF-α)-induced apoptosis. Glucosylceramide synthase (GCS) catalyzes glycosylation of ceramide and produces glucosylceramide. By introduction of the GCS gene, cytotoxic resistance to TNF-α has been conferred in human breast cancer cells. MCF-7/GCS-transfected cells expressed 4.1-fold higher levels of GCS activity and exhibited a 15-fold (P < 0.0005) greater EC₅₀ for TNF-α, compared with the parental MCF-7 cell line. DNA fragmentation and DNA synthesis studies showed that TNF-α had little influence on the induction of apoptosis or on growth arrest in MCF-7/GCS cells, compared to MCF-7 cells. These studies reveal that TNF-α resistance in MCF-7/GCS cells is closely related to ceramide hyperglycosylation, a hallmark of this transfected cell line, and resistance was not aligned with changes in TNF receptor 1 expression. This work demonstrates that GCS, which catalyzes ceramide glycosylation, potentiates cytotoxic resistance to TNF-α.     

Jab1/Cops5 contributes to chemoresistance in breast cancer by regulating Rad51

Jab1 overexpression correlates with poor prognosis in breast cancer patients, suggestting that targeting the aberrant Jab1 signaling in breast cancer could be a promising strategy. In the current study, we investigate the hypothesis that Jab1 positively regulates the DNA repair protein Rad51 and, in turn, the cellular response of breast cancer to chemotherapy with adriamycin and cisplatin. High-throughput mRNA sequencing (RNA-Seq) data from 113 normal and 1109 tumor tissues (obtained from TCGA) were integrated to our analysis to give further support to our findings. We found that Jab1 was overexpressed in adriamycin-resistant breast cancer cell MCF-7R compared with parental MCF-7 cells, and that knockdown of Jab1 expression conferred cellular sensitivity to adriamycin and cisplatin both in vivo and in vitro. By contrast, exogenous Jab1 expression enhanced the resistance of breast cancer cells to adriamycin and cisplatin. Moreover, we discovered that Jab1 positively regulated Rad51 in p53-dependent manner and that overexpression of Rad51 conferred cellular resistance to adriamycin and cisplatin in Jab1-deficient cells. Data from TCGA further validated an correlation between Jab1 and Rad51 in breast cancer, and elevated Jab1 and Rad51 associated with poor survival in breast cancer patients. Our findings indicate that Jab1 association with Rad51 plays an important role in cellular response to chemotherapy in breast cancer.     

The opposite effects of doxorubicin on bone marrow stem cells versus breast cancer stem cells depend on glucosylceramide synthase

Myelosuppression and drug resistance are common adverse effects in cancer patients with chemotherapy, and those severely limit the therapeutic efficacy and lead treatment failure. It is unclear by which cellular mechanism anticancer drugs suppress bone marrow, while drug-resistant tumors survive. We report that due to the difference of glucosylceramide synthase (GCS), catalyzing ceramide glycosylation, doxorubicin (Dox) eliminates bone marrow stem cells (BMSCs) and expands breast cancer stem cells (BCSCs). It was found that Dox decreased the numbers of BMSCs (ABCG2(+)) and the sphere formation in a dose-dependent fashion in isolated bone marrow cells. In tumor-bearing mice, Dox treatments (5mg/kg, 6 days) decreased the numbers of BMSCs and white blood cells; conversely, those treatments increased the numbers of BCSCs (CD24(-)/CD44(+)/ESA(+)) more than threefold in the same mice. Furthermore, therapeutic-dose of Dox (1mg/kg/week, 42 days) decreased the numbers of BMSCs while it increased BCSCs in vivo. Breast cancer cells, rather than bone marrow cells, highly expressed GCS, which was induced by Dox and correlated with BCSC pluripotency. These results indicate that Dox may have opposite effects, suppressing BMSCs versus expanding BCSCs, and GCS is one determinant of the differentiated responsiveness of bone marrow and cancer cells.     

