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Regulated necrosis, termed necroptosis, represents a potential therapeutic target for refractory cancer. Ceramide nanoliposomes (CNLs), considered potential chemotherapeutic agents, induce necroptosis by targeting the activating protein mixed lineage kinase domain-like protein (MLKL). In the present study, we examined the potential of pronecroptotic therapy using CNLs for refractory triple-negative breast cancer (TNBC), for which there is a lack of definite and effective therapeutic targets among the various immunohistological subtypes of breast cancer. MLKL mRNA expression in tumor tissues was significantly higher in TNBC patients than in those with non-TNBC subtypes. Similarly, among the 50 breast cancer cell lines examined, MLKL expression was higher in TNBC-classified cell lines. TNBC cell lines were more susceptible to the therapeutic effects of CNLs than the non-TNBC subtypes of breast cancer cell lines. In TNBC-classified MDA-MB-231 cells, the knockdown of MLKL suppressed cell death induced by CNLs or the active substance short-chain C6-ceramide. Accordingly, TNBC cells were prone to CNL-evoked necroptotic cell death. These results will contribute to the development of CNL-based pronecroptotic therapy for TNBC.
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Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/patologia , Linhagem Celular Tumoral , Apoptose , Necrose , Ceramidas/farmacologiaRESUMO
Cachexia is a life-threatening disease characterized by chronic, inflammatory muscle wasting and systemic metabolic impairment. Despite its high prevalence, there are no efficacious therapies for cachexia. Mice chronically infected with the protozoan parasite Toxoplasma gondii represent a novel animal model recapitulating the chronic kinetics of cachexia. To understand how perturbations to metabolic tissue homeostasis influence circulating metabolite availability we used mass spectrometry analysis. Despite the significant reduction in circulating triacylglycerides, non-esterified fatty acids, and glycerol, sphingolipid long-chain bases and a subset of phosphatidylcholines (PCs) were significantly increased in the sera of mice with T. gondii infection-induced cachexia. In addition, the TCA cycle intermediates α-ketoglutarate, 2-hydroxyglutarate, succinate, fumarate, and malate were highly depleted in cachectic mouse sera. Sphingolipids and their de novo synthesis precursors PCs are the major components of the mitochondrial membrane and regulate mitochondrial function consistent with a causal relationship in the energy imbalance driving T. gondii-induced chronic cachexia.
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Sparse scan partial thermal ablation (TA) with focused ultrasound (FUS) may be deployed to treat solid tumors and increase delivery of systemically administered therapeutics. Furthermore, C6-ceramide-loaded nanoliposomes (CNLs), which rely upon the enhanced-permeation and retention (EPR) effect for delivery, have shown promise for treating solid tumors and are being tested in clinical trials. Here, our objective was to determine whether CNLs synergize with TA in the control of 4T1 breast tumors. CNL monotherapy of 4T1 tumors yielded significant intratumoral bioactive C6 accumulation by the EPR effect, but tumor growth was not controlled. TA increased bioactive C6 accumulation by ~ 12.5-fold over the EPR effect. In addition, TA + CNL caused shifts in long-chain to very-long-chain ceramide ratios (i.e., C16/24 and C18/C24) that could potentially contribute to tumor control. Nonetheless, these changes in intratumoral ceramide levels were still insufficient to confer tumor growth control beyond that achieved when combining with TA with control "ghost" nanoliposomes (GNL). While this lack of synergy could be due to increased "pro-tumor" sphingosine-1-phosphate (S1P) levels, this is unlikely because S1P levels exhibited only a moderate and statistically insignificant increase with TA + CNL. In vitro studies showed that 4T1 cells are highly resistant to C6, offering the most likely explanation for the inability of TA to synergize with CNL. Thus, while our results show that sparse scan TA is a powerful approach for markedly enhancing CNL delivery and generating "anti-tumor" shifts in long-chain to very-long-chain ceramide ratios, resistance of the tumor to C6 can still be a rate-limiting factor for some solid tumor types.
