Lansoprazole and carbonic anhydrase IX inhibitors sinergize against human melanoma cells.

AbstractContext: Proton Pump Inhibitors (PPIs) reduce tumor acidity and therefore resistance of tumors to drugs. Carbonic Anhydrase IX (CA IX) inhibitors have proven to be effective against tumors, while tumor acidity might impair their full effectiveness.Objective: To analyze the effect of PPI/CA IX inhibitors combined treatment against human melanoma cells.Methods: The combination of Lansoprazole (LAN) and CA IX inhibitors (FC9-399A and S4) has been investigated in terms of cell proliferation inhibition and cell death in human melanoma cells.Results: The combination of these inhibitors was more effective than the single treatments in both inhibiting cell proliferation and in inducing cell death in human melanoma cells.Discussion: These results represent the first successful attempt in combining two different proton exchanger inhibitors.Conclusion: This is the first evidence on the effectiveness of a new approach against tumors based on the combination of PPI and CA IX inhibitors, thus providing an alter..

TRAIL-R4 Promotes Tumor Growth and Resistance to Apoptosis in Cervical Carcinoma HeLa Cells through AKT

International audienceBACKGROUND: TRAIL/Apo2L is a pro-apoptotic ligand of the TNF family that engages the apoptotic machinery through two pro-apoptotic receptors, TRAIL-R1 and TRAIL-R2. This cell death program is tightly controlled by two antagonistic receptors, TRAIL-R3 and TRAIL-R4, both devoid of a functional death domain, an intracellular region of the receptor, required for the recruitment and the activation of initiator caspases. Upon TRAIL-binding, TRAIL-R4 forms a heteromeric complex with the agonistic receptor TRAIL-R2 leading to reduced caspase-8 activation and apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: We provide evidence that TRAIL-R4 can also exhibit, in a ligand independent manner, signaling properties in the cervical carcinoma cell line HeLa, through Akt. Ectopic expression of TRAIL-R4 in HeLa cells induced morphological changes, with cell rounding, loss of adherence and markedly enhanced cell proliferation in vitro and tumor growth in vivo. Disruption of the PI3K/Akt pathway using the pharmacological inhibitor LY294002, siRNA targeting the p85 regulatory subunit of phosphatidylinositol-3 kinase, or by PTEN over-expression, partially restored TRAIL-mediated apoptosis in these cells. Moreover, the Akt inhibitor, LY294002, restituted normal cell proliferation index in HeLa cells expressing TRAIL-R4. CONCLUSIONS/SIGNIFICANCE: Altogether, these results indicate that, besides its ability to directly inhibit TRAIL-induced cell death at the membrane, TRAIL-R4 can also trigger the activation of signaling pathways leading to cell survival and proliferation in HeLa cells. Our findings raise the possibility that TRAIL-R4 may contribute to cervical carcinogenesis

High Levels of Exosomes Expressing CD63 and Caveolin-1 in Plasma of Melanoma Patients

BACKGROUND: Metastatic melanoma is an untreatable cancer lacking reliable and non-invasive markers of disease progression. Exosomes are small vesicles secreted by normal as well as tumor cells. Human tumor-derived exosomes are involved in malignant progression and we evaluated the presence of exosomes in plasma of melanoma patients as a potential tool for cancer screening and follow-up. METHODOLOGY/PRINCIPAL FINDINGS: We designed an in-house sandwich ELISA (Exotest) to capture and quantify exosomes in plasma based on expression of housekeeping proteins (CD63 and Rab-5b) and a tumor-associated marker (caveolin-1). Western blot and flow cytometry analysis of exosomes were used to confirm the Exotest-based findings. The Exotest allowed sensitive detection and quantification of exosomes purified from human tumor cell culture supernatants and plasma from SCID mice engrafted with human melanoma. Plasma levels of exosomes in melanoma-engrafted SCID mice correlated to tumor size. We evaluated the levels of plasma exosomes expressing CD63 and caveolin-1 in melanoma patients (n = 90) and healthy donors (n = 58). Consistently, plasma exosomes expressing CD63 (504+/-315) or caveolin-1 (619+/-310) were significantly increased in melanoma patients as compared to healthy donors (223+/-125 and 228+/-102, respectively). While the Exotest for CD63+ plasma exosomes had limited sensitivity (43%) the Exotest for detection of caveolin-1+ plasma exosomes showed a higher sensitivity (68%). Moreover, caveolin-1+ plasma exosomes were significantly increased with respect to CD63+ exosomes in the patients group. CONCLUSIONS/SIGNIFICANCE: We describe a new non-invasive assay allowing detection and quantification of human exosomes in plasma of melanoma patients. Our results suggest that the Exotest for detection of plasma exosomes carrying tumor-associated antigens may represent a novel tool for clinical management of cancer patients