Doxorubicin Influences the Expression of Glucosylceramide Synthase in Invasive Ductal Breast Cancer

Introduction

Glucosylceramide synthase (GCS) is one enzyme that provides a major route for ceramide clearance. Recent evidence has indicated an important role for GCS in multidrug resistance (MDR) tumors. Doxorubicin (DOX)can modulate the expression of GCS in leukemia and ovary cell lines. However, few studies have investigated their relationship in breast cancer;

Methods

We collected 84 excision biopsies from patients with invasive ductal breast cancer of whom 33 patients had undergone preoperative chemotherapy. Immunohistochemistry was used to analyze the expression of GCS protein and significantly showed that the expression of GCS was higher in the samples from patients treated with preoperative chemotherapy(p = 0.018). In order to investigate the underlying mechanism, breast cancer cell lines were cultured with different concentrations of DOX, and mRNA and protein levels of GCS were then detected;

Results

DOX significantly upregulated the expression of GCS at both the mRNA and protein level in ERα-positive MCF-7 cells.We then block down the Sp1 site of GCS promoter, which inhibited the DOX-mediated increase in GCS expression; and after Erα was inhibited in MCF-7 cells, the up-regulation of GCS by DOX also been inhibited.

Conclusions

In conclusion, our data demonstrated the novel finding that DOX could modulate the expression of GCS through the Sp1 site of GCS promoter in ERα-positive breast cancer cells.     

The inhibitory effect of kokusaginine on the growth of human breast cancer cells and MDR-resistant cells is mediated by the inhibition of tubulin assembly

The emergence of multidrug resistance (MDR) is a significant challenge in breast carcinoma chemotherapy. Kokusaginine isolated from Dictamnus dasycarpus Turcz. has been reported to show cytotoxicity in several human cancer cell lines including breast cancer cells MCF-7. In this study, kokusaginine showed the potent inhibitory effect on MCF-7 multidrug resistant subline MCF-7/ADR and MDA-MB-231 multidrug resistant subline MDA-MB-231/ADR. Kokusaginine markedly induced apoptosis in a concentration-dependent manner in MCF-7/ADR cells. Furthermore, kokusaginine reduced P-gp mRNA and protein levels, and suppressed P-gp function especially in MCF-7/ADR cells. In addition, kokusaginine showed to inhibit tubulin assembly and the binding of colchicine to tubulin by binding directly to tubulin and affects tubulin formation in vitro. Taken together, these results support the potential therapeutic value of kokusaginine as an anti-MDR agent in chemotherapy for breast carcinoma.     

Metformin reverses multidrug resistance and epithelial–mesenchymal transition (EMT) via activating AMP-activated protein kinase (AMPK) in human breast cancer cells

Breast cancer is the most frequently diagnosed tumor type and the primary leading cause of cancer deaths in women worldwide and multidrug resistance is the major obstacle for breast cancer treatment improvement. Emerging evidence suggests that metformin, the most widely used antidiabetic drug, resensitizes and cooperates with some anticancer drugs to exert anticancer effect. However, there are no data regarding the reversal effect of metformin on chemoresistance in breast cancer. In the present study, we investigated the resistance reversal effect of metformin on acquired multidrug-resistant breast cancer cells MCF-7/5-Fu derived from MCF-7 breast cancer cells and innate multidrug-resistant MDA-MB-231 breast cancer cells, and we found that metformin resensitized MCF7/5-FU and MDA-MB-231 to 5-fluorouracil (5-FU), adriamycin, and paclitaxel. We also observed that metformin reversed epithelial–mesenchymal transition (EMT) phenotype and decreased the invasive capacity of MCF7/5-FU and MDA-MB-231 cells. However, there were no significant changes upon metformin-treated MCF7 cells. Moreover, we found metformin treatment activated AMPK signal pathway in MCF7/5-FU and MDA-MB-231 cells and compound C, the AMPK inhibitor, could partly abolish the resensitization and EMT reversal effect of metformin. To the best of our knowledge, we are the first to report that metformin can resensitize multidrug-resistant breast cancer cells due to activating AMPK signal pathway. Our study will help elucidate the mechanism of chemoresistance and establish new strategies of chemotherapy for human breast cancer.     

