Fullerenes are used across scientific disciplines because of their diverse properties gained by altering encapsulated or surface bound components. In this study, the recently developed theranostic agent based on a radiolabeled functionalized metallofullerene (177Lu-DOTA-f-Gd3N@C80) was synthesized with high radiochemical yield and purity. The efficacy of this agent was demonstrated in two orthotopic xenograft brain tumor models of glioblastoma multiforme (GBM). A dose-dependent improvement in survival was also shown. The in vivo stability of the agent was verified through dual label measurements of biological elimination from the tumor. Overall, these results provide evidence that nanomaterial platforms can be used to deliver effective interstitial brachytherapy.
fullerene; nanomedicine; brachytherapy; glioblastoma; metallofullerene
Chronic inflammation has long been appreciated to play a critical role in tumor development and maintenance. Among the mechanisms involved in coordinating the initiation and resolution of inflammation are those responsible for modifying mRNA stability and/or translation. Several studies have linked the RNA-binding protein HuR, which increases mRNA stability, with malignant transformation. However, in this issue of the JCI, Yiakouvaki et al. compellingly demonstrate in mice that increased HuR activity in myeloid cells has a protective role in the onset of pathologic intestinal inflammation (i.e., colitis) and colitis-associated cancer (CAC). These observations highlight the need to understand the roles of HuR in distinct cell populations in vivo and suggest that enhancing HuR activity may be of clinical benefit in protecting against pathologic inflammation and cancer.
Lapatinib is characterized as an ErbB1/ErbB2 dual inhibitor and has recently been approved for the treatment of metastatic breast cancer. In this study, we examined mechanisms associated with enhancing the activity of lapatinib via combination with other therapies.
In the present studies, estrogen receptor (ER) positive and ER negative breast cancer cells were genetically manipulated to up- or downregulate eIF2-alpha, its phospho-mutant, Nck1, or Nck2, then treated with OSU-03012, lapatinib or the combination and assayed for cytotoxicity/cytostaticity using clonogenic assays.
Treatment of breast cancer cell lines with lapatinib and OSU-03012 (a small molecule derivative of the Cox-2 inhibitor celecoxib) induced synergistic cytotoxic/cytostatic effects. This combination therapy corresponded to an increase in the phosphorylation of eIF2-α at serine51 and a decrease in Nck1 expression. Ectopic expression of phospho-mutant eIF2-α (Ser51Ala) or downregulation of eIF2-α in addition to downregulation of the eIF2-α kinase PERK inhibited the synergistic and cytotoxic effects. Furthermore, ectopic expression of Nck1, but not Nck2 abolished the decrease in cell viability observed in combination-treated cells. Downregulation of Nck1 failed to “rescue” the ablation of the cytotoxic/cytostatic effects by the phospho-mutant of eIF2-α (Ser51Ala) demonstrating that Nck1 downregulation is upstream of eIF2-α phosphorylation in the anti-survival pathway activated by lapatinib and OSU-03012 treatment. Finally, co-immunoprecipitation assays indicated that eIF2-α dissociates from the Nck1/PP1 complex after OSU-03012 and lapatinib co-treatment.
These data indicate that OSU-03012 and lapatinib co-treatment is an effective combination therapy, which functions to enhance cell killing through the Nck1/eIF2 complex. Hence, this complex is a novel target for the treatment of metastatic breast cancer.
