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

Methylation of WTH3, a possible drug resistant gene, inhibits p53 regulated expression

<p>Abstract</p> <p>Background</p> <p>Previous results showed that over-expression of the <it>WTH3 </it>gene in MDR cells reduced <it>MDR1 </it>gene expression and converted their resistance to sensitivity to various anticancer drugs. In addition, the <it>WTH3 </it>gene promoter was hypermethylated in the MCF7/AdrR cell line and primary drug resistant breast cancer epithelial cells. <it>WTH3 </it>was also found to be directly targeted and up regulated by the <it>p53 </it>gene. Furthermore, over expression of the <it>WTH3 </it>gene promoted the apoptotic phenotype in various host cells.</p> <p>Methods</p> <p>To further confirm <it>WTH3</it>'s drug resistant related characteristics, we recently employed the small hairpin RNA (shRNA) strategy to knockdown its expression in HEK293 cells. In addition, since the <it>WTH3 </it>promoter's p53-binding site was located in a CpG island that was targeted by methylation, we were interested in testing the possible effect this epigenetic modification had on the <it>p53 </it>transcription factor relative to <it>WTH3 </it>expression. To do so, the <it>in vitro </it>methylation method was utilized to examine the <it>p53 </it>transgene's influence on either the methylated or non-methylated <it>WTH3 </it>promoter.</p> <p>Results</p> <p>The results generated from the gene knockdown strategy showed that reduction of <it>WTH3 </it>expression increased <it>MDR1 </it>expression and elevated resistance to Doxorubicin as compared to the original control cells. Data produced from the methylation studies demonstrated that DNA methylation adversely affected the positive impact of <it>p53 </it>on <it>WTH3 </it>promoter activity.</p> <p>Conclusion</p> <p>Taken together, our studies provided further evidence that <it>WTH3 </it>played an important role in MDR development and revealed one of its transcription regulatory mechanisms, DNA methylation, which antagonized <it>p53</it>'s positive impact on <it>WTH3 </it>expression.</p

C16 ceramide is crucial for triacylglycerol-induced apoptosis in macrophages

Triacylglycerol (TG) accumulation caused by adipose triglyceride lipase (ATGL) deficiency or very low-density lipoprotein (VLDL) loading of wild-type (Wt) macrophages results in mitochondrial-mediated apoptosis. This phenotype is correlated to depletion of Ca2+ from the endoplasmic reticulum (ER), an event known to induce the unfolded protein response (UPR). Here, we show that ER stress in TG-rich macrophages activates the UPR, resulting in increased abundance of the chaperone GRP78/BiP, the induction of pancreatic ER kinase-like ER kinase, phosphorylation and activation of eukaryotic translation initiation factor 2A, the translocation of activating transcription factor (ATF)4 and ATF6 to the nucleus and the induction of the cell death executor CCAAT/enhancer-binding protein homologous protein. C16:0 ceramide concentrations were increased in Atgl–/– and VLDL-loaded Wt macrophages. Overexpression of ceramide synthases was sufficient to induce mitochondrial apoptosis in Wt macrophages. In accordance, inhibition of ceramide synthases in Atgl–/– macrophages by fumonisin B1 (FB1) resulted in specific inhibition of C16:0 ceramide, whereas intracellular TG concentrations remained high. Although the UPR was still activated in Atgl–/– macrophages, FB1 treatment rescued Atgl–/– macrophages from mitochondrial dysfunction and programmed cell death. We conclude that C16:0 ceramide elicits apoptosis in Atgl–/– macrophages by activation of the mitochondrial apoptosis pathway

C6-ceramide synergistically potentiates the anti-tumor effects of histone deacetylase inhibitors via AKT dephosphorylation and α-tubulin hyperacetylation both in vitro and in vivo

Histone deacetylase inhibitors (HDACIs) have shown promising anti-tumor effects for a variety of malignancies, however, many tumors are reportedly resistant to them. In this study, we made a novel discovery that co-administration of HDACIs (Trichostatin A (TSA) and others) and exogenous cell-permeable short-chain ceramide (C6) results in striking increase in cancer cell death and apoptosis in multiple cancer cells. These events are associated with perturbations in diverse cell signaling pathways, including inactivation of Akt/mTOR and increase in α-tubulin acetylation (both in vivo and in vitro). TSA interacts in a highly synergistic manner with C6-ceramide to disrupt HDAC6/protein phosphatase 1 (PP1)/tubulin complex, to induce α-tubulin hyperacetylation, and to release and activate PP1, which then leads to AKT dephosphorylation and eventually causes cancer cell death. Interestingly, TSA itself results in short-term ceramide accumulation, which as a result of metabolic (glycosylation) removal, does not result in evident increase of cancer cell death. However, adding C6-ceramide led to a very pronounced increase in ceramide level and marked increase in cell death. Importantly, the effective synergistic anti-tumor activity of TSA plus C6-ceramide is also seen in in vivo mice xenograft pancreatic and ovarian cancer models, indicating that this regimen (HDACI plus C6-ceramide) may represent a more effective form of therapy against pancreatic and ovarian carcinoma