Knockdown of dual specificity phosphatase 4 enhances the chemosensitivity of MCF-7 and MCF-7/ADR breast cancer cells to doxorubicin

BackgroundBreast cancer is the major cause of cancer-related deaths in females world-wide. Doxorubicin-based therapy has limited efficacy in breast cancer due to drug resistance, which has been shown to be associated with the epithelial-to-mesenchymal transition (EMT). However, the molecular mechanisms linking the EMT and drug resistance in breast cancer cells remain unclear. Dual specificity phosphatase 4 (DUSP4), a member of the dual specificity phosphatase family, is associated with cellular proliferation and differentiation; however, its role in breast cancer progression is controversial.MethodsWe used cell viability assays, Western blotting and immunofluorescent staining, combined with siRNA interference, to evaluate chemoresistance and the EMT in MCF-7 and adriamycin-resistant MCF-7/ADR breast cancer cells, and investigate the underlying mechanisms.ResultsKnockdown of DUSP4 significantly increased the chemosensitivity of MCF-7 and MCF-7/ADR breast cancer cells to doxorubicin, and MCF-7/ADR cells which expressed high levels of DUSP4 had a mesenchymal phenotype. Furthermore, knockdown of DUSP4 reversed the EMT in MCF-7/ADR cells, as demonstrated by upregulation of epithelial biomarkers and downregulation of mesenchymal biomarkers, and also increased the chemosensitivity of MCF-7/ADR cells to doxorubicin.ConclusionsDUSP4 might represent a potential drug target for inhibiting drug resistance and regulating the process of the EMT during the treatment of breast cancer.     

Paeonol reverses paclitaxel resistance in human breast cancer cells by regulating the expression of transgelin 2

Paclitaxel (PTX) is a first-line antineoplastic drug that is commonly used in clinical chemotherapy for breast cancer treatment. However, the occurrence of drug resistance in chemotherapeutic treatment has greatly restricted its use. There is thus an urgent need to find ways of reversing paclitaxel chemotherapy resistance in breast cancer. Plant-derived agents have great potential in preventing the onset of the carcinogenic process and enhancing the efficacy of mainstream antitumor drugs. Paeonol, a main compound derived from the root bark of Paeonia suffruticosa, has various biological activities, and is reported to have reversal drug resistance effects. This study established a paclitaxel-resistant human breast cancer cell line (MCF-7/PTX) and applied the dual-luciferase reporter gene assay, MTT assay, flow cytometry, transfection assay, Western blotting and the quantitative real-time polymerase chain reaction (qRT-PCR) to investigate the reversing effects of paeonol and its underlying mechanisms. It was found that transgelin 2 may mediate the resistance of MCF-7/PTX cells to paclitaxel by up-regulating the expressions of the adenosine-triphosphate binding cassette transporter proteins, including P-glycoprotein (P-gp), multidrug resistance associated protein 1 (MRP1), and breast cancer resistance protein (BCRP). Furthermore, the ability of paeonol to reverse paclitaxel resistance in breast cancer was confirmed, with a superior 8.2-fold reversal index. In addition, this study found that paeonol down-regulated the transgelin 2-mediated paclitaxel resistance by reducing the expressions of P-gp, MRP1, and BCRP in MCF-7/PTX cells. These results not only provide insight into the potential application of paeonol to the reversal of paclitaxel resistance, thus facilitating the sensitivity of breast cancer chemotherapy, but also highlight a potential role of transgelin 2 in the development of paclitaxel resistance in breast cancer.     

Small-molecule synthetic compound norcantharidin reverses multi-drug resistance by regulating Sonic hedgehog signaling in human breast cancer cells

