RESUMO
The three arms of the unfolded protein response (UPR) surveil the luminal environment of the endoplasmic reticulum (ER) and transmit information through the lipid bilayer to the cytoplasm to alert the cell of stress conditions within the ER lumen. That same lipid bilayer is the site of de novo synthesis of phospholipids and sphingolipids. Thus, it is no surprise that lipids are modulated by and are modulators of ER stress. Given that sphingolipids have both prosurvival and proapoptotic effects, they also exert opposing effects on life/death decisions in the face of prolonged ER stress detected by the UPR. In this review, we will focus on several recent studies that demonstrate how sphingolipids affect each arm of the UPR. We will also discuss the role of sphingolipids in the process of immunogenic cell death downstream of the protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic initiating factor 2α (eIF2α) arm of the UPR. Furthermore, we will discuss strategies to target the sphingolipid metabolic pathway that could potentially act synergistically with agents that induce ER stress as novel anticancer treatments. SIGNIFICANCE STATEMENT: This review provides the readers with a brief discussion of the sphingolipid metabolic pathway and the unfolded protein response. The primary focus of the review is the mechanism(s) by which sphingolipids modulate the endoplasmic reticulum (ER) stress response pathways and the critical role of sphingolipids in the process of immunogenic cell death associated with the ER stress response.
Assuntos
Morte Celular Imunogênica , Neoplasias , Humanos , Bicamadas Lipídicas/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , Estresse do Retículo Endoplasmático , Resposta a Proteínas não Dobradas , Retículo Endoplasmático/metabolismo , Neoplasias/metabolismo , Esfingolipídeos/metabolismoRESUMO
The Rho associated coiled-coil containing protein kinase (ROCK1 and ROCK2) and myotonic dystrophy-related Cdc-42 binding kinases (MRCKα and MRCKß) are critical regulators of cell proliferation and cell plasticity, a process intimately involved in cancer cell migration and invasion. Previously, we reported the discovery of a novel small molecule (DJ4) selective multi-kinase inhibitor of ROCK1/2 and MRCKα/ß. Herein, we further characterized the anti-proliferative and apoptotic effects of DJ4 in non-small cell lung cancer and triple-negative breast cancer cells. To further optimize the ROCK/MRCK inhibitory potency of DJ4, we generated a library of 27 analogs. Among the various structural modifications, we identified four additional active analogs with enhanced ROCK/MRCK inhibitory potency. The anti-proliferative and cell cycle inhibitory effects of the active analogs were examined in non-small cell lung cancer, breast cancer, and melanoma cell lines. The anti-proliferative effectiveness of DJ4 and the active analogs was further demonstrated against a wide array of cancer cell types using the NCI-60 human cancer cell line panel. Lastly, these new analogs were tested for anti-migratory effects in highly invasive MDA-MB-231 breast cancer cells. Together, our results demonstrate that selective inhibitors of ROCK1/2 (DJE4, DJ-Allyl) inhibited cell proliferation and induced cell cycle arrest at G2/M but were less effective in cell death induction compared with dual ROCK1/2 and MRCKα/ß (DJ4 and DJ110).
RESUMO
We recently identified the sphingosine kinases (SphK1/2) as key intracellular regulators of immunogenic cell death (ICD) in colorectal cancer (CRC) cells. To better understand the mechanism by which SphK inhibition enhances ICD, we focused on the intracellular signaling pathways leading to cell surface exposure of calreticulin (ectoCRT). Herein, we demonstrate that ABT-263 and AZD-5991, inhibitors of Bcl-2/Bcl-XL and Mcl-1, respectively, induce the production of ectoCRT, indicative of ICD. Inhibition of SphK1 significantly enhanced ABT/AZD-induced ectoCRT production, in a caspase 8-dependent manner. Mechanistically, we demonstrate that ABT/AZD-induced Bak/Bax activation stimulates pro-survival SphK1/sphingosine-1-phosphate (S1P) signaling, which attenuates ectoCRT production. Additionally, we identified a regulatory role for ceramide synthase 6 (CerS6)/C16:0 ceramide in transporting of ectoCRT to the cell surface. Together, these results indicate that the sphingolipid metabolic regulators of the sphingolipid rheostat, S1P and C16:0 ceramide, influence survival/death decisions of CRC cells in response to ICD-inducing chemotherapeutic agents. Importantly, SphK1, which produces S1P, is a stress-responsive pro-survival lipid kinase that suppresses ICD. While ceramide, produced by the inhibition of SphK1 is required for production of the cell surface marker of ICD, ectoCRT. Thus, inhibition of SphK1 represents a means to enhance the therapeutic efficacy of ICD-inducing agents.