Assuntos
Ceramidas , Neoplasias , Humanos , Ceramidas/uso terapêutico , Neoplasias/tratamento farmacológico , EsfingosinaRESUMO
Targeted therapies such as venetoclax (VEN) (Bcl-2 inhibitor) have revolutionized the treatment of chronic lymphocytic leukemia (CLL). We previously reported that persister CLL cells in treated patients overexpress multiple antiapoptotic proteins and display resistance to proapoptotic agents. Here, we demonstrated that multidrug-resistant CLL cells in vivo exhibited apoptosis restriction at a pre-mitochondrial level due to insufficient activation of the Bax and Bak (Bax/Bak) proteins. Co-immunoprecipitation analyses with selective BH domain antagonists revealed that the pleiotropic proapoptotic protein (Bim) was prevented from activating Bax/Bak by "switching" interactions to other upregulated antiapoptotic proteins (Mcl-1, Bcl-xL, Bcl-2). Hence, treatments that bypass Bax/Bak restriction are required to deplete these resistant cells in patients. Protein phosphatase 2A (PP2A) contributes to oncogenesis and treatment resistance. We observed that small-molecule activator of PP2A (SMAP) induced cytotoxicity in multiple cancer cell lines and CLL samples, including multidrug-resistant leukemia and lymphoma cells. The SMAP (DT-061) activated apoptosis in multidrug-resistant CLL cells through induction of mitochondrial permeability transition pores, independent of Bax/Bak. DT-061 inhibited the growth of wild-type and Bax/Bak double-knockout, multidrug-resistant CLL cells in a xenograft mouse model. Collectively, we discovered multidrug-resistant CLL cells in patients and validated a pharmacologically tractable pathway to deplete this reservoir.
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Leucemia Linfocítica Crônica de Células B , Humanos , Animais , Camundongos , Proteína X Associada a bcl-2/metabolismo , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteína Fosfatase 2/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/metabolismo , Resistência a Múltiplos MedicamentosRESUMO
Acute myeloid leukemia (AML) is an aggressive disease with complex and heterogeneous biology. Although several genomic classifications have been proposed, there is a growing interest in going beyond genomics to stratify AML. In this study, we profile the sphingolipid family of bioactive molecules in 213 primary AML samples and 30 common human AML cell lines. Using an integrative approach, we identify two distinct sphingolipid subtypes in AML characterized by a reciprocal abundance of hexosylceramide (Hex) and sphingomyelin (SM) species. The two Hex-SM clusters organize diverse samples more robustly than known AML driver mutations and are coupled to latent transcriptional states. Using transcriptomic data, we develop a machine-learning classifier to infer the Hex-SM status of AML cases in TCGA and BeatAML clinical repositories. The analyses show that the sphingolipid subtype with deficient Hex and abundant SM is enriched for leukemic stemness transcriptional programs and comprises an unappreciated high-risk subgroup with poor clinical outcomes. Our sphingolipid-focused examination of AML identifies patients least likely to benefit from standard of care and raises the possibility that sphingolipidomic interventions could switch the subtype of AML patients who otherwise lack targetable alternatives.
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Sparse scan partial thermal ablation (TA) with focused ultrasound (FUS) may be deployed to treat solid tumors and increase delivery of systemically administered therapeutics. Further, C6-ceramide-loaded nanoliposomes (CNLs), which rely upon the enhanced permeation and retention (EPR) effect for delivery, have shown promise for treating solid tumors and are being tested in clinical trials. Here, our objective was to determine whether CNLs synergize with TA in the control of 4T1 breast tumors. CNL-monotherapy of 4T1 tumors yielded significant intratumoral bioactive C6 accumulation by the EPR effect, but tumor growth was not controlled. TA increased bioactive C6 accumulation by â¼12.5-fold over the EPR effect. In addition, TA+CNL caused shifts in long-chain to very-long-chain ceramide ratios (i.e., C16/24 and C18/C24) that could potentially contribute to tumor control. Nonetheless, these changes in intratumoral ceramide levels were still insufficient to confer tumor growth control beyond that achieved when combining with TA with control "ghost" nanoliposomes (GNL). While this lack of synergy could be due to increased "pro-tumor" sphingosine-1-phosphate (S1P) levels, this is unlikely because S1P levels exhibited only a moderate and statistically insignificant increase with TA+CNL. In vitro studies showed that 4T1 cells are highly resistant to C6, offering the most likely explanation for the inability of TA to synergize with CNL. Thus, while our results show that sparse scan TA is a powerful approach for markedly enhancing CNL delivery and generating "anti-tumor" shifts in long-chain to very-long-chain ceramide ratios, resistance of the tumor to C6 can still be a rate-limiting factor for some solid tumor types.