Multi-drug resistance (MDR), an unfavorable factor compromising treatment efficacy of anticancer drugs, involves upregulated ATP binding cassette (ABC) transporters and activated Sonic hedgehog (Shh) signaling. By preparing human breast cancer MCF-7 cells resistant to doxorubicin (DOX), we examined the effect and mechanism of norcantharidin (NCTD), a small-molecule synthetic compound, on reversing multidrug resistance. The DOX-prepared MCF-7R cells also possessed resistance to vinorelbine, characteristic of MDR. At suboptimal concentration, NCTD significantly inhibited the viability of DOX-sensitive (MCF-7S) and DOX-resistant (MCF-7R) cells and reversed the resistance to DOX and vinorelbine. NCTD increased the intracellular accumulation of DOX in MCF-7R cells and suppressed the upregulated the mdr-1 mRNA, P-gp and BCRP protein expression, but not the MRP-1. The role of P-gp was strengthened by partial reversal of the DOX and vinorelbine resistance by cyclosporine A. NCTD treatment suppressed the upregulation of Shh expression and nuclear translocation of Gli-1, a hallmark of Shh signaling activation in the resistant clone. Furthermore, the Shh ligand upregulated the expression of P-gp and attenuated the growth inhibitory effect of NCTD. The knockdown of mdr-1 mRNA had not altered the expression of Shh and Smoothened in both MCF-7S and MCF-7R cells. This indicates that the role of Shh signaling in MDR might be upstream to mdr-1/P-gp, and similar effect was shown in breast cancer MDA-MB-231 and BT-474 cells. This study demonstrated that NCTD may overcome multidrug resistance through inhibiting Shh signaling and expression of its downstream mdr-1/P-gp expression in human breast cancer cells.     

BCL-2 antisense and cisplatin combination treatment of MCF-7 breast cancer cells with or without functional p53

Summary Chemotherapy has been used for treatment of breast cancer but with limited success. We characterized the effects of bcl-2 antisense and cisplatin combination therapy in two human isogenic breast carcinoma cells p53(+)MCF-7 and p53(−)MCF-7/E6. The transferrin-facilitated lipofection strategy we have developed yielded same transfection efficiency in both cells. Bcl-2 antisense delivered with this strategy significantly induced more cell death, apoptosis, and cytochrome c release in MCF-7/E6 than in MCF-7, but did not affect Fas level in both cells and activated caspase-8 equally. Cisplatin exerted same effects on cell viability and apoptosis in both cells, but released smaller amounts of cytochrome c while activated more caspase-8 in MCF-7/E6. The combination treatment yielded greater effects on cell viability, apoptosis, cytochrome c release, and caspase-8 activation than individual treatments in both cells although p53(−) cells were more sensitive. The potentiated activation of caspase-8 in the combination treatment suggested that caspase-8-mediated (but cytochrome c-independent) apoptotic pathway is the major contributor of the enhanced cell killing. Thus, bcl-2 antisense delivered with transferrin-facilitated lipofection can achieve the efficacy of killing breast cancer cells and sensitizing them to chemotherapy. Bcl-2 antisense and cisplatin combination treatment is a potentially useful therapeutic strategy for breast cancer irrespective of p53 status. Hesham Basma and Hesham El-Refaey contributed equally     

Synthesis and biological evaluation of a cyclic ether fluorinated noscapine analog

We present here a novel semi-synthetic cyclic ether fluorinated noscapine analog (CEFNA) that shows potent antiproliferative and anticancer activity in both hormone-responsive (MCF-7) and hormone non-responsive (MDA-MB-231) breast cancer cells. Interestingly, it is also effective against MCF-7/Adr, an adriamycin-resistant variant of MCF-7 cells. Immunofluorescence experiments showed numerous micronuclei, indicative of apoptotic cell death triggered by this novel analog. Mechanistically, CEFNA exerts a strong antimitotic effect as revealed by cell-cycle studies that show a dose-dependent increase in G2/M population preceding a rising sub-G1 population, suggesting apoptosis.     

