RESUMO
Pancreas ductal adenocarcinoma (PDAC) remains a malignant tumor with very poor prognosis and low 5-year overall survival. Here, we aimed to simultaneously target mitochondria and lysosomes as a new treatment paradigm of malignant pancreas cancer in vitro and in vivo. We demonstrate that the clinically used sphingosine analog FTY-720 together with PAPTP, an inhibitor of mitochondrial Kv1.3, induce death of pancreas cancer cells in vitro and in vivo. The combination of both drugs results in a marked inhibition of the acid sphingomyelinase and accumulation of cellular sphingomyelin in vitro and in vivo in orthotopic and flank pancreas cancers. Mechanistically, PAPTP and FTY-720 cause a disruption of both mitochondria and lysosomes, an alteration of mitochondrial bioenergetics and accumulation of cytoplasmic Ca2+, events that collectively mediate cell death. Our findings point to an unexpected cross-talk between lysosomes and mitochondria mediated by sphingolipid metabolism. We show that the combination of PAPTP and FTY-720 induces massive death of pancreas cancer cells, thereby leading to a substantially delayed and reduced PDAC growth in vivo. KEY MESSAGES: FTY-720 inhibits acid sphingomyelinase in pancreas cancer cells (PDAC). FTY-720 induces sphingomyelin accumulation and lysosomal dysfunction. The mitochondrial Kv1.3 inhibitor PAPTP disrupts mitochondrial functions. PAPTP and FTY-720 synergistically kill PDAC in vitro. The combination of FTY-720 and PAPTP greatly delays PDAC growth in vivo.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Esfingomielina Fosfodiesterase , Esfingomielinas/metabolismo , Cloridrato de Fingolimode , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Linhagem Celular Tumoral , Ductos Pancreáticos/metabolismo , Ductos Pancreáticos/patologia , Neoplasias PancreáticasRESUMO
Despite several new developments in the treatment of multiple myeloma, all available therapies are only palliative without curative potential and all patients ultimately relapse. Thus, novel therapeutic options are urgently required to prolong survival of or to even cure myeloma. Here, we show that multiple myeloma cells express the potassium channel Kv1.3 in their mitochondria. The mitochondrial Kv1.3 inhibitors PAPTP and PCARBTP are efficient against two tested human multiple myeloma cell lines (L-363 and RPMI-8226) and against ex vivo cultured, patient-derived myeloma cells, while healthy bone marrow cells are spared from toxicity. Cell death after treatment with PAPTP and PCARBTP occurs via the mitochondrial apoptotic pathway. In addition, we identify up-regulation of the multidrug resistance pump MDR-1 as the main potential resistance mechanism. Combination with ABT-199 (venetoclax), an inhibitor of Bcl2, has a synergistic effect, suggesting that mitochondrial Kv1.3 inhibitors could potentially be used as combination partner to venetoclax, even in the treatment of t(11;14) negative multiple myeloma, which represent the major part of cases and are rather resistant to venetoclax alone. We thus identify mitochondrial Kv1.3 channels as druggable targets against multiple myeloma.
RESUMO
The potassium channel Kv1.3, involved in several important pathologies, is the target of a family of psoralen-based drugs whose mechanism of action is not fully understood. Here we provide evidence for a physical interaction of the mitochondria-located Kv1.3 (mtKv1.3) and Complex I of the respiratory chain and show that this proximity underlies the death-inducing ability of psoralenic Kv1.3 inhibitors. The effects of PAP-1-MHEG (PAP-1, a Kv1.3 inhibitor, with six monomeric ethylene glycol units attached to the phenyl ring of PAP-1), a more soluble novel derivative of PAP-1 and of its various portions on mitochondrial physiology indicate that the psoralenic moiety of PAP-1 bound to mtKv1.3 facilitates the diversion of electrons from Complex I to molecular oxygen. The resulting massive production of toxic Reactive Oxygen Species leads to death of cancer cells expressing Kv1.3. In vivo, PAP-1-MHEG significantly decreased melanoma volume. In summary, PAP-1-MHEG offers insights into the mechanisms of cytotoxicity of this family of compounds and may represent a valuable clinical tool.