Increased tumour dihydroceramide production after Photofrin-PDT alone and improved tumour response after the combination with the ceramide analogue LCL29. Evidence from mouse squamous cell carcinomas

Photodynamic therapy (PDT) has been proven effective for treatment of several types of cancer. Photodynamic therapy alone, however, attains limited cures with some tumours and there is need for its improved efficacy in such cases. Sphingolipid (SL) analogues can promote tumour response in combination with anticancer drugs. In this study, we used mouse SCCVII squamous cell carcinoma tumours to determine the impact of Photofrin-PDT on the in vivo SL profile and the effect of LCL29, a C6-pyridinium ceramide, on PDT tumour response. Following PDT, the levels of dihydroceramides (DHceramides), in particular C20-DHceramide, were elevated in tumours. Similarly, increases in DHceramides, in addition to C20:1-ceramide, were found in PDT-treated SCCVII cells. These findings indicate the importance of the de novo ceramide pathway in Photofrin-PDT response not only in cells but also in vivo. Notably, co-exposure of SCCVII tumours to Photofrin-PDT and LCL29 led to enhanced tumour response compared with PDT alone. Thus, we show for the first time that Photofrin-PDT has a distinct signature effect on the SL profile in vitro and in vivo, and that the combined treatment advances PDT therapeutic gain, implying translational significance of the combination

WTH3 is a direct target of the p53 protein

Previous results showed that overexpression of the WTH3 gene in multidrug resistance (MDR) cells reduced MDR1 gene expression and converted their resistance to sensitivity to various anticancer drugs. The WTH3 gene promoter was found to be differentially regulated in paired MDR vs non-MDR MCF7 cells owing to epigenetic modifications and transcription factor modulations. To understand further the mechanisms that govern WTH3's differential expression, we uncovered a p53-binding site in its promoter, which indicated that WTH3 could be regulated by the p53 gene. This hypothesis was then tested by different strategies. The resulting data revealed that (1) the WTH3 promoter was upregulated by the p53 transgene in diverse host cells; (2) there was a correlation between WTH3 expression levels and p53 gene status in a cell line panel; (3) a WTH3 promoter region was directly targeted by the p53 protein in vitro and in vivo. In addition, overexpression of the WTH3 gene promoted the apoptotic phenotype in host cells. On the basis of these findings, we believe that the negative role played by the WTH3 gene in MDR development is through its proapoptotic potential that is regulated by multiple mechanisms at the transcription level, and one of these mechanisms is linked to the p53 gene

Comparative effects of RRR-alpha- and RRR-gamma-tocopherol on proliferation and apoptosis in human colon cancer cell lines

BACKGROUND: Mediterranean societies, with diets rich in vitamin E isoforms, have a lower risk for colon cancer than those of northern Europe and the Americas. Vitamin E rich diets may neutralize free radicals generated by fecal bacteria in the gut and prevent DNA damage, but signal transduction activities can occur independent of the antioxidant function. The term vitamin E represents eight structurally related compounds, each differing in their potency and mechanisms of chemoprevention. The RRR-γ-tocopherol isoform is found primarily in the US diet, while RRR-α-tocopherol is highest in the plasma. METHODS: The effectiveness of RRR-α- and RRR-γ-tocopherol at inhibiting cell growth and inducing apoptosis in colon cancer cell lines with varying molecular characteristics (SW480, HCT-15, HCT-116 and HT-29) and primary colon cells (CCD-112CoN, nontransformed normal phenotype) was studied. Colon cells were treated with and without RRR-α- or RRR-γ-tocopherol using varying tocopherol concentrations and time intervals. Cell proliferation and apoptosis were measured using the trypan blue assay, annexin V staining, DNA laddering and caspase activation. RESULTS: Treatment with RRR-γ-tocopherol resulted in significant cell death for all cancer cell lines tested, while RRR-α-tocopherol did not. Further, RRR-γ-tocopherol treatment showed no cytotoxicity to normal colon cells CCD-112CoN at the highest concentration and time point tested. RRR-γ-tocopherol treatment resulted in cleavage of PARP, caspase 3, 7, and 8, but not caspase 9. Differences in the percentage cell death and apoptosis were observed in different cell lines suggesting that molecular differences in these cell lines may influence the ability of RRR-γ-tocopherol to induce cell death. CONCLUSION: This is the first study to demonstrate that multiple colon cancer cell lines containing varying genetic alterations will under go growth reduction and apoptosis in the presence of RRR-γ-tocopherol without damage to normal colon cells. The amount growth reduction was dependent upon the molecular signatures of the cell lines. Since RRR-γ-tocopherol is effective at inhibition of cell proliferation at both physiological and pharmacological concentrations dietary RRR-γ-tocopherol may be chemopreventive, while pharmacological concentrations of RRR-γ-tocopherol may aid chemotherapy without toxic effects to normal cells demonstrated by most chemotherapeutic agents