上皮细胞黏附分子增强细胞间紧密连接促进乳腺癌细胞耐药

乳腺癌是致死率很高的恶性肿瘤,由ABCG2 (ATP-binding cassette G2)介导的多药耐药(multidrug resistance,MDR)是导致其化疗失败的重要原因,探讨ABCG2介导的耐药机制并探寻其关键分子是当前亟待解决的难题。上皮细胞黏附分子(epithelial cell adhesion molecule,EpCAM)参与多种肿瘤耐药,且与乳腺癌MDR密切相关,但它在ABCG2介导的乳腺癌耐药中的作用尚未阐明。本研究目的在于探究EpCAM对于ABCG2介导的乳腺癌细胞的多药耐药的调节作用及其机制。CCK8细胞毒性结果证实,相对于人乳腺癌药物敏感株MCF-7,耐药株MCF-7/MX对米托蒽醌(mitoxantrone,MX)的耐药性显著增强;Western 印迹结果显示,与MCF-7相比,MCF-7/MX细胞中ABCG2高表达,EpCAM表达上调。siRNA法敲低MCF-7/MX细胞中EpCAM可下调其ABCG2表达,并恢复对MX的敏感性。倒置显微镜观察细胞形态,发现敲低EpCAM可减少MCF-7/MX细胞间连接。免疫荧光双染法观察到EpCAM与密封蛋白1(claudin 1)在MCF-7/MX细胞共定位;进一步Western 印迹结果表明,敲低EpCAM减少MCF-7/MX细胞中密封蛋白1表达。综上所述,EpCAM可能通过与密封蛋白1相互作用,增强细胞间紧密连接,促进ABCG2介导的乳腺癌多药耐药。     

Development of stealth liposome coencapsulating doxorubicin and fluoxetine

Stealth liposomes form an important subset of liposomes, demonstrating prolonged circulation half-life and improved safety in vivo. Caelyx? (liposomal doxorubicin; Merck & Co., Whitehouse Station, New Jersey, USA) is a successful example of the application of stealth liposomes in anticancer treatment. However, multidrug resistance (MDR) to chemotherapy still remains a critical problem, accounting for more than 90% of treatment failure in patients with advanced cancer. To circumvent MDR, fluoxetine and doxorubicin were tested in combination for synergistic activity in MCF-7 (human breast carcinoma) and MCF-7/adr (doxorubicin-resistant human breast carcinoma) cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell-viability assay. Coencapsulation of doxorubicin and fluoxetine, using an ammonium sulphate gradient, was investigated, and a factorial experiment was designed to determine the optimal drug-to-lipid (D/L) ratio for coencapsulation. Drug release from Dox-Flu-SL (stealth liposome coencapsulating doxorubicin and fluoxetine) under both in vitro and in vivo conditions was determined. In MCF-7 cells, synergism was demonstrated at specific doxorubicin:fluoxetine ratios of between 0.09 and 0.5 (molar ratio), while MCF/7/adr cells demonstrated synergism across all drug ratios. Coencapsulation of doxorubicin and fluoxetine (Dox-Flu-SL) was successfully achieved (optimal doxorubicin:fluoxetine:lipid molar ratio of 0.02:0.05:1), obtaining a mean concentration of 257 ± 12.1 and 513 ± 29.3 μM for doxorubicin and fluoxetine, respectively. Most important, Dox-Flu-SL demonstrated drug release in synergistic ratios in cell-culture media, accounting for the improved cytotoxicity of Dox-Flu-SL over liposomal doxorubicin (LD) in both MCF-7 and MCF-7/adr cells. Pharmacokinetic studies also revealed that Dox-Flu-SL effectively prolonged drug-circulation time and reduced tissue biodistribution. Dox-Flu-SL presents a promising anticancer formulation, capable of effective reversal of drug resistance, and may constitute a novel approach for cancer therapy.     

Ceramide and glucosylceramide upregulate expression of the multidrug resistance gene MDR1 in cancer cells

In the present study we used human breast cancer cell lines to assess the influence of ceramide and glucosylceramide (GC) on expression of MDR1, the multidrug resistance gene that codes for P-glycoprotein (P-gp), because GC has been shown to be a substrate for P-gp. Acute exposure (72 h) to C8-ceramide (5 microg/ml culture medium), a cell-permeable ceramide, increased MDR1 mRNA levels by 3- and 5-fold in T47D and in MDA-MB-435 cells, respectively. Acute exposure of MCF-7 and MDA-MB-231 cells to C8-GC (10 microg/ml culture medium), a cell-permeable analog of GC, increased MDR1 expression by 2- and 4- fold, respectively. Chronic exposure of MDA-MB-231 cells to C8-ceramide for extended periods enhanced MDR1 mRNA levels 45- and 390-fold at passages 12 and 22, respectively, and also elicited expression of P-gp. High-passage C8-ceramide-grown MDA-MB-231 (MDA-MB-231/C8cer) cells were more resistant to doxorubicin and paclitaxel. Incubation with [1-(14)C]C6-ceramide showed that cells converted short-chain ceramide into GC, lactosylceramide, and sphingomyelin. When challenged with 5 mug/ml [1-(14)C]C6-ceramide, MDA-MB-231, MDA-MB-435, MCF-7, and T47D cells took up 31, 17, 21, and 13%, respectively, and converted 82, 58, 62, and 58% of that to short-chain GC. Exposing cells to the GCS inhibitor, ethylenedioxy-P4, a substituted analog of 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol, prevented ceramide's enhancement of MDR1 expression. These experiments show that high levels of ceramide and GC enhance expression of the multidrug resistance phenotype in cancer cells. Therefore, ceramide's role as a messenger of cytotoxic response might be linked to the multidrug resistance pathway.     

