n-caproylsphingosine and Breast-Neoplasms

Here we reported that co-administration of docetaxel and a cell-permeable short-chain ceramide (C6) resulted in a striking increase in growth inhibition and apoptosis in primary and transformed breast cells (MCF-7 and MDA-231), which were associated with mitochondrial permeability transition pore (mPTP) opening, a significant reactive oxygen species (ROS) production and the pro-apoptotic AMP-Protein Kinase (AMPK) as well as c-Jun N-terminal kinases (JNK) activations. Contrarily, the mPTP blocker sanglifehrin A (SfA) or the ROS scavenger N-acetyl-l-cysteine (NAC) largely inhibited co-administration-induced cytotoxicity. Further, cyclosporin A (CsA), the inhibitor of cyclophilin-D (Cyp-D, the key mPTP component), as well as Cyp-D RNA silencing also suppressed breast cancer cell death by the co-treatment, while cells overexpressing Cyp-D showed hypersensitivity to docetaxel. Meanwhile, JNK and AMPK inhibition alleviated cell death induced by the co-administration in cultured breast cancer cells. Significantly, C6 ceramide plus docetaxel caused dramatic human epidermal growth factor receptor (HER)-1/-2 degradation and downstream Akt/Erk inhibition in HER-2 expressing MDA-231 cells. These in vitro findings provide confidence in support of further development of C6 ceramide as an adjunct of docetaxel for the treatment of the metastatic breast cancer.

Topics: Adenylate Kinase; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Survival; Ceramides; Cisplatin; Docetaxel; Drug Synergism; ErbB Receptors; Female; HEK293 Cells; Humans; Hydroxamic Acids; MAP Kinase Kinase 4; MCF-7 Cells; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Paclitaxel; Proteolysis; Taxoids; TOR Serine-Threonine Kinases; Vorinostat

Breast cancer stem cells (CSC) are thought responsible for tumor growth and relapse, metastization and active evasion to standard chemotherapy. The recognition that CSC may originate from non-stem cancer cells (non-SCC) through plastic epithelial-to-mesenchymal transition turned these into relevant cell targets. Of crucial importance for successful therapeutic intervention is the identification of surface receptors overexpressed in both CSC and non-SCC. Cell surface nucleolin has been described as overexpressed in cancer cells as well as a tumor angiogenic marker. Herein we have addressed the questions on whether nucleolin was a common receptor among breast CSC and non-SCC and whether it could be exploited for targeting purposes. Liposomes functionalized with the nucleolin-binding F3 peptide, targeted simultaneously, nucleolin-overexpressing putative breast CSC and non-SCC, which was paralleled by OCT4 and NANOG mRNA levels in cells from triple negative breast cancer (TNBC) origin. In murine embryonic stem cells, both nucleolin mRNA levels and F3 peptide-targeted liposomes cellular association were dependent on the stemness status. An in vivo tumorigenic assay suggested that surface nucleolin overexpression per se, could be associated with the identification of highly tumorigenic TNBC cells. This proposed link between nucleolin expression and the stem-like phenotype in TNBC, enabled 100% cell death mediated by F3 peptide-targeted synergistic drug combination, suggesting the potential to abrogate the plasticity and adaptability associated with CSC and non-SCC. Ultimately, nucleolin-specific therapeutic tools capable of simultaneous debulk multiple cellular compartments of the tumor microenvironment may pave the way towards a specific treatment for TNBC patient care.

Topics: Amino Acid Sequence; Animals; Antibiotics, Antineoplastic; Breast; Breast Neoplasms; Cell Line, Tumor; Ceramides; Doxorubicin; Drug Delivery Systems; Drug Synergism; Female; Humans; Liposomes; Mice; Molecular Sequence Data; Neoplastic Stem Cells; Nucleolin; Peptides; Phosphoproteins; RNA-Binding Proteins; Triple Negative Breast Neoplasms; Up-Regulation