RESUMO
Background: Endogenous and synthetic cannabinoids have been shown to induce cancer cell death through the accumulation of the sphingolipid, ceramide (Cer). Recently, we have demonstrated that Cer accumulation enhances the induction of immunogenic cell death (ICD). Objectives: The primary objective of this study was to demonstrate that (±) 5-epi CP 55,940 (5-epi), a by-product of the chemical synthesis of the synthetic cannabinoid CP 55,940, induces ICD in colorectal cancer (CRC) cells, and that modulation of the sphingolipid metabolic pathway through inhibition of the sphingosine kinases (SphKs) enhances these effects. Methods: A cell culture model system of human CRC cell lines was employed to measure the cell surface and intracellular production of markers of ICD. The effects of 5-epi, alone and in combination with SphK inhibitors, on production of Cer through the de novo sphingolipid synthesis pathway were measured by Liquid Chromatography - Tandem Mass Spectrometry (LC/MS/MS)-based sphingolipidomic analysis. Cell surface exposure of calreticulin (ectoCRT), a hallmark of ICD, was measured by flow cytometry. Examination of the effects of 5-epi, alone and in combination with SphK inhibitors, on the intracellular signaling pathway associated with ICD was conducted by immunoblot analysis of human CRC cell lines. Results: Sphingolipidomic analysis indicated that 5-epi induces the de novo sphingolipid synthetic pathway. 5-epi dose dependently induces cell surface exposure of ectoCRT, and inhibition of Cer metabolism through inhibition of the SphKs significantly enhances 5-epi-induced ectoCRT exposure in multiple CRC cell lines. 5-epi induces and SphK inhibition enhances activation of the cell death signaling pathway associated with ICD. Conclusions: This study is the first demonstration that cannabinoids can induce the cell surface expression of ectoCRT, and potentially induce ICD. Moreover, this study reinforces our previous observation of a role for Cer accumulation in the induction of ICD and extends this observation to the cannabinoids, agents not typically associated with ICD. Inhibition of SphKs enhanced the 5-epi-induced signaling pathways leading to ICD and production of ectoCRT. Overexpression of SphK1 has previously been associated with chemotherapy resistance. Thus, targeting the SphKs has the potential to reverse chemotherapy resistance and simultaneously enhance the antitumor immune response through enhancement of ICD induction.
Assuntos
Canabinoides , Esfingosina , Humanos , Calreticulina/metabolismo , Ceramidas/farmacologia , Esfingolipídeos/metabolismo , Espectrometria de Massas em TandemRESUMO
Agents that induce immunogenic cell death (ICD) alter the cellular localization of calreticulin (CRT), causing it to become cell surface-exposed within the plasma membrane lipid raft microdomain [cell surface-exposed CRT (ectoCRT)] where it serves as a damage associated-molecular pattern that elicits an antitumor immune response. We have identified the sphingolipid metabolic pathway as an integral component of the process of ectoCRT exposure. Inhibition of the sphingosine kinases (SphKs) enhances mitoxantrone-induced production of hallmarks of ICD, including ectoCRT production, with an absolute mean difference of 40 MFI (95% CI: 19-62; P = 0.0014) and 1.3-fold increase of ATP secretion with an absolute mean difference of 87 RLU (95% CI: 55-120; P < 0.0001). Mechanistically, sphingosine kinase inhibition increases mitoxantrone-induced accumulation of ceramide species, including C16:0 ceramide 2.8-fold with an absolute mean difference of 1.390 pmol/nmol Pi (95% CI: 0.798-1.983; P = 0.0023). We further examined the localization of ectoCRT to the lipid raft microdomain and demonstrate that ectoCRT forms disulfide-bridged dimers. Together, our findings suggest that ceramide accumulation impinges on the homeostatic function of the endoplasmic reticulum to induce ectoCRT exposure and that structural alterations of ectoCRT may underlie its immunogenicity. Our findings further suggest that inhibition of the SphKs may represent a means to enhance the therapeutic immunogenic efficacy of ICD-inducing agents while reducing overt toxicity/immunosuppressive effects by allowing for the modification of dosing regimens or directly lowering the dosages of ICD-inducing agents employed in therapeutic regimens. SIGNIFICANCE STATEMENT: This study demonstrates that inhibition of sphingosine kinase enhances the mitoxantrone-induced cell surface exposure of a dimeric form of the normally endoplasmic reticulum resident chaperone calreticulin as part of the process of a unique form of regulated cell death termed immunogenic cell death. Importantly, inhibition of sphingosine kinase may represent a means to enhance the therapeutic efficacy of immunogenic cell death-inducing agents, such as mitoxantrone, while reducing their overt toxicity and immunosuppressive effects, leading to better therapeutic outcomes for patients.