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Acute myelogenous leukemia (AML), the most prevalent acute and aggressive leukemia diagnosed in adults, often recurs as a difficult-to-treat, chemotherapy-resistant disease. Because chemotherapy resistance is a major obstacle to successful treatment, novel therapeutic intervention is needed. Upregulated ceramide clearance via accelerated hydrolysis and glycosylation has been shown to be an element in chemotherapy-resistant AML, a problem considering the crucial role ceramide plays in eliciting apoptosis. Herein we employed agents that block ceramide clearance to determine if such a "reset" would be of therapeutic benefit. SACLAC was utilized to limit ceramide hydrolysis, and D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-threo-PDMP) was used to block the glycosylation route. The SACLAC D-threo-PDMP inhibitor combination was synergistically cytotoxic in drug-resistant, P-glycoprotein-expressing (P-gp) AML but not in wt, P-gp-poor cells. Interestingly, P-gp antagonists that can limit ceramide glycosylation via depression of glucosylceramide transit also synergized with SACLAC, suggesting a paradoxical role for P-gp in the implementation of cell death. Mechanistically, cell death was accompanied by a complete drop in ceramide glycosylation, concomitant, striking increases in all molecular species of ceramide, diminished sphingosine 1-phosphate levels, resounding declines in mitochondrial respiratory kinetics, altered Akt, pGSK-3ß, and Mcl-1 expression, and caspase activation. Although ceramide was generated in wt cells upon inhibitor exposure, mitochondrial respiration was not corrupted, suggestive of mitochondrial vulnerability in the drug-resistant phenotype, a potential therapeutic avenue. The inhibitor regimen showed efficacy in an in vivo model and in primary AML cells from patients. These results support the implementation of SL enzyme targeting to limit ceramide clearance as a therapeutic strategy in chemotherapy-resistant AML, inclusive of a novel indication for the use of P-gp antagonists.
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Ceramides are an emerging class of anti-inflammatory lipids, and nanoscale ceramide-delivery systems are potential therapeutic strategies for inflammatory diseases. This study investigated the therapeutic effects of ceramide nanoliposomes (CNL) on type 2 inflammation-based asthma, induced by repeated ovalbumin (OVA) challenges. Asthmatic mice intratracheally treated with ceramide-free liposomes (Ghost) displayed typical airway remodeling including mucosal accumulation and subepithelial fibrosis, whereas, in CNL-treated mice, the degree of airway remodeling was significantly decreased. Compared to the Ghost group, CNL treatment unexpectedly failed to significantly influence formation of type 2 cytokines, including IL-5 and IL-13, known to facilitate pathogenic production of airway mucus predominantly comprising MUC5AC mucin. Interestingly, CNL treatment suppressed OVA-evoked hyperplasia of MUC5AC-generating goblet cells in the airways. This suggests that CNL suppressed goblet cell hyperplasia and airway mucosal accumulation independently of type 2 cytokine formation. Mechanistically, CNL treatment suppressed cell growth and EGF-induced activation of Akt, but not ERK1/2, in a human lung epithelial cell culture system recapitulating airway goblet cell hyperplasia. Taken together, CNL is suggested to have therapeutic effects on airway remodeling in allergic asthma by targeting goblet cell hyperplasia. These findings raise the potential of ceramide-based therapies for airway diseases, such as asthma.
Assuntos
Antineoplásicos , Asma , Humanos , Animais , Camundongos , Hiperplasia/patologia , Remodelação das Vias Aéreas , Líquido da Lavagem Broncoalveolar , Asma/patologia , Pulmão/patologia , Citocinas/farmacologia , Antineoplásicos/farmacologiaRESUMO
Classical MCL (cMCL) constitutes 6-8% of all B cell NHL. Despite recent advances, MCL is incurable except with allogeneic stem cell transplant. Blastic mantle cell lymphoma (bMCL) is a rarer subtype of cMCL associated with an aggressive clinical course and poor treatment response, frequent relapse and poor outcomes. We treated 13 bMCL patients with combined epigenetic and immunotherapy treatment consisting of vorinostat, cladribine and rituximab (SCR). We report an increased OS greater than 40 months with several patients maintaining durable remissions without relapse for longer than 5 years. This is remarkably better then current treatment regimens which in bMCL range from 14.5-24 months with conventional chemotherapy regimens. We demonstrate that the G/A870 CCND1 polymorphism is predictive of blastic disease, nuclear localization of cyclinD1 and response to SCR therapy. The major resistance mechanisms to SCR therapy are loss of CD20 expression and evasion of treatment by sanctuary in the CNS. These data indicate that administration of epigenetic agents improves efficacy of anti-CD20 immunotherapies. This approach is promising in the treatment of MCL and potentially other previously treatment refractory cancers.