Breast cancer; Lapatinib; Combination therapy; Nck; eIF2-alpha
Two splice variants derived from the BCL-x gene via alternative 5′ splice site selection (5′SS) are pro-apoptotic Bcl-x(s) and anti-apoptotic Bcl-x(L). Previously, our laboratory demonstrated that apoptotic signaling pathways regulated the alternative 5′SS selection via protein phosphatase-1 and de novo ceramide. In this study, we examined the elusive pro-survival signaling pathways that regulate the 5′SS selection of Bcl-x pre-mRNA in cancer cells. Taking a broad-based approach by utilizing a number of small molecule inhibitors of various mitogenic/survival pathways, we found that only treatment of non-small cell lung cancer (NSCLC) cell lines with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (50 μM) or the pan-PKC inhibitor GÖ6983 (25 μM) decreased the Bcl-x(L)/Bcl-x(s) mRNA ratio. Pan-PKC inhibitors that did not target the atypical PKCs, PKCι and PKCζ, had no effect on the Bcl-x(L)/Bcl-x(s) mRNA ratio. Additional studies demonstrated that downregulation of the proto-oncogene, PKCι, in contrast to PKCζ, also resulted in a decrease in the Bcl-x(L)/Bcl-x(s) mRNA ratio. Furthermore, downregulation of PKCι correlated with a dramatic decrease in the expression of SAP155, an RNA trans-acting factor that regulates the 5′SS selection of Bcl-x pre-mRNA. Inhibition of the PI3K or atypical PKC pathway induced a dramatic loss of SAP155 complex formation at ceramide-responsive RNA cis-element 1. Lastly, forced expression of Bcl-x(L) “rescued” the loss of cell survival induced by PKCι siRNA. In summary, the PI3K/PKCι regulates the alternative splicing of Bcl-x pre-mRNA with implications in the cell survival of NSCLC cells.
Bcl-x; Bcl-x(L); Bcl-x(s); alternative splicing; non-small cell lung cancer; protein kinase C iota; SAP155
Increasing evidence points to the functional importance of alternative splice variations in cancer pathophysiology with the alternative pre-mRNA processing of caspase 9 as one example. In this study, we delve into the underlying molecular mechanisms that regulate the alternative splicing of caspase 9. Specifically, the pre-mRNA sequence of caspase 9 was analyzed for RNA cis-elements known to interact with SRSF1, a required enhancer for caspase 9 RNA splicing. This analysis revealed thirteen possible RNA cis-elements for interaction with SRSF1 with mutagenesis of these RNA cis-elements identifying a strong intronic splicing enhancer located in intron 6 (C9-I6/ISE). SRSF1 specifically interacted with this sequence, which was required for SRSF1 to act as a splicing enhancer of the inclusion of the four exon cassette. To further determine the biological importance of this mechanism, we employed RNA oligonucleotides to redirect caspase 9 pre-mRNA splicing in favor of caspase 9b expression, which resulted in an increase in the IC50 of non-small cell lung cancer (NSCLC) cells to daunorubicin, cisplatinum, and paclitaxel. In contrast, downregulation of caspase 9b induced a decrease in the the IC50 of these chemotherapeutic drugs. Lastly, these studies demonstrated that caspase 9 RNA splicing was a major mechanism for the synergistic effects of combination therapy with daunorubicin and erlotinib. Overall, we have identified a novel intronic splicing enhancer that regulates caspase 9 RNA splicing and specifically interacts with SRSF1. Furthermore, we demonstrate that the alternative splicing of caspase 9 is an important molecular mechanism with therapeutic relevance to NSCLCs.
ceramide; non-small cell lung cancer; RNA trans-factor; tumor repressor; oncogene; ASF/SF2; SRp30a; SRSF1; chemotherapy; erlotinib; daunorubicin; cisplatinum; paclitaxel
Prostaglandin E2 (PGE2) is an important mediator of the inflammatory response. Phospho-ceramide analogue-1 (PCERA-1), a synthetic phospholipid-like molecule, was previously reported to modulate pro- and anti-inflammatory cytokine production. We show here that PCERA-1 inhibited LPS-stimulated PGE2 production in RAW264.7 macrophages, without affecting COX-2 expression. Furthermore, PCERA-1 efficiently suppressed arachidonic acid (AA) release in response to LPS. The dephosphorylated derivative of PCERA-1, ceramide analogue-1 (CERA-1), mimicked the inhibitory effect of PCERA-1 on AA release and PGE2 production in macrophages. Inhibition of PGE2 production by CERA-1 was completely rescued by addition of exogenous AA. Importantly, PCERA-1 and ceramide-1-phosphate (C1P) stimulated the enzymatic activity of cPLA2α in an in-vitro assay, whereas CERA-1 and ceramide inhibited both basal and C1P-stimulated cPLA2α activity. Collectively, these results indicate that CERA-1 suppresses AA release and subsequent PGE2 production in LPS-stimulated macrophages by direct interaction with cPLA2, and suggest that ceramide may similarly counteract C1P effect on cPLA2 activity in cells. The suppression of PGE2 production is suggested to contribute to the anti-inflammatory action of PCERA-1.