Neuronal Conduction of Excitation without Action Potentials Based on Ceramide Production

International audienceBACKGROUND: Action potentials are the classic mechanism by which neurons convey a state of excitation throughout their length, leading, after synaptic transmission, to the activation of other neurons and consequently to network functioning. Using an in vitro integrated model, we found previously that peripheral networks in the autonomic nervous system can organise an unconventional regulatory reflex of the digestive tract motility without action potentials. METHODOLOGY/PRINCIPAL FINDINGS: In this report, we used combined neuropharmacological and biochemical approaches to elucidate some steps of the mechanism that conveys excitation along the nerves fibres without action potentials. This mechanism requires the production of ceramide in membrane lipid rafts, which triggers in the cytoplasm an increase in intracellular calcium concentration, followed by activation of a neuronal nitric oxide synthase leading to local production of nitric oxide, and then to guanosine cyclic monophosphate. This sequence of second messengers is activated in cascade from rafts to rafts to ensure conduction of the excitation along the nerve fibres. CONCLUSIONS/SIGNIFICANCE: Our results indicate that second messengers are involved in neuronal conduction of excitation without action potentials. This mechanism represents the first evidence-to our knowledge-that excitation is carried along nerves independently of electrical signals. This unexpected ceramide-based conduction of excitation without action potentials along the autonomic nerve fibres opens up new prospects in our understanding of neuronal functioning

Role of Sphingosine Kinase 1 and Sphingosine-1-Phosphate Axis in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is primarily diagnosed in the latter stages of disease progression and is the third leading cause of cancer deaths worldwide. Thus, there is a need to find biomarkers of early HCC as well as the development of more effective treatments for the disease. Sphingosine-1-phosphate (S1P) is a pleiotropic lipid signaling molecule produced by two isoforms of sphingosine kinase (SphK1 and SphK2) that is involved in regulation of many aspects of mammalian physiology and pathophysiology, including inflammation, epithelial and endothelial barrier function, cancer, and metastasis, among many others. Abundant evidence indicates that SphK1 and S1P promote cancer progression and metastasis in multiple types of cancers. However, the role of SphK/S1P in HCC is less well studied. Here, we review the current state of knowledge of SphKs and S1P in HCC, including evidence for the correlation of SphK1 expression and S1P levels with progression of HCC and negative outcomes, and discuss how this information could lead to the design of more effective diagnostic and treatment modalities for HCC

Identification of aberrant forms of alkaline sphingomyelinase (NPP7) associated with human liver tumorigenesis

Alkaline sphingomyelinase (alk-SMase) is expressed in the intestine and human liver. It may inhibit colonic tumorigenesis, and loss of function mutations have been identified in human colon cancer. The present study investigates its expression in human liver cancer. In HepG2 liver cancer cells, RT–PCR identified three transcripts with 1.4, 1.2 and 0.4 kb, respectively. The 1.4 kb form is the wild-type cDNA with five translated exons, the 1.2 kb product lacks exon 4 and the 0.4 kb form is a combination of exons 1 and 5. Genomic sequence showed that these aberrant transcripts were products of alternative splicing. Transient expression of the 1.2 kb form showed no alk-SMase activity. In HepG2 cells, the alk-SMase activity is low in monolayer condition and increased with cell polarisation. Coexistence of 1.4 and 1.2 kb forms was also identified in one hepatoma biopsy. GenBank search identified a cDNA clone from human liver tumour, which codes a protein containing full length of alk-SMase plus a 73-amino-acid tag at the N terminus. The aberrant form was translated by an alternative starting codon upstream of the wild-type mRNA. Expression study showed that linking the tag markedly reduced the enzyme activity. We also analysed human liver biopsy samples and found relatively low alk-SMase activity in diseases with increased risk of liver tumorigenesis. In conclusion, expression of alk-SMase is changed in hepatic tumorigenesis, resulting in loss or marked reduction of the enzyme function