Targeting the Interplay between Cancer Metabolic Reprogramming and Cell Death Pathways as a Viable Therapeutic Path

In cancer cells, metabolic adaptations are often observed in terms of nutrient absorption, biosynthesis of macromolecules, and production of energy necessary to meet the needs of the tumor cell such as uncontrolled proliferation, dissemination, and acquisition of resistance to death processes induced by both unfavorable environmental conditions and therapeutic drugs. Many oncogenes and tumor suppressor genes have a significant effect on cellular metabolism, as there is a close relationship between the pathways activated by these genes and the various metabolic options. The metabolic adaptations observed in cancer cells not only promote their proliferation and invasion, but also their survival by inducing intrinsic and acquired resistance to various anticancer agents and to various forms of cell death, such as apoptosis, necroptosis, autophagy, and ferroptosis. In this review we analyze the main metabolic differences between cancer and non-cancer cells and how these can affect the various cell death pathways, effectively determining the susceptibility of cancer cells to therapy-induced death. Targeting the metabolic peculiarities of cancer could represent in the near future an innovative therapeutic strategy for the treatment of those tumors whose metabolic characteristics are known

Régulation de la signalisation TRAIL par l'ezrine

Background and Aim: TRAIL has sparked a growing interest in oncology due to its ability to selectively trigger cancer cell death while sparing normal cells. The Fas/actin association through ezrin, a member of the ERM protein family, has been reported to regulate early steps of Fas-mediated apoptosis. In this project, we addressed the role of ezrin regarding TRAIL-induced cell death in B lymphoma cell lines, or adherent cancer cell lines (HeLa WT, HCT116, SW480). Methods: Molecular and biochemical approaches were employed to study the relevance of ezrin and its phosphorylation status in TRAIL signaling. Results: We found that ezrin displays a negative function towards TRAIL- and Fas-mediated apoptosis and that the ezrin-mediated TRAIL-induced cell death inhibition led to ezrin activation through phosphorylation/dephosphorylation events at serine 66 and tyrosine 353, but is mainly independent of TRAIL DISC (Death Inducing Signalling Complex) formation or activation. Mutations of these residues to alanine (S66A) or aspartic acid (Y353D) selectively enhanced TRAIL-induced cell death, whereas point mutations mimicking ezrin phosphorylation on S66 (S66D) or a nonphosphorylable variant on Y353 (Y353F) strongly protected cancer cells from apoptosis induced by TRAIL. Moreover, inhibition of the ezrin serine 66 PKA target site, using H89, increased cancer cell sensitivity to TRAIL, while treatment with 8bromocyclic AMP, a PKA activator, decreased TRAIL-induced cell death. In addition, combined TRAIL/cisplatin treatments abrogated ezrin-mediated inhibition of TRAIL-induced apoptosis.Objectifs: La cytokine TRAIL (TNF Related Apoptosis Inducing Ligand) suscite un intérêt majeur en thérapie anti-cancéreuse grâce à sa capacité à induire l’apoptose des cellules cancéreuses tout en épargnant les cellules saines. L’association du récepteur Fas et de l’actine via l’ezrine, une protéine de la famille ERM (Ezrin, Moesin, Radixin), régule les premières étapes de l’induction de l’apoptose par FasL. Au cours de mon projet de thèse, nous avons voulu déterminer le rôle que pouvait jouer l’ezrine au cours de l’apoptose induite par TRAIL, dans des lymphomes B ou des cellules cancéreuses adhérentes (HeLa, HCT116 et SW480). Matériel et Méthodes: Des approches biochimiques et moléculaires nous ont permis d’étudier et de déterminer l’implication de l’ezrine et sa phosphorylation dans la régulation de la mort induite par TRAIL. Résultats: Ce travail démontre que l’ezrine peut réguler de manière négative l’apoptose induite par TRAIL et FasL. Cette activité inhibitrice est régulée par la phosphorylation/déphosphorylation sur la serine 66 ainsi que sur la thréonine 353. Néanmoins cette régulation n’affecte ni la formation, ni l’activation du DISC (Death Inducing Signalling Complex). Des mutations de ces résidus par une alanine (S66A) ou un acide aspartique (Y353D) augmente sélectivement la capacité de TRAIL à induire l’apoptose. Au contraire, des mutations ponctuelles de ces résidus permettant de mimer la phosphorylation de l’ezrine sur la serine 66 (S66D) ou l’expression d’un variant non phosphorylable sur la thréonine 353 (Y353F) protègent les cellules cancéreuses de l’apoptose induite par TRAIL. De manière concordante, l’utilisation du H89, un inhibiteur de PKA, kinase responsable de la phosphorylation de la serine 66 augmente la sensibilité des cellules cancéreuses à TRAIL, alors qu’au contraire, un activateur de PKA (8bromocyclic AMP) rend ces mêmes cellules plus résistantes à TRAIL. Enfin, l’association de TRAIL et du cisplatine permet de dépasser l’inhibition de l’apoptose par l’ezrine