phospholipase A2; arachidonic acid; prostaglandin E2; LPS; ceramide; ceramide-1-phosphate
We have previously shown that essential hypertension in humans and spontaneously hypertensive rats (SHR), is associated with increased levels of ceramide and marked alterations in sphingolipid biology. Pharmacological elevation of ceramide in isolated carotid arteries of SHR leads to vasoconstriction via a calcium-independent phospholipase A2, cyclooxygenase-1 and thromboxane synthase-dependent release of thromboxane A2. This phenomenon is almost absent in vessels from normotensive Wistar Kyoto (WKY) rats. Here we investigated whether lowering of blood pressure can reverse elevated ceramide levels and reduce ceramide-mediated contractions in SHR.
Methods and Findings
For this purpose SHR were treated for 4 weeks with the angiotensin II type 1 receptor antagonist losartan or the vasodilator hydralazine. Both drugs decreased blood pressure equally (SBP untreated SHR: 191±7 mmHg, losartan: 125±5 mmHg and hydralazine: 113±14 mmHg). The blood pressure lowering was associated with a 20–25% reduction in vascular ceramide levels and improved endothelial function of isolated carotid arteries in both groups. Interestingly, losartan, but not hydralazine treatment, markedly reduced sphingomyelinase-induced contractions. While both drugs lowered cyclooxygenase-1 expression, only losartan and not hydralazine, reduced the endothelial expression of calcium-independent phospholipase A2. The latter finding may explain the effect of losartan treatment on sphingomyelinase-induced vascular contraction.
In summary, this study corroborates the importance of sphingolipid biology in blood pressure control and specifically shows that blood pressure lowering reduces vascular ceramide levels in SHR and that losartan treatment, but not blood pressure lowering per se, reduces ceramide-mediated arterial contractions.
Increasing evidence points to the functional importance of alternative splice variations in cancer pathophysiology. Two splice variants are derived from the CASP9 gene via the inclusion (Casp9a) or exclusion (Casp9b) of a four exon cassette. Here we show that alternative splicing of Casp9 is dysregulated in non-small cell lung cancers (NSCLC) regardless of their pathological classification. Based on these findings we hypothesized that survival pathways activated by oncogenic mutation regulated this mechanism. In contrast to K-RasV12 expression, EGFR overexpression or mutation dramatically lowered the Casp9a/9b splice isoform ratio. Moreover, Casp9b downregulation blocked the ability of EGFR mutations to induce anchorage-independent growth. Furthermore, Casp9b expression blocked inhibition of clonogenic colony formation by erlotinib. Interrogation of oncogenic signaling pathways showed that inhibition of PI3K or Akt dramatically increased the Casp9a/9b ratio in NSCLC cells. Finally, Akt was found to mediate exclusion of the exon 3,4,5,6 cassette of Casp9 via the phosphorylation state of the RNA splicing factor SRp30a via serines 199, 201, 227 and 234. Taken together, our findings demonstrate that oncogenic factors activating the PI3Kinase/Akt pathway can regulate alternative splicing of Casp9 via a coordinated mechanism involving the phosphorylation of SRp30a.
SRp30a; alternative splicing; erlotinib; Akt
Background: Pro-TNFα is transformed into the active/soluble form through proteolysis by TNFα-converting enzyme (TACE).
Results: Genetic ablation of ceramide kinase induces an increase in TACE activity and secreted TNFα.
Conclusion: Ceramide 1-phosphate (C1P) negatively regulates the activity of TACE.
Significance: The TACE/C1P interaction is a viable drug target for the treatment of heart disease and sepsis.
Tumor necrosis factor α (TNFα) is a well known cytokine involved in systemic and acute inflammation. In this study, we demonstrate that ceramide 1-phosphate (C1P) produced by ceramide kinase (CERK) is a negative regulator of LPS-induced TNFα secretion. Specifically, bone marrow-derived macrophages isolated from CERK knock-out mice (CERK−/−) generated higher levels of TNFα than the wild-type mice (CERK+/+) in response to LPS. An increase in basal TNFα secretion was also observed in CERK−/− murine embryonic fibroblasts, which was rescued by re-expression of wild-type CERK. This effect was due to increased secretion and not transcription. The secretion of TNFα is regulated by TNFα-converting enzyme (TACE also known as ADAM17), and importantly, the activity of TACE was higher in cell extracts from CERK−/− as compared with wild type. In vitro analysis also demonstrated that C1P is a potent inhibitor of this enzyme, in stark contrast to ceramide and sphingosine 1-phosphate. Furthermore, TACE specifically bound C1P with high affinity. Finally, several putative C1P-binding sites were identified via homology throughout the protein sequence of TACE. These results indicate that C1P produced by CERK has a negative effect on the processing/secretion of TNFα via modulation of TACE activity.