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Epigênese Genética , Imunoterapia , Linfoma de Célula do Manto , Adulto , Antígenos CD20/imunologia , Cladribina , Humanos , Fatores Imunológicos/uso terapêutico , Linfoma de Célula do Manto/tratamento farmacológico , Linfoma de Célula do Manto/terapia , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/terapia , Rituximab/uso terapêutico , Vorinostat/uso terapêuticoRESUMO
Aim: Despite the huge advancements in cancer therapies and treatments over the past decade, most patients with metastasized melanoma still die from the disease. This poor prognosis largely results from resistance to conventional chemotherapies and other cytotoxic drugs. We have previously identified 6 antigenic peptides derived from melanomas that have proven efficacious for activating CD4+ T cells in clinical trials for melanoma. Our aim was to improve pharmacodynamics, pharmacokinetic and toxicological parameters by individually encapsulating each of the 6 melanoma helper peptides within their own immunogenic nanoliposomes. Methods: We modified these liposomes as necessary to account for differences in the peptides' chemical properties, resulting in 3 distinct formulations. To further enhance immunogenicity, we also incorporated KDO2, a TLR4 agonist, into the lipid bilayer of all nanoliposome formulations. We then conducted in vivo imaging studies in mice and ex vivo cell studies from 2 patient samples who both strongly expressed one of the identified peptides. Results: We demonstrate that these liposomes, loaded with the different melanoma helper peptides, can be readily mixed together and simultaneously delivered without toxicity in vivo. These liposomes are capable of being diffused to the secondary lymphoid organs very quickly and for at least 6 days. In addition, we show that these immunogenic liposomes enhance immune responses to specific peptides ex vivo. Conclusion: Lipid-based delivery systems, including nanoliposomes and lipid nanoparticles, have now been validated for pharmacological (small molecules, bioactive lipids) and molecular (mRNA, siRNA) therapeutic approaches. However, the utility of these formulations as cancer vaccines, delivering antigenic peptides, has not yet achieved the same degree of commercial success. Here, we describe the novel and successful development of a nanoliposome-based cancer vaccine for melanoma. These vaccines help to circumvent drug resistance by increasing a patient's T cell response, making them more susceptible to checkpoint blockade therapy.
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Despite several new therapeutic options for acute myeloid leukemia (AML), disease relapse remains a significant challenge. We have previously demonstrated that augmenting ceramides can counter various drug-resistance mechanisms, leading to enhanced cell death in cancer cells and extended survival in animal models. Using a nanoscale delivery system for ceramide (ceramide nanoliposomes, CNL), we investigated the effect of CNL within a standard of care venetoclax/cytarabine (Ara-C) regimen. We demonstrate that CNL augmented the efficacy of venetoclax/cytarabine in in vitro, ex vivo, and in vivo models of AML. CNL treatment induced non-apoptotic cytotoxicity, and augmented cell death induced by Ara-C and venetoclax. Mechanistically, CNL reduced both venetoclax (Mcl-1) and cytarabine (Chk1) drug-resistant signaling pathways. Moreover, venetoclax and Ara-C augmented the generation of endogenous pro-death ceramide species, which was intensified with CNL. Taken together, CNL has the potential to be utilized as an adjuvant therapy to improve outcomes, potentially extending survival, in patients with AML.