Assuntos
Fosfotransferases (Aceptor do Grupo Álcool) , Calreticulina , Membrana Celular , Microdomínios da Membrana , MitoxantronaRESUMO
The recently renewed interest in scientific rigor and reproducibility is of critical importance for both scientists developing new targeted small-molecule inhibitors and those employing these molecule in cellular studies, alike. While off-target effects are commonly considered as limitations for any given small-molecule inhibitor, the ability of a given compound to distinguish between enzyme isoforms is often neglected when employing compounds in cellular studies. To call attention to this issue, we have compared the results of an assay for "direct target engagement", the Cellular Thermal Shift Assay (CETSA), to the published isoform selectivity of 12 commercially available sphingosine kinase 1 and 2 (SphK 1 and SphK2) inhibitors. Our results suggest that, at the concentrations commonly employed in cellular assay systems, none of the tested SKIs can be considered isoform selective. Thus, caution and complimentary assay strategies must be employed to fully discern isoform selectivity for the SphKs. Moreover, caution must be employed by the scientific community as a whole when designing experiments that aim to discern the effects of one enzyme isoform versus another to ensure that the concentration ranges used are able to distinguish isoform selectivity.
Assuntos
Bioensaio/métodos , Descoberta de Drogas/métodos , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , HumanosRESUMO
Our sphingosine kinase inhibitor (SKI) optimization studies originated with the optimization of the SKI-I chemotype by replacement of the substituted benzyl rings with substituted phenyl rings giving rise to the discovery of SKI-178. We have recently reported that SKI-178 is a dual-targeted inhibitor of both sphingosine kinase isoforms (SphK1/2) and a microtubule disrupting agent (MDA). In mechanism-of-action studies, we have shown that these two separate actions synergize to induce cancer cell death in acute myeloid leukemia (AML) cell and animal models. Owning to the effectiveness of SKI-178, we sought to further refine the chemotype while maintaining "on-target" SKI and MDA activities. Herein, we modified the "linker region" between the substituted phenyl rings of SKI-178 through a structure guided approach. These studies have yielded the discovery of an SKI-178 congener, SKI-349, with log-fold enhancements in both SphK inhibition and cytotoxic potency. Importantly, SKI-349 also demonstrates log-fold improvements in therapeutic efficacy in a retro-viral transduction model of MLL-AF9 AML as compared to previous studies with SKI-178. Together, our results strengthen the hypothesis that simultaneous targeting of the sphingosine kinases (SphK1/2) and the induction of mitotic spindle assembly checkpoint arrest, via microtubule disruption, might be an effective therapeutic strategy for hematological malignancies including AML.