Influenza dell'acidità microambientale sulla omeostasi dei tumori maligni compresi quelli del tratto gastro-enterico

Evidenze sperimentali ottenute finora mettono in luce una nuova caratteristica delle cellule tumorali: il loro microambiente acido, dovuto probabilmente ad una accumulazione di acidi prodotti durante il metabolismo cellulare e la glicolisi (acido lattico e protoni). Pompe protoniche quali la H+-ATPasi vacuolare (V-ATPasi) sono coinvolte nel controllo del pH cellulare sia in cellule sane che nei tumori, dove vi è una up-regolazione. Gli inibitori delle pompe protoniche (PPI) sono una classe di proteine che inibiscono direttamente la V-ATPasi. È inoltre noto che un pH acido è la condizione ottimale per una piena attivazione dei PPI. In questo lavoro si è cercato di capire se i PPI, normalmente usati come anti-acidi in caso di gastriti o ulcere, siano in grado di inibire l'acidificazione del microambiente tumorale e possano aumentare la sensibilità delle cellule tumorali ai farmaci citotossici. Si è poi cercato anche di capire se il trattamento coi PPI in condizioni di terreno non tamponato sia in grado di interferire con la sopravvivenza e la proliferazione dei tumori umani. I risultati ottenuti hanno mostrato che il trattamento coi PPI sensibilizza diverse linee cellulari agli effetti citotossici del cisplatino, 5-fluorouracile e della vinblastina. Il trattamento coi PPI induceva anche un marcato aumento nel citoplasma della ritenzione dei farmaci citotossici, del pH extracellulare e di quello degli organuli lisosomiali. Negli esperimenti in vivo, il pre-trattamento per via orale con l'omeprazolo era stato in grado di sensibilizzare tumori solidi agli effetti del cisplatino. I PPI, inoltre, inducevano in differenti tumori una inibizione della proliferazione in maniera dose-dipendente, che si era dimostrata essere associata ad una citotossicità dose- e tempo-dipendente simile ad apoptosi. L'attività proapoptotica dei PPI è stata consistente con una chiara inibizione in vivo della crescita tumorale in seguito a trattamento coi PPI in tumori a cellule B. Questo studio mette in luce l'importanza dell'acidità e del gradiente di pH nell'omeostasi dei tumori e suggerisce un nuovo approccio terapeutico per i tumori basato sull'utilizzo dei PPI.Evidences have shown that tumor cells exist within an acidic microenvironment, probably due to accumulation and poor removal of metabolic acids such as lactic acid and protons derived from glycolysis. Proton pumps like the vacuolar H+- ATPases (V-ATPases) are involved in the control of cellular pH in normal and tumor cells, where are up-regulated. Proton pump inhibitors (PPI) are a class of H+-ATPase inhibitors that inhibit directly the V-ATPase. It is also known that low pH is the most suitable condition for a full PPI activation. Here, we investigated whether PPIs, currently used in the anti-acid treatment of peptic desease, could inhibit the acidification of the tumor microenvironment and increase the sensitivity of tumor cells to cytotoxic agents. We tested, also, whether PPI treatment in unbuffered culture conditions could affect survival and proliferation of human tumors. The results shown that PPI treatment sensitized tumor cell lines to the effects of cisplatin, 5-fluorouracil and vinblastine. PPI treatment induced a marked increase in the cytoplasmic retention of the cytotoxic drugs, in both extracellular pH and in the pH of lysosomal organelles. In in vivo experiments, oral pretreatment with omeprazole was able to induce sensitivity of human solid tumors to cisplatin. PPI, also, induced a dose-dependent inhibition of proliferation of different human tumors, which was associated with a dose- and time-dependent apoptotic like cytotoxicity. The proapoptotic activity of PPI was consistent with a clear inhibition in vivo of tumor growth following PPI treatment of B-cell lymphoma. This study supports the importance of acidity and pH gradients in tumor cell homeostasis and suggest new therapeutic approaches for human tumors based on PPI