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Antineoplásicos , Leucemia Mieloide Aguda , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Ceramidas , Citarabina/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , SulfonamidasRESUMO
Acute myeloid leukemia (AML) is an aggressive, heterogenous malignancy characterized by clonal expansion of bone marrow-derived myeloid progenitor cells. While our current understanding of the molecular and genomic landscape of AML has evolved dramatically and opened avenues for molecularly targeted therapeutics to improve upon standard intensive induction chemotherapy, curative treatments are elusive, particularly in older patients. Responses to current AML treatments are transient and incomplete, necessitating the development of novel treatment strategies to improve outcomes. To this end, harnessing the power of bioactive sphingolipids to treat cancer shows great promise. Sphingolipids are involved in many hallmarks of cancer of paramount importance in AML. Leukemic blast survival is influenced by cellular levels of ceramide, a bona fide pro-death molecule, and its conversion to signaling molecules such as sphingosine-1-phosphate and glycosphingolipids. Preclinical studies demonstrate the efficacy of therapeutics that target dysregulated sphingolipid metabolism as well as their combinatorial synergy with clinically-relevant therapeutics. Thus, increased understanding of sphingolipid dysregulation may be exploited to improve AML patient care and outcomes. This review summarizes the current knowledge of dysregulated sphingolipid metabolism in AML, evaluates how pro-survival sphingolipids promote AML pathogenesis, and discusses the therapeutic potential of targeting these dysregulated sphingolipid pathways.
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Leucemia Mieloide Aguda , Esfingolipídeos , Idoso , Ceramidas/metabolismo , Ceramidas/uso terapêutico , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/terapia , Transdução de Sinais , Esfingolipídeos/metabolismo , Esfingolipídeos/uso terapêuticoRESUMO
Combination therapy represents an effective therapeutic approach to overcome hepatocellular cancer (HCC) resistance to immune checkpoint blockade (ICB). Based upon previous work demonstrating that nanoliposome C6-ceramide (LipC6) not only induces HCC apoptosis but also prevents HCC-induced immune tolerance, we now investigate the potential of LipC6 in combination with ICB in HCC treatment. We generated orthotopic HCC-bearing mice, which have typical features in common with human patients, and then treated them with LipC6 in combination with the antibodies (Abs) for programmed cell death protein 1 (PD-1) or cytotoxic T-lymphocyte antigen 4 (CTLA4). The tumor growth was monitored by magnetic resonance imaging (MRI) and the intrahepatic immune profiles were checked by flow cytometry in response to the treatments. Realtime PCR (qPCR) was used to detect the expression of target genes. The results show that LipC6 in combination with anti-CTLA4 Ab, but not anti-PD-1 Ab, significantly slowed tumor growth, enhanced tumor-infiltrating CD8+ T cells, and suppressed tumor-resident CD4+ CD25+ FoxP3+ Tregs. Further molecular investigation indicates that the combinational treatment suppressed transcriptional factor Krüppel-like Factor 2 (KLF2), forkhead box protein P3 (FoxP3), and CTLA4. Our studies suggest that LipC6 in combination with anti-CTLA4 Ab represents a novel therapeutic approach with significant potential in activating anti-HCC immune response and suppressing HCC growth.
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Antineoplásicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Antineoplásicos/farmacologia , Linfócitos T CD8-Positivos , Antígeno CTLA-4 , Carcinoma Hepatocelular/metabolismo , Ceramidas , Fatores de Transcrição Forkhead/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , CamundongosRESUMO
Modifications in sphingolipid (SL) metabolism and mitochondrial bioenergetics are key factors implicated in cancer cell response to chemotherapy, including chemotherapy resistance. In the present work, we utilized acute myeloid leukemia (AML) cell lines, selected to be refractory to various chemotherapeutics, to explore the interplay between SL metabolism and mitochondrial biology supportive of multidrug resistance (MDR). In agreement with previous findings in cytarabine or daunorubicin resistant AML cells, relative to chemosensitive wildtype controls, HL-60 cells refractory to vincristine (HL60/VCR) presented with alterations in SL enzyme expression and lipidome composition. Such changes were typified by upregulated expression of various ceramide detoxifying enzymes, as well as corresponding shifts in ceramide, glucosylceramide, and sphingomyelin (SM) molecular species. With respect to mitochondria, despite consistent increases in both basal respiration and maximal respiratory capacity, direct interrogation of the oxidative phosphorylation (OXPHOS) system revealed intrinsic deficiencies in HL60/VCR, as well as across multiple MDR model systems. Based on the apparent requirement for augmented SL and mitochondrial flux to support the MDR phenotype, we explored a combinatorial therapeutic paradigm designed to target each pathway. Remarkably, despite minimal cytotoxicity in peripheral blood mononuclear cells (PBMC), co-targeting SL metabolism, and respiratory complex I (CI) induced synergistic cytotoxicity consistently across multiple MDR leukemia models. Together, these data underscore the intimate connection between cellular sphingolipids and mitochondrial metabolism and suggest that pharmacological intervention across both pathways may represent a novel treatment strategy against MDR.