Assuntos
Canal de Potássio Kv1.3 , Mitocôndrias , Animais , Linhagem Celular Tumoral , Dissecação , Humanos , Canal de Potássio Kv1.3/antagonistas & inibidores , Canal de Potássio Kv1.3/genética , Camundongos Endogâmicos C57BL , Espécies Reativas de OxigênioRESUMO
Inhibition of acid sphingomyelinase (ASM), a lysosomal enzyme that catalyzes the hydrolysis of sphingomyelin into ceramide and phosphorylcholine, may serve as an investigational tool or a therapeutic intervention to control many diseases. Specific ASM inhibitors are currently not sufficiently characterized. Here, we found that 1-aminodecylidene bis-phosphonic acid (ARC39) specifically and efficiently (>90%) inhibits both lysosomal and secretory ASM in vitro. Results from investigating sphingomyelin phosphodiesterase 1 (SMPD1/Smpd1) mRNA and ASM protein levels suggested that ARC39 directly inhibits ASM's catalytic activity in cultured cells, a mechanism that differs from that of functional inhibitors of ASM. We further provide evidence that ARC39 dose- and time-dependently inhibits lysosomal ASM in intact cells, and we show that ARC39 also reduces platelet- and ASM-promoted adhesion of tumor cells. The observed toxicity of ARC39 is low at concentrations relevant for ASM inhibition in vitro, and it does not strongly alter the lysosomal compartment or induce phospholipidosis in vitro. When applied intraperitoneally in vivo, even subtoxic high doses administered short-term induced sphingomyelin accumulation only locally in the peritoneal lavage without significant accumulation in plasma, liver, spleen, or brain. These findings require further investigation with other possible chemical modifications. In conclusion, our results indicate that ARC39 potently and selectively inhibits ASM in vitro and highlight the need for developing compounds that can reach tissue concentrations sufficient for ASM inhibition in vivo.
Assuntos
Inibidores Enzimáticos/farmacologia , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Linhagem Celular , Humanos , Lisossomos/metabolismoRESUMO
BACKGROUND/AIMS: Glioblastoma (GBM) is one of the most aggressive cancers, counting for a high number of the newly diagnosed patients with central nervous system (CNS) cancers in the United States and Europe. Major features of GBM include aggressive and invasive growth as well as a high resistance to treatment. Kv1.3, a potassium channel of the shaker family, is expressed in the inner mitochondrial membrane of many cancer cells. Inhibition of mitochondrial Kv1.3 was shown to induce apoptosis in several tumor cells at doses that were not lethal for normal cells. METHODS: We investigated the expression of Kv1.3 in different glioma cell lines by immunocytochemistry, western blotting and electron microscopy and analyzed the effect of newly synthesized, mitochondria-targeted, Kv1.3 inhibitors on the induction of cell death in these cells. Finally, we performed in vivo studies on glioma bearing mice. RESULTS: Here, we report that Kv1.3 is expressed in mitochondria of human and murine GL261, A172 and LN308 glioma cells. Treatment with the novel Kv1.3 inhibitors PAPTP or PCARBTP as well as with clofazimine induced massive cell death in glioma cells, while Psora-4 and PAP-1 were almost without effect. However, in vivo experiments revealed that the drugs had no effect on orthotopic brain tumors in vivo. CONCLUSION: These data serve as proof of principle that Kv1.3 inhibitors kills GBM cells, but drugs that act in vivo against glioblastoma must be developed to translate these findings in vivo.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Canal de Potássio Kv1.3/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/farmacologia , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Clofazimina/farmacologia , Clofazimina/uso terapêutico , Cumarínicos/farmacologia , Cumarínicos/uso terapêutico , Humanos , Imuno-Histoquímica , Camundongos , Compostos Organofosforados/farmacologia , Compostos Organofosforados/uso terapêuticoRESUMO
The potassium channel Kv1.3 is highly expressed in the mitochondria of various cancerous cells. Here we show that direct inhibition of Kv1.3 using two mitochondria-targeted inhibitors alters mitochondrial function and leads to reactive oxygen species (ROS)-mediated death of even chemoresistant cells independently of p53 status. These inhibitors killed 98% of ex vivo primary chronic B-lymphocytic leukemia tumor cells while sparing healthy B cells. In orthotopic mouse models of melanoma and pancreatic ductal adenocarcinoma, the compounds reduced tumor size by more than 90% and 60%, respectively, while sparing immune and cardiac functions. Our work provides direct evidence that specific pharmacological targeting of a mitochondrial potassium channel can lead to ROS-mediated selective apoptosis of cancer cells in vivo, without causing significant side effects.
Assuntos
Antineoplásicos/farmacologia , Canal de Potássio Kv1.3/antagonistas & inibidores , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Bloqueadores dos Canais de Potássio/farmacologia , Idoso , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/patologia , Estudos de Casos e Controles , Cumarínicos/farmacologia , Estabilidade de Medicamentos , Feminino , Humanos , Canal de Potássio Kv1.3/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Melanoma/tratamento farmacológico , Melanoma/patologia , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Terapia de Alvo Molecular , Compostos Organofosforados/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Bloqueadores dos Canais de Potássio/síntese química , Bloqueadores dos Canais de Potássio/químicaRESUMO
Although chemotherapy is able to cure many patients with malignancies, it still also often fails. Therefore, novel approaches and targets for chemotherapeutic treatment of malignancies are urgently required. Recent studies demonstrated the expression of several potassium channels in the inner mitochondrial membrane. Among them the voltage gated potassium channel Kv1.3 and the big-potassium (BK) channel were shown to directly function in cell death by serving as target for pro-apoptotic Bax and Bak proteins. Here, we discuss the role of mitochondrial potassium channel Kv1.3 (mitoKv1.3) in cell death and its potential function in treatment of solid tumors, leukemia and lymphoma. Bax and Bak inhibit mitoKv1.3 by directly binding into the pore of the channel, by a toxin-like mechanism. Inhibition of mitoKv1.3 results in an initial hyperpolarization of the inner mitochondrial membrane that triggers the production of reactive oxygen species (ROS). ROS in turn induce a release of cytochrome c from the cristae of the inner mitochondrial membrane and an activation of the permeability transition pore, resulting in opening of the intrinsic apoptotic cell death. Since mitoKv1.3 functions downstream of pro-apoptotic Bax and Bak, compounds that directly inhibit mitoKv1.3 may serve as a new class of drugs for treatment of tumors, even with an altered expression of either pro- or anti-apoptotic Bcl-2 protein family members. This was successfully proven by the in vivo treatment of mouse melanoma and ex vivo human chronic leukemia B cells with inhibitors of mitoKv1.3.