3-Ketone-4,6-diene ceramide analogs exclusively induce apoptosis in chemo-resistant cancer cells

Multidrug-resistance is a major cause of cancer chemotherapy failure in clinical treatment. Evidence shows that multidrug-resistant cancer cells are as sensitive as corresponding regular cancer cells under the exposure to anticancer ceramide analogs. In this work we designed five new ceramide analogs with different backbones, in order to test the hypothesis that extending the conjugated system in ceramide analogs would lead to an increase of their anticancer activity and selectivity towards resistant cancer cells. The analogs with the 3-ketone-4,6-diene backbone show the highest apoptosis-inducing efficacy. The most potent compound, analog 406, possesses higher pro-apoptotic activity in chemo-resistant cell lines MCF-7TN-R and NCI/ADR-RES than the corresponding chemo-sensitive cell lines MCF-7 and OVCAR-8, respectively. However, this compound shows the same potency in inhibiting the growth of another pair of chemo-sensitive and chemo-resistant cancer cells, MCF-7 and MCF-7/Dox. Mechanism investigations indicate that analog 406 can induce apoptosis in chemo-resistant cancer cells through the mitochondrial pathway. Cellular glucosylceramide synthase assay shows that analog 406 does not interrupt glucosylceramide synthase in chemo-resistant cancer cell NCI/ADR-RES. These findings suggest that due to certain intrinsic properties, ceramide analogs’ pro-apoptotic activity is not disrupted by the normal drug-resistance mechanisms, leading to their potential use for overcoming cancer multidrug-resistance.     

A New Mixed-Backbone Oligonucleotide against Glucosylceramide Synthase Sensitizes Multidrug-Resistant Tumors to Apoptosis

Enhanced ceramide glycosylation catalyzed by glucosylceramide synthase (GCS) limits therapeutic efficiencies of antineoplastic agents including doxorubicin in drug-resistant cancer cells. Aimed to determine the role of GCS in tumor response to chemotherapy, a new mixed-backbone oligonucleotide (MBO-asGCS) with higher stability and efficiency has been generated to silence human GCS gene. MBO-asGCS was taken up efficiently in both drug-sensitive and drug-resistant cells, but it selectively suppressed GCS overexpression, and sensitized drug-resistant cells. MBO-asGCS increased doxorubicin sensitivity by 83-fold in human NCI/ADR-RES, and 43-fold in murine EMT6/AR1 breast cancer cells, respectively. In tumor-bearing mice, MBO-asGCS treatment dramatically inhibited the growth of multidrug-resistant NCI/ADR-RE tumors, decreasing tumor volume to 37%, as compared with scrambled control. Furthermore, MBO-asGCS sensitized multidrug-resistant tumors to chemotherapy, increasing doxorubicin efficiency greater than 2-fold. The sensitization effects of MBO-asGCS relied on the decreases of gene expression and enzyme activity of GCS, and on the increases of C₁₈-ceramide and of caspase-executed apoptosis. MBO-asGCS was accumulation in tumor xenografts was greater in other tissues, excepting liver and kidneys; but MBO-asGCS did not exert significant toxic effects on liver and kidneys. This study, for the first time in vivo, has demonstrated that GCS is a promising therapeutic target for cancer drug resistance, and MBO-asGCS has the potential to be developed as an antineoplastic agent.