Assuntos
Antineoplásicos/farmacologia , Desenvolvimento de Medicamentos , Inibidores Enzimáticos/farmacologia , Microtúbulos/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Microtúbulos/metabolismo , Estrutura Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Polimerização/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Despite progress, our understanding of psychiatric and neurological illnesses remains poor, at least in part due to the inability to access neurons directly from patients. Currently, there are in vitro models available but significant work remains, including the search for a less invasive, inexpensive and rapid method to obtain neuronal-like cells with the capacity to deliver reproducible results. Here, we present a new protocol to transdifferentiate human circulating monocytes into neuronal-like cells in 20 days and without the need for viral insertion or reprograming. We have thoroughly characterized these monocyte-derived-neuronal-like cells (MDNCs) through various approaches including immunofluorescence (IF), flow cytometry, qRT-PCR, single cell mRNA sequencing, electrophysiology and pharmacological techniques. These MDNCs resembled human neurons early in development, expressed a variety of neuroprogenitor and neuronal genes as well as several neuroprogenitor and neuronal proteins and also presented electrical activity. In addition, when these neuronal-like cells were exposed to either dopamine or colchicine, they responded similarly to neurons by retracting their neuronal arborizations. More importantly, MDNCs exhibited reproducible differentiation rates, arborizations and expression of dopamine 1 receptors (DR1) on separate sequential samples from the same individual. Differentiation efficiency measured by cell morphology was on average 11.9 ± 1.4% (mean, SEM, n = 38,819 cells from 15 donors). To provide context and help researchers decide which in vitro model of neuronal development is best suited to address their scientific question,we compared our results with those of other in vitro models currently available and exposed advantages and disadvantages of each paradigm.
RESUMO
AIM: To further characterize the selectivity, mechanism-of-action and therapeutic efficacy of the novel small molecule inhibitor, SKI-178. METHODS: Using the state-of-the-art Cellular Thermal Shift Assay (CETSA) technique to detect "direct target engagement" of proteins intact cells, in vitro and in vivo assays, pharmacological assays and multiple mouse models of acute myeloid leukemia (AML). RESULTS: Herein, we demonstrate that SKI-178 directly target engages both Sphingosine Kinase 1 and 2. We also present evidence that, in addition to its actions as a Sphingosine Kinase Inhibitor, SKI-178 functions as a microtubule network disrupting agent both in vitro and in intact cells. Interestingly, we separately demonstrate that simultaneous SphK inhibition and microtubule disruption synergistically induces apoptosis in AML cell lines. Furthermore, we demonstrate that SKI-178 is well tolerated in normal healthy mice. Most importantly, we demonstrate that SKI-178 has therapeutic efficacy in several mouse models of AML. CONCLUSION: SKI-178 is a multi-targeted agent that functions both as an inhibitor of the SphKs as well as a disruptor of the microtubule network. SKI-178 induced apoptosis arises from a synergistic interaction of these two activities. SKI-178 is safe and effective in mouse models of AML, supporting its further development as a multi-targeted anti-cancer therapeutic agent.
RESUMO
Sphingosine kinase 1 (Sphk1) associates with early endocytic membranes during endocytosis; however, the role of sphingosine or sphingosine-1-phosphate as the critical metabolite in endocytic trafficking has not been established. Here, we demonstrate that the recruitment of Sphk1 to sphingosine-enriched endocytic vesicles and the generation of sphingosine-1-phosphate facilitate membrane trafficking along the endosomal pathway. Exogenous sphingosine and sphingosine-based Sphk1 inhibitors induce the Sphk1-dependent fusion of endosomal membranes to accumulate enlarged late endosomes and amphisomes enriched in sphingolipids. Interestingly, Sphk1 also appears to facilitate endosomal fusion independent of its catalytic activity, given that catalytically inactive Sphk1G82D is recruited to endocytic membranes by sphingosine or sphingosine-based Sphk1 inhibitor and promotes membrane fusion. Furthermore, we reveal that the clearance of enlarged endosomes is dependent on the activity of ceramide synthase, lysosomal biogenesis, and the restoration of autophagic flux. Collectively, these studies uncover intersecting roles for Sphk1, sphingosine, and autophagic machinery in endocytic membrane trafficking.
Assuntos
Autofagia , Endocitose , Endossomos/metabolismo , Membranas Intracelulares/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Amino Álcoois/farmacologia , Animais , Autofagia/efeitos dos fármacos , Proteína 5 Relacionada à Autofagia/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Esfingolipídeos/metabolismo , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismoRESUMO
Metastatic cancer cells show great plasticity in their migratory mechanisms. In this review we briefly describe the signal transduction pathways associated with the ROCK and MRCK kinases and their roles in cancer cell migration and in its plasticity. With respect to therapeutic strategies targeting metastatic cancers, selectively blocking a single target, such as ROCK or MRCK, can induce alternate modes of cancer cell migration (i.e. plasticity) making the treatment ineffective. To address the problem of plasticity, we will discuss the strategy of simultaneous targeting of both ROCK and MRCK as an effective anti-metastatic therapeutics.