Lipids; Lipid-binding Protein; Lipid Synthesis; Sphingolipid; Tumor Necrosis Factor (TNF)
Hypertension is, amongst others, characterized by endothelial dysfunction and vascular remodeling. As sphingolipids have been implicated in both the regulation of vascular contractility and growth, we investigated whether sphingolipid biology is altered in hypertension and whether this is reflected in altered vascular function.
Methods and Findings
In isolated carotid arteries from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats, shifting the ceramide/S1P ratio towards ceramide dominance by administration of a sphingosine kinase inhibitor (dimethylsphingosine) or exogenous application of sphingomyelinase, induced marked endothelium-dependent contractions in SHR vessels (DMS: 1.4±0.4 and SMase: 2.1±0.1 mN/mm; n = 10), that were virtually absent in WKY vessels (DMS: 0.0±0.0 and SMase: 0.6±0.1 mN/mm; n = 9, p<0.05). Imaging mass spectrometry and immunohistochemistry indicated that these contractions were most likely mediated by ceramide and dependent on iPLA2, cyclooxygenase-1 and thromboxane synthase. Expression levels of these enzymes were higher in SHR vessels. In concurrence, infusion of dimethylsphingosine caused a marked rise in blood pressure in anesthetized SHR (42±4%; n = 7), but not in WKY (−12±10%; n = 6). Lipidomics analysis by mass spectrometry, revealed elevated levels of ceramide in arterial tissue of SHR compared to WKY (691±42 vs. 419±27 pmol, n = 3–5 respectively, p<0.05). These pronounced alterations in SHR sphingolipid biology are also reflected in increased plasma ceramide levels (513±19 pmol WKY vs. 645±25 pmol SHR, n = 6–12, p<0.05). Interestingly, we observed similar increases in ceramide levels (correlating with hypertension grade) in plasma from humans with essential hypertension (185±8 pmol vs. 252±23 pmol; n = 18 normotensive vs. n = 19 hypertensive patients, p<0.05).
Hypertension is associated with marked alterations in vascular sphingolipid biology such as elevated ceramide levels and signaling, that contribute to increased vascular tone.
Because ceramide accumulates in several forms of cardiovascular disease and ceramide-induced apoptosis may involve the volume-sensitive Cl− current, ICl,swell, we assessed whether ceramide activates ICl,swell.
Methods and results
ICl,swell was measured in rabbit ventricular myocytes by whole-cell patch clamp after isolating anion currents. Exogenous C2-ceramide (C2-Cer), a membrane-permeant short-chain ceramide, elicited an outwardly rectifying Cl− current in both physiological and symmetrical Cl− solutions that was fully inhibited by DCPIB, a specific ICl,swell blocker. In contrast, the metabolically inactive C2-Cer analogue C2-dihydroceramide (C2-H2Cer) failed to activate Cl− current. Bacterial sphingomyelinase (SMase), which generates endogenous long-chain ceramides as was confirmed by tandem mass spectrometry, also elicited an outwardly rectifying Cl− current that was inhibited by DCPIB and tamoxifen, another ICl,swell blocker. Bacterial SMase-induced current was partially reversed by osmotic shrinkage and fully suppressed by ebselen, a scavenger of reactive oxygen species. Outward rectification with physiological and symmetrical Cl− gradients, block by DCPIB and tamoxifen, and volume sensitivity are characteristics that identify ICl,swell. Insensitivity to C2-H2Cer and block by ebselen suggest involvement of ceramide signalling rather than direct lipid-channel interaction.
Exogenous and endogenous ceramide elicited ICl,swell in ventricular myocytes. This may contribute to persistent activation of ICl,swell and aspects of altered myocyte function in cardiovascular diseases associated with by ceramide accumulation.