Assuntos
Mitocôndrias/metabolismo , Canais de Potássio/metabolismo , Animais , Apoptose/efeitos dos fármacos , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Potássio/metabolismo , Bloqueadores dos Canais de Potássio/uso terapêutico , Bloqueadores dos Canais de Potássio/toxicidade , Canais de Potássio/química , Canais de Potássio/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Melanogenesis is the vital response to protect skin cells against UVB-induced DNA damage. Melanin is produced by melanocytes, which transfer it to surrounding keratinocytes. Recently, we have shown that the aryl hydrocarbon receptor (AhR) is part of the UVB-stress response in epidermal keratinocytes. UVB triggers AhR signaling by generating the AhR ligand 6-formylindolo(3,2-b)carbazole from tryptophan. We show here that normal murine melanocytes express functional AhR. Using standard UVB tanning protocols, AhR-deficient mice were shown to tan significantly weaker than wild-type mice; in these mice, tyrosinase activity in the epidermis was lower as well. Tanning responses and tyrosinase activity, however, were normal in keratinocyte-specific conditional AhR knockout mice, indicating that release of melanogenic keratinocyte factors is unaffected by the UVB-AhR signaling pathway and that the diminished tanning response in AhR(-/-) mice is confined to the level of melanocytes. Accordingly, the number of dihydroxyphenylalanin-positive melanocytes increased significantly less on UVB irradiation in AhR(-/-) mice than in wild-type mice. This difference in melanocyte number was associated with a significantly reduced expression of stem cell factor-1 and c-kit in melanocytes of AhR(-/-) mice. Thus, the environmental signal sensor AhR links solar UVB radiation to skin pigmentation.
Assuntos
Melanócitos , Receptores de Hidrocarboneto Arílico/fisiologia , Pigmentação da Pele/fisiologia , Pigmentação da Pele/efeitos da radiação , Raios Ultravioleta , Animais , Contagem de Células , Diferenciação Celular/fisiologia , Diferenciação Celular/efeitos da radiação , Células Cultivadas , Células Epidérmicas , Epiderme/fisiologia , Epiderme/efeitos da radiação , Queratinócitos/citologia , Queratinócitos/fisiologia , Queratinócitos/efeitos da radiação , Melaninas/metabolismo , Melanócitos/citologia , Melanócitos/fisiologia , Melanócitos/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Hidrocarboneto Arílico/genéticaRESUMO
The toxic environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent immunomodulatory chemical. TCDD activates the aryl hydrocarbon receptor (AhR) and suppresses peripheral humoral and cellular adaptive immune responses. Though the major route of uptake is via food, little is known until now on the immunotoxic effects of TCDD on the gut-associated lymphoid tissue. We show here that AhR is strongly expressed along the small intestine, especially in intestinal epithelial cells (IEC). The AhR marker gene cyp1a1 is induced in IEC by oral TCDD exposure. We asked how TCDD affects oral tolerance, a unique function of mucosal immunity. C57BL/6 mice were injected with 10 µg/kg body weight TCDD and fed with ovalbumin (OVA) in a high-dose tolerization protocol. Mice were immunized and boosted with OVA on days 12, 23, and 55 after tolerization. Five of 14, 6 of 15, and 13 of 14 TCDD-treated mice generated OVA-specific immunoglobulin (Ig)G1 antibodies after the first, second, and third immunization with OVA, respectively. Only one mouse harbored anti-OVA IgG1 antibodies in the control group even after the third immunization with OVA. OVA-specific IgA in fecal samples of tolerized and TCDD-exposed mice could be detected at the levels of nontolerized mice, whereas completely absent in tolerant control mice. Correlated to this, we found in TCDD-treated mice an increase in interleukin-6 producing CD103+ dendritic cells (DC) present in the gut-draining mesenteric lymph nodes (MLN) and a small increase in the frequency of Th17 cells. Neither the frequencies nor the absolute numbers of immune cells in the lamina propria (LP) or in intraepithelial lymphocytes were changed by TCDD treatment. Our data not only have implications for food allergies in settings of environmental exposure but also raise concerns regarding the harmlessness of overdosing potential AhR agonist in food, which needs to be studied further.