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Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Leucemia/metabolismo , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Esfingolipídeos/metabolismo , Citarabina/farmacologia , Daunorrubicina/farmacologia , Células HL-60 , Humanos , Leucemia/patologia , Mitocôndrias/patologia , Vincristina/farmacologiaRESUMO
The poor prognosis of acute myeloid leukemia (AML) and the highly heterogenous nature of the disease motivates targeted gene therapeutic investigations. Rho-associated protein kinases (ROCKs) are crucial for various actin cytoskeletal changes, which have established malignant consequences in various cancers, yet are still not being successfully utilized clinically towards cancer treatment. This work establishes the therapeutic activity of ROCK inhibitor (5Z)-2-5-(1H-pyrrolo[2,3-b]pyridine-3-ylmethylene)-1,3-thiazol-4(5H)-one (DJ4) in both in vitro and in vivo preclinical models of AML to highlight the potential of this class of inhibitors. Herein, DJ4 induced cytotoxic and proapoptotic effects in a dose-dependent manner in human AML cell lines (IC50: 0.05-1.68 µM) and primary patient cells (IC50: 0.264-13.43 µM); however, normal hematopoietic cells were largely spared. ROCK inhibition by DJ4 disrupts the phosphorylation of downstream targets, myosin light chain (MLC2) and myosin-binding subunit of MLC phosphatase (MYPT), yielding a potent yet selective treatment response at micromolar concentrations, from 0.02 to 1 µM. Murine models injected with luciferase-expressing leukemia cell lines subcutaneously or intravenously and treated with DJ4 exhibited an increase in overall survival and reduction in disease progression relative to the vehicle-treated control mice. Overall, DJ4 is a promising candidate to utilize in future investigations to advance the current AML therapy.
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Anti-androgens are a common therapy in prostate cancer (PCa) targeting androgen receptor (AR) signaling. However, these therapies fail due to selection of highly aggressive AR-negative cancer cells that have no therapeutic options available. We demonstrate that elevating endogenous ceramide levels with administration of exogenous ceramide nanoliposomes (CNLs) was efficacious in AR-negative cell lines with limited efficacy in AR-positive cells. This effect is mediated through reduced de novo sphingolipid synthesis in AR-positive cells. We show that anti-androgens elevate de novo generation of sphingolipids via SPTSSB, a rate-limiting mediator of sphingolipid generation. Moreover, pharmacological inhibition of AR increases the efficacy of CNL in AR-positive cells through de novo synthesis, while SPTSSB knockdown limited CNL's efficacy in AR-negative cells. Alluding to clinical relevance, SPTSSB is upregulated in patients with advanced PCa after anti-androgens treatment. These findings emphasize the relevance of AR regulation upon sphingolipid metabolism and the potential of CNL as a PCa therapeutic.
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Therapies for head and neck squamous cell carcinoma (HNSCC) are, at best, moderately effective, underscoring the need for new therapeutic strategies. Ceramide treatment leads to cell death as a consequence of mitochondrial damage by generating oxidative stress and causing mitochondrial permeability. However, HNSCC cells are able to resist cell death through mitochondria repair via mitophagy. Through the use of the C6-ceramide nanoliposome (CNL) to deliver therapeutic levels of bioactive ceramide, we demonstrate that the effects of CNL are mitigated in drug-resistant HNSCC via an autophagic/mitophagic response. We also demonstrate that inhibitors of lysosomal function, including chloroquine (CQ), significantly augment CNL-induced death in HNSCC cell lines. Mechanistically, the combination of CQ and CNL results in dysfunctional lysosomal processing of damaged mitochondria. We further demonstrate that exogenous addition of methyl pyruvate rescues cells from CNL + CQ-dependent cell death by restoring mitochondrial functionality via the reduction of CNL- and CQ-induced generation of reactive oxygen species and mitochondria permeability. Taken together, inhibition of late-stage protective autophagy/mitophagy augments the efficacy of CNL through preventing mitochondrial repair. Moreover, the combination of inhibitors of lysosomal function with CNL may provide an efficacious treatment modality for HNSCC.