Assuntos
Movimento Celular/fisiologia , Neoplasias/enzimologia , Neoplasias/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Quinases Associadas a rho/metabolismo , Humanos , Neoplasias/genética , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Quinases Associadas a rho/genéticaRESUMO
We previously developed SKI-178 (N'-[(1E)-1-(3,4-dimethoxyphenyl)ethylidene]-3-(4-methoxxyphenyl)-1H-pyrazole-5-carbohydrazide) as a novel sphingosine kinase-1 (SphK1) selective inhibitor and, herein, sought to determine the mechanism-of-action of SKI-178-induced cell death. Using human acute myeloid leukemia (AML) cell lines as a model, we present evidence that SKI-178 induces prolonged mitosis followed by apoptotic cell death through the intrinsic apoptotic cascade. Further examination of the mechanism of action of SKI-178 implicated c-Jun NH2-terminal kinase (JNK) and cyclin-dependent protein kinase 1 (CDK1) as critical factors required for SKI-178-induced apoptosis. In cell cycle synchronized human AML cell lines, we demonstrate that entry into mitosis is required for apoptotic induction by SKI-178 and that CDK1, not JNK, is required for SKI-178-induced apoptosis. We further demonstrate that the sustained activation of CDK1 during prolonged mitosis, mediated by SKI-178, leads to the simultaneous phosphorylation of the prosurvival Bcl-2 family members, Bcl-2 and Bcl-xl, as well as the phosphorylation and subsequent degradation of Mcl-1. Moreover, multidrug resistance mediated by multidrug-resistant protein1 and/or prosurvival Bcl-2 family member overexpression did not affect the sensitivity of AML cells to SKI-178. Taken together, these findings highlight the therapeutic potential of SKI-178 targeting SphK1 as a novel therapeutic agent for the treatment of AML, including multidrug-resistant/recurrent AML subtypes.
Assuntos
Apoptose/fisiologia , Hidrazinas/farmacologia , Leucemia Mieloide Aguda/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Pirazóis/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Células HL-60 , Humanos , Hidrazinas/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Pirazóis/uso terapêutico , Células U937RESUMO
Two structurally related protein kinase families, the Rho kinases (ROCK) and the myotonic dystrophy kinase-related Cdc42-binding kinases (MRCK) are required for migration and invasion of cancer cells. We hypothesized that simultaneous targeting of these two kinase families might represent a novel therapeutic strategy to block the migration and invasion of metastatic cancers. To this end, we developed DJ4 as a novel small molecule inhibitor of these kinases. DJ4 potently inhibited activities of ROCK and MRCK in an ATP competitive manner. In cellular functional assays, DJ4 treatment significantly blocked stress fiber formation and inhibited migration and invasion of multiple cancer cell lines in a concentration dependent manner. Our results strongly indicate that DJ4 may be further developed as a novel anti-metastatic chemotherapeutic agent for multiple cancers.
Assuntos
Miotonina Proteína Quinase/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Tiazolidinas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Humanos , Invasividade Neoplásica , Neoplasias/enzimologia , Neoplasias/patologiaRESUMO
Cardiac function is regulated in part by the sympathetic branch of the autonomic nervous system via the stellate ganglion (SG) neurons. Neurotransmitters, such as noradrenaline (NA), and neuropeptides, including nociceptin (Noc), influence the excit ability of SG neurons by modulating Ca(2+) channel function following activation of the adrenergic and nociceptin/orphanin FQ peptide (NOP) opioid receptors, respectively. The regulation of Ca(2+) channels is mediated by Gßγ, but the specific Gß subunit that modulates the channels is not known. In the present study, small interference RNA (siRNA) was employed to silence the natively expressed Gß proteins in rat SG tissue and to examine the coupling specificity of adrenergic and NOP opioid receptors to Ca(2+) channels employing the whole-cell variant of the patch-clamp technique.Western blotting analysis showed that Gß1, Gß2 and Gß4 are natively expressed. The knockdown of Gß2 or Gß4 led to a significant decrease of the NA- and Noc-mediated Ca(2+)current inhibition, while Gß1 silencing was without effect. However, sustaining low levels of Gß2 resulted in an increased expression of Gß4 and a concomitant compensation of both adrenergic and opioid signalling pathways modulating Ca(2+) channels. Conversely, Gß4-directed siRNA was not accompanied with a compensation of the signalling pathway. Finally, the combined silencing of Gß2 and Gß4 prevented any additional compensatory mechanisms.Overall, our studies suggest that in SG neurons, Gß2 and Gß4 normally maintain the coupling of Ca(2+) channels with the receptors, with the latter subtype responsible for maintaining the integrity of both pathways.