Régulation de la signalisation TRAIL par l'ezrine

Objectifs: La cytokine TRAIL (TNF Related Apoptosis Inducing Ligand) suscite un intérêt majeur en thérapie anti-cancéreuse grâce à sa capacité à induire l apoptose des cellules cancéreuses tout en épargnant les cellules saines. L association du récepteur Fas et de l actine via l ezrine, une protéine de la famille ERM (Ezrin, Moesin, Radixin), régule les premières étapes de l induction de l apoptose par FasL. Au cours de mon projet de thèse, nous avons voulu déterminer le rôle que pouvait jouer l ezrine au cours de l apoptose induite par TRAIL, dans des lymphomes B ou des cellules cancéreuses adhérentes (HeLa, HCT116 et SW480). Matériel et Méthodes: Des approches biochimiques et moléculaires nous ont permis d étudier et de déterminer l implication de l ezrine et sa phosphorylation dans la régulation de la mort induite par TRAIL. Résultats: Ce travail démontre que l ezrine peut réguler de manière négative l apoptose induite par TRAIL et FasL. Cette activité inhibitrice est régulée par la phosphorylation/déphosphorylation sur la serine 66 ainsi que sur la thréonine 353. Néanmoins cette régulation n affecte ni la formation, ni l activation du DISC (Death Inducing Signalling Complex). Des mutations de ces résidus par une alanine (S66A) ou un acide aspartique (Y353D) augmente sélectivement la capacité de TRAIL à induire l apoptose. Au contraire, des mutations ponctuelles de ces résidus permettant de mimer la phosphorylation de l ezrine sur la serine 66 (S66D) ou l expression d un variant non phosphorylable sur la thréonine 353 (Y353F) protègent les cellules cancéreuses de l apoptose induite par TRAIL. De manière concordante, l utilisation du H89, un inhibiteur de PKA, kinase responsable de la phosphorylation de la serine 66 augmente la sensibilité des cellules cancéreuses à TRAIL, alors qu au contraire, un activateur de PKA (8bromocyclic AMP) rend ces mêmes cellules plus résistantes à TRAIL. Enfin, l association de TRAIL et du cisplatine permet de dépasser l inhibition de l apoptose par l ezrine.Background and Aim: TRAIL has sparked a growing interest in oncology due to its ability to selectively trigger cancer cell death while sparing normal cells. The Fas/actin association through ezrin, a member of the ERM protein family, has been reported to regulate early steps of Fas-mediated apoptosis. In this project, we addressed the role of ezrin regarding TRAIL-induced cell death in B lymphoma cell lines, or adherent cancer cell lines (HeLa WT, HCT116, SW480). Methods: Molecular and biochemical approaches were employed to study the relevance of ezrin and its phosphorylation status in TRAIL signaling. Results: We found that ezrin displays a negative function towards TRAIL- and Fas-mediated apoptosis and that the ezrin-mediated TRAIL-induced cell death inhibition led to ezrin activation through phosphorylation/dephosphorylation events at serine 66 and tyrosine 353, but is mainly independent of TRAIL DISC (Death Inducing Signalling Complex) formation or activation. Mutations of these residues to alanine (S66A) or aspartic acid (Y353D) selectively enhanced TRAIL-induced cell death, whereas point mutations mimicking ezrin phosphorylation on S66 (S66D) or a nonphosphorylable variant on Y353 (Y353F) strongly protected cancer cells from apoptosis induced by TRAIL. Moreover, inhibition of the ezrin serine 66 PKA target site, using H89, increased cancer cell sensitivity to TRAIL, while treatment with 8bromocyclic AMP, a PKA activator, decreased TRAIL-induced cell death. In addition, combined TRAIL/cisplatin treatments abrogated ezrin-mediated inhibition of TRAIL-induced apoptosis.DIJON-BU Doc.électronique (212319901) / SudocSudocFranceF

Targeting the Interplay between Cancer Metabolic Reprogramming and Cell Death Pathways as a Viable Therapeutic Path

In cancer cells, metabolic adaptations are often observed in terms of nutrient absorption, biosynthesis of macromolecules, and production of energy necessary to meet the needs of the tumor cell such as uncontrolled proliferation, dissemination, and acquisition of resistance to death processes induced by both unfavorable environmental conditions and therapeutic drugs. Many oncogenes and tumor suppressor genes have a significant effect on cellular metabolism, as there is a close relationship between the pathways activated by these genes and the various metabolic options. The metabolic adaptations observed in cancer cells not only promote their proliferation and invasion, but also their survival by inducing intrinsic and acquired resistance to various anticancer agents and to various forms of cell death, such as apoptosis, necroptosis, autophagy, and ferroptosis. In this review we analyze the main metabolic differences between cancer and non-cancer cells and how these can affect the various cell death pathways, effectively determining the susceptibility of cancer cells to therapy-induced death. Targeting the metabolic peculiarities of cancer could represent in the near future an innovative therapeutic strategy for the treatment of those tumors whose metabolic characteristics are known