Cl channel; Ceramide; Sphingomyelinase; ICl,swell; VRAC
Caspase-9 is involved in the intrinsic apoptotic pathway and suggested to play a role as a tumor suppressor. Little is known about the mechanisms governing caspase-9 expression, but post-transcriptional pre-mRNA processing generates 2 splice variants from the caspase-9 gene, pro-apoptotic caspase-9a and anti-apoptotic caspase-9b. Here we demonstrate that the ratio of caspase-9 splice variants is dysregulated in non–small cell lung cancer (NSCLC) tumors. Mechanistic analysis revealed that an exonic splicing silencer (ESS) regulated caspase-9 pre-mRNA processing in NSCLC cells. Heterogeneous nuclear ribonucleoprotein L (hnRNP L) interacted with this ESS, and downregulation of hnRNP L expression induced an increase in the caspase-9a/9b ratio. Although expression of hnRNP L lowered the caspase-9a/9b ratio in NSCLC cells, expression of hnRNP L produced the opposite effect in non-transformed cells, suggesting a post-translational modification specific for NSCLC cells. Indeed, Ser52 was identified as a critical modification regulating the caspase-9a/9b ratio. Importantly, in a mouse xenograft model, downregulation of hnRNP L in NSCLC cells induced a complete loss of tumorigenic capacity that was due to the changes in caspase-9 pre-mRNA processing. This study therefore identifies a cancer-specific mechanism of hnRNP L phosphorylation and subsequent lowering of the caspase-9a/9b ratio, which is required for the tumorigenic capacity of NSCLC cells.
Elevated TNFα levels are associated with insulin resistance, but the molecular mechanisms linking cytokine signaling to impaired insulin function remain elusive. We previously demonstrated a role for Akt in insulin regulation of PKCβII alternative splicing through phosphorylation of SRp40, a required mechanism for insulin-stimulated glucose uptake. We hypothesized that TNFα attenuated insulin signaling by dephosphorylating Akt and its targets via ceramide-activated protein phosphatase (CAPP). Western blot analysis of L6 cell lysates demonstrated impaired insulin–stimulated phosphorylation of Akt, SRp40, and GSK3β in response to TNFα and the short chain C6 ceramide analog. TNFα increased serine/threonine phosphatase activity of PP1 in response to C6, but not insulin, suggesting a ceramide-specific effect. Myriocin, an inhibitor of de novo ceramide synthesis, blocked stimulation of the PP1 activity. Ceramide species measurement by LC-MS showed consistent increases in C24:1 and C16 ceramides. Effects of TNFα and C6 on insulin–stimulated phosphorylation of GSK3β were prevented by myriocin and tautomycin, a PP1 inhibitor, further implicating a de novo ceramide-PP1 pathway. Alternative splicing assays demonstrated that TNFα abolished insulin-mediated inclusion of the PKCβII exon. Collectively, our work demonstrates a role for PP1-like CAPP in mediating TNFα effects blocking insulin phosphorylation cascades involved in glycogen metabolism and alternative splicing.
TNFα; Ceramide activated protein phosphatase (CAPP); Akt; SR proteins; PKCβII; alternative splicing
The RNA-binding protein Sam68 is involved in apoptosis, but its cellular mRNA targets and its mechanism of action remain unknown. We demonstrate that Sam68 binds the mRNA for Bcl-x and affects its alternative splicing. Depletion of Sam68 by RNA interference caused accumulation of antiapoptotic Bcl-x(L), whereas its up-regulation increased the levels of proapoptotic Bcl-x(s). Tyrosine phosphorylation of Sam68 by Fyn inverted this effect and favored the Bcl-x(L) splice site selection. A point mutation in the RNA-binding domain of Sam68 influenced its splicing activity and subnuclear localization. Moreover, coexpression of ASF/SF2 with Sam68, or fusion with an RS domain, counteracted Sam68 splicing activity toward Bcl-x. Finally, Sam68 interacted with heterogenous nuclear RNP (hnRNP) A1, and depletion of hnRNP A1 or mutations that impair this interaction attenuated Bcl-x(s) splicing. Our results indicate that Sam68 plays a role in the regulation of Bcl-x alternative splicing and that tyrosine phosphorylation of Sam68 by Src-like kinases can switch its role from proapoptotic to antiapoptotic in live cells.