Assuntos
Canais de Cálcio/fisiologia , Subunidades beta da Proteína de Ligação ao GTP/fisiologia , Receptores Adrenérgicos/fisiologia , Receptores Opioides/fisiologia , Gânglio Estrelado/fisiologia , Animais , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Masculino , Neurônios/fisiologia , RNA Interferente Pequeno/genética , Ratos , Ratos WistarRESUMO
A host of beneficial effects have been attributed to the red wine polyphenol, resveratrol. Foremost, among these are its anti-cancer properties. Yet, the mechanism by which resveratrol achieves these effects are unknown. In this issue of the BJP, Lim et al. report that resveratrol and its higher order oligomers inhibit sphingosine kinase 1 (SphK1). SphK1 is a key regulator of sphingolipid metabolism and alterations of this key metabolic pathway have been linked to many hyperproliferative diseases. This study identifies a target for the action of resveratrol and its higher order oligomers and opens the door to evaluation of SphK1 as a target for chemo-prevention of cancer.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Estilbenos/farmacologia , Humanos , ResveratrolRESUMO
Resistance to therapies develops rapidly for melanoma leading to more aggressive disease. Therefore, agents are needed that specifically inhibit proteins or pathways controlling the development of this disease, which can be combined, dependent on genes deregulated in a particular patient's tumors. This study shows that elevated sphingosine-1-phosphate (S-1-P) levels resulting from increased activity of sphingosine kinase-1 (SPHK1) occur in advanced melanomas. Targeting SPHK1 using siRNA decreased anchorage-dependent and -independent growth as well as sensitized melanoma cells to apoptosis-inducing agents. Pharmacological SPHK1 inhibitors SKI-I but not SKI-II decreased S-1-P content, elevated ceramide levels, caused a G2-M block and induced apoptotic cell death in melanomas. Targeting SPHK1 using siRNA or the pharmacological agent called SKI-I decreased the levels of pAKT. Furthermore, SKI-I inhibited the expression of CYCLIN D1 protein and increased the activity of caspase-3/7, which in turn led to the degradation of PARP. In animals, SKI-I but not SKI-II retarded melanoma growth by 25-40%. Thus, targeting SPHK1 using siRNAs or SKI-I has therapeutic potential for melanoma treatment either alone or in combination with other targeted agents.
Assuntos
Melanoma/tratamento farmacológico , Melanoma/patologia , Terapia de Alvo Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclina D1/metabolismo , Regulação para Baixo/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Lisofosfolipídeos/metabolismo , Melanócitos/efeitos dos fármacos , Melanócitos/patologia , Melanoma/enzimologia , Camundongos , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/metabolismo , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/patologia , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Estaurosporina/farmacologia , Tiazóis/farmacologia , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ceramidases play a critical role in generating sphingosine-1-phosphate by hydrolyzing ceramide into sphingosine, a substrate for sphingosine kinase. In order to elucidate its transcriptional regulation, we identify here a putative promoter region in the 5'-UTR of the human neutral CDase (nCDase) gene. Using human genomic DNA, we cloned a 3000 bp region upstream of the translational start site of the nCDase gene. Luciferase reporter analyses demonstrated that this 3000 bp region had promoter activity, with the strongest induction occurring within the first 200 bp. Computational analysis revealed the 200 bp essential promoter region contained several well-characterized promoter elements, lacked a conical TATA box, but did contain a reverse oriented CCAAT box, a feature common to housekeeping genes. Electrophoretic mobility shift assays demonstrated that the identified candidate transcriptional response elements (TRE) bind their respective transcription factors, including NF-Y, AP-2, Oct-1, and GATA. Mutagenic analyses of the TRE revealed that these sites regulated promoter activity and mutating an individual site decreased promoter reporter activity by up to 50%. Together, our findings suggest that regulation of nCDase expression involves coordinated TATA-less transcriptional activity.