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Ceramidas/administração & dosagem , Lipossomos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Mitofagia/efeitos dos fármacos , Nanopartículas , Trifosfato de Adenosina/metabolismo , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Citometria de Fluxo , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Piruvatos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
Enterotoxigenic Bacteroides fragilis (ETBF) is a commensal bacterium of great importance to human health due to its ability to induce colitis and cause colon tumor formation in mice through the production of B. fragilis toxin (BFT). The formation of tumors is dependent on a pro-inflammatory signaling cascade, which begins with the disruption of epithelial barrier integrity through cleavage of E-cadherin. Here, we show that BFT increases levels of glucosylceramide, a vital intestinal sphingolipid, both in mice and in colon organoids (colonoids) generated from the distal colons of mice. When colonoids are treated with BFT in the presence of an inhibitor of glucosylceramide synthase (GCS), the enzyme responsible for generating glucosylceramide, colonoids become highly permeable, lose structural integrity, and eventually burst, releasing their contents into the extracellular matrix. By increasing glucosylceramide levels in colonoids via an inhibitor of glucocerebrosidase (GBA, the enzyme that degrades glucosylceramide), colonoid permeability was reduced, and bursting was significantly decreased. In the presence of BFT, pharmacological inhibition of GCS caused levels of tight junction protein 1 (TJP1) to decrease. However, when GBA was inhibited, TJP1 levels remained stable, suggesting that BFT-induced production of glucosylceramide helps to stabilize tight junctions. Taken together, our data demonstrate a glucosylceramide-dependent mechanism by which the colon epithelium responds to BFT.
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Toxinas Bacterianas/toxicidade , Bacteroides fragilis/metabolismo , Colo/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Glucosilceramidas/metabolismo , Metaloendopeptidases/toxicidade , Transdução de Sinais/efeitos dos fármacos , Animais , Colite/induzido quimicamente , Colite/metabolismo , Colo/metabolismo , Células Epiteliais/metabolismo , Glucosilceramidase/metabolismo , Glucosiltransferases/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Permeabilidade/efeitos dos fármacos , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
In non-alcoholic steatohepatitis (NASH), many lines of investigation have reported a dysregulation in lipid homeostasis, leading to intrahepatic lipid accumulation. Recently, the role of dysfunctional sphingolipid metabolism has also been proposed. Human and animal models of NASH have been associated with elevated levels of long chain ceramides and pro-apoptotic sphingolipid metabolites, implicated in regulating fatty acid oxidation and inflammation. Importantly, inhibition of de novo ceramide biosynthesis or knock-down of ceramide synthases reverse some of the pathology of NASH. In contrast, cell permeable, short chain ceramides have shown anti-inflammatory actions in multiple models of inflammatory disease. Here, we investigated non-apoptotic doses of a liposome containing short chain C6-Ceramide (Lip-C6) administered to human hepatic stellate cells (hHSC), a key effector of hepatic fibrogenesis, and an animal model characterized by inflammation and elevated liver fat content. On the basis of the results from unbiased liver transcriptomic studies from non-alcoholic fatty liver disease patients, we chose to focus on adenosine monophosphate activated kinase (AMPK) and nuclear factor-erythroid 2-related factor (Nrf2) signaling pathways, which showed an abnormal profile. Lip-C6 administration inhibited hHSC proliferation while improving anti-oxidant protection and energy homeostasis, as indicated by upregulation of Nrf2, activation of AMPK and an increase in ATP. To confirm these in vitro data, we investigated the effect of a single tail-vein injection of Lip-C6 in the methionine-choline deficient (MCD) diet mouse model. Lip-C6, but not control liposomes, upregulated phospho-AMPK, without inducing liver toxicity, apoptosis, or exacerbating inflammatory signaling pathways. Alluding to mechanism, mass spectrometry lipidomics showed that Lip-C6-treatment reversed the imbalance in hepatic phosphatidylcholines and diacylglycerides species induced by the MCD-fed diet. These results reveal that short-term Lip-C6 administration reverses energy/metabolic depletion and increases protective anti-oxidant signaling pathways, possibly by restoring homeostatic lipid function in a model of liver inflammation with fat accumulation.