The sphingolipid ceramide is known to play a central role in chemo- and radiation-induced cell death. Acid ceramidase (AC) hydrolyzes ceramide, and thus reduces intracellular levels of this proapoptotic lipid. The role of AC as a putative anticancer target is supported by reports of upregulation in prostate cancer and in some breast tumors. In this study, we determined whether the introduction of an AC inhibitor would enhance the apoptosis-inducing effects of C6-ceramide (C6-cer) in breast cancer cells. Cultured breast cancer cells were treated with DM102 [(2R,3Z)-N-(1-hydroxyoctadec-3-en-2-yl)pivalamide, C6-cer, or the combination. Cell viability and cytotoxic synergy were assessed. Activation of apoptotic pathways, generation of reactive oxygen species, and mitochondrial transmembrane potential were determined. DM102 was a more effective AC inhibitor than N-oleoylethanolamine (NOE) and (1R,2R)-2-N-(tetradecanoylamino)-1-(4'-nitrophenyl)-1,3-propandiol (B-13) in MDA-MB-231, MCF-7, and BT-474 cells. As single agents, C6-cer (IC(50) 5-10 μM) and DM102 (IC(50) 20 μM) were only moderately cytotoxic in MDA-MB-231, MCF-7, and SK-BR-3 cells. Co-administration, however, produced synergistic decreases in viability (combination index <0.5) in all cell lines. Apoptosis was confirmed in MDA-MB-231 cells by detection of caspase 3 cleavage and a >3-fold increase in caspase 3/7 activation, PARP cleavage, and a >70% increase in Annexin-V positive cells. C6-cer/DM102 increased ROS levels 4-fold in MDA-MB-231 cells, shifted the ratio of Bax:Bcl-2 to >9-fold that of control cells, and resulted in mitochondrial membrane depolarization. DM102 also increased the synthesis of (3)H-palmitate-labeled long-chain ceramides by 2-fold when C6-cer was present. These data support the effectiveness of targeting AC in combination with exogenous short-chain ceramide as an anticancer strategy, and warrant continued investigation into the utility of the C6-cer/DM102 drug duo in human breast cancer.

Topics: Acid Ceramidase; Amides; Apoptosis; Breast Neoplasms; Cell Proliferation; Cell Survival; Ceramides; Drug Synergism; Enzyme Inhibitors; Fatty Acids, Unsaturated; Female; Humans; Membrane Potential, Mitochondrial; Reactive Oxygen Species

The synthesis of a small number of ceramide analogues containing a combination of linear and highly branched alkyl chains on either the d-sphingosine or the N-acyl core of the molecule is reported. Regardless of location, the presence of the branched chain improves potency relative to the positive control, C2 ceramide; however, the most potent compound (4) has the branched side chain as part of the d-sphingosine core. The induction of apoptosis by 4 in terms of Annexin V binding and DiOC(6) labeling was superior to that achieved with C2 ceramide.

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Ceramides; Humans; Molecular Structure; Signal Transduction; Sphingolipids; Structure-Activity Relationship; T-Lymphocytes

It is therapeutically desirable to effectively deliver ceramide, an antimitogenic and proapoptotic lipid second messenger, to transformed cell types. However, the targeted delivery of cell-permeable ceramide analogs, including C6-ceramide, to cells may be impeded by the hydrophobicity of these bioactive lipids, resulting in reduced efficacy. The objective of this study is to develop and optimize liposomal vehicles to augment ceramide delivery to a breast adenocarcinoma cell line. We designed conventional, cationic, and pegylated drug release vesicles to efficaciously deliver ceramide to MDA-MB-231 breast adenocarcinoma cells. In vitro pharmacokinetic analysis demonstrated that liposomal ceramide delivery resulted in significantly greater accumulation of ceramide in MDA-MB-231 cells. Ceramide-formulated liposomes significantly inhibited MDA-MB-231 cell proliferation as compared with nonliposomal administration of ceramide. Ceramide-induced apoptosis correlated with the pharmacokinetic profile and the diminished proliferation in this highly aggressive, metastatic cell line. Liposomal ceramide formulations inhibited phosphorylated Akt levels and stimulated caspase-3/7 activity more effectively than nonliposomal ceramide, events consistent with apoptosis. Together, these results indicate that bioactive ceramide analogs can be incorporated into conventional, cationic, or pegylated liposomal vehicles for improved drug delivery and release.

Topics: Apoptosis; Breast Neoplasms; Cell Division; Ceramides; Chemistry, Pharmaceutical; Drug Carriers; Drug Delivery Systems; Female; Humans; Liposomes; Signal Transduction; Tumor Cells, Cultured