Assuntos
Ceramidase Neutra/genética , Regiões 5' não Traduzidas , Animais , Sequência de Bases , Sítios de Ligação/genética , Clonagem Molecular , Primers do DNA/genética , Regulação Enzimológica da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Elementos de Resposta , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
Synthesis and anti-melanoma activity of various naphthalimide analogs, rationally modified by introducing isothiocyanate (ITC) and thiourea (TU) functionalities, found in well-known anti-cancer agents, is described. The structure-activity relationship comparison of the novel agents in inhibiting cancer cell growth was evaluated in various melanoma cell lines. Both ITC and TU analogs effectively inhibited cell viability and induced apoptosis in various human melanoma cells. Nitro substitution and increase in alkyl chain length, in general, enhanced the apoptotic activity of ITC derivatives. All the new compounds were well tolerated when injected intraperitoneal (i.p.) in mice at effective doses at which both the ITC and TU derivatives inhibited melanoma tumor growth in mice following i.p. xenograft. The nitro substituted naphthalimide-ITC derivative 3d was found to be the most effective in inducing apoptosis, and in inhibiting melanoma cell and tumor growth.
Assuntos
Antineoplásicos/síntese química , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Melanoma/tratamento farmacológico , Naftalimidas/síntese química , Naftalimidas/uso terapêutico , Neoplasias Cutâneas/tratamento farmacológico , Animais , Anexina A5/análise , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Injeções Intraperitoneais , Isotiocianatos/química , Melanoma/patologia , Camundongos , Camundongos Nus , Naftalimidas/farmacologia , Transplante de Neoplasias , Neoplasias Cutâneas/patologia , Relação Estrutura-Atividade , Tioureia/químicaRESUMO
Many forms of cellular stress cause an elevation of endogenous ceramide levels leading to growth arrest or apoptosis. Ceramidases (CDase) play a critical role in regulating apoptosis by hydrolyzing ceramide into sphingosine, a precursor for promitogenic sphingosine-1-phosphate. Growth factor induction of neutral CDase (nCDase) has been shown to have a cytoprotective effect against cytokine-induced increases in ceramide levels. To further define the physiological regulation of nCDase, we identified a 200 bp promoter region and demonstrated that serum activated this proximal promoter, which correlated with a serum-induced increase in human nCDase mRNA expression. Computational analysis revealed a putative cis-element for AP-1, a transcription factor activated by serum. Electrophoretic mobility shift assays demonstrated that the identified transcriptional response element binds to AP-1 transcription factors. RNA interference-mediated knockdown of the AP-1 subunit, c-Jun, inhibited the activity of the human nCDase proximal promoter, whereas, c-Jun overexpression increased promoter activity, which directly correlated with human nCDase mRNA transcription, decreased ceramide mass, and protection against caspase 3/7-dependent apoptosis. Taken together, our findings suggest that c-Jun/AP-1 signaling may, in part, regulate serum-induced human nCDase gene transcription.
Assuntos
Ceramidase Neutra/genética , Fator de Transcrição AP-1/metabolismo , Apoptose/genética , Apoptose/fisiologia , Sequência de Bases , Sítios de Ligação/genética , Ceramidas/metabolismo , Meios de Cultura , Primers do DNA/genética , Regulação Enzimológica da Expressão Gênica , Técnicas de Silenciamento de Genes , Genes fos , Células HEK293 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Regiões Promotoras Genéticas , Ligação Proteica , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos Reguladores de Transcrição , Transdução de Sinais , Fator de Transcrição AP-1/antagonistas & inibidores , Fator de Transcrição AP-1/genéticaRESUMO
The sphingolipid metabolic pathway represents a potential source of new therapeutic targets for numerous hyperproliferative/inflammatory diseases. Targets such as the sphingosine kinases (SphKs) have been extensively studied and numerous strategies have been employed to develop inhibitors against these enzymes. Herein, we report on the optimization of our novel small-molecule inhibitor SKI-I (N'-[(2-hydroxy-1-naphthyl)methylene]-3-(2-naphthyl)-1H-pyrazole-5-carbohydrazide) and the identification of a SphK1-specific analog, SKI-178, that is active in vitro and in vivo. This SphK1 specific small-molecule, non-lipid like, inhibitor will be of use to elucidate the roles of SphK1 and SphK2 in the development/progression of hyperproliferative and/or inflammatory diseases.