RESUMO
Calcium (Ca2+) is a universal second messenger involved in synaptogenesis and cell survival; consequently, its regulation is important for neurons. ATPase plasma membrane Ca2+ transporting 1 (ATP2B1) belongs to the family of ATP-driven calmodulin-dependent Ca2+ pumps that participate in the regulation of intracellular free Ca2+. Here, we clinically describe a cohort of 12 unrelated individuals with variants in ATP2B1 and an overlapping phenotype of mild to moderate global development delay. Additional common symptoms include autism, seizures, and distal limb abnormalities. Nine probands harbor missense variants, seven of which were in specific functional domains, and three individuals have nonsense variants. 3D structural protein modeling suggested that the variants have a destabilizing effect on the protein. We performed Ca2+ imaging after introducing all nine missense variants in transfected HEK293 cells and showed that all variants lead to a significant decrease in Ca2+ export capacity compared with the wild-type construct, thus proving their pathogenicity. Furthermore, we observed for the same variant set an incorrect intracellular localization of ATP2B1. The genetic findings and the overlapping phenotype of the probands as well as the functional analyses imply that de novo variants in ATP2B1 lead to a monogenic form of neurodevelopmental disorder.
Assuntos
Deficiência Intelectual , Malformações do Sistema Nervoso , Transtornos do Neurodesenvolvimento , Células HEK293 , Humanos , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Mutação de Sentido Incorreto/genética , Transtornos do Neurodesenvolvimento/genética , Fenótipo , ATPases Transportadoras de Cálcio da Membrana Plasmática/genéticaRESUMO
The naturally occurring dipeptide carnosine (ß-alanyl-L-histidine) specifically attenuates tumor growth. Here, we ask whether other small imidazole-containing compounds also affect the viability of tumor cells without affecting non-malignant cells and whether the formation of histamine is involved. Patient-derived fibroblasts and glioblastoma cells were treated with carnosine, L-alanyl-L-histidine (LA-LH), ß-alanyl-L-alanine, L-histidine, histamine, imidazole, ß-alanine, and L-alanine. Cell viability was assessed by cell-based assays and microscopy. The intracellular release of L-histidine and formation of histamine was investigated by high-performance liquid chromatography coupled to mass spectrometry. Carnosine and LA-LH inhibited tumor cell growth with minor effects on fibroblasts, and L-histidine, histamine, and imidazole affected viability in both cell types. Compounds without the imidazole moiety did not diminish viability. In the presence of LA-LH but not in the presence of carnosine, a significant rise in intracellular amounts of histidine was detected in all cells. The formation of histamine was not detectable in the presence of carnosine, LA-LH, or histidine. In conclusion, the imidazole moiety of carnosine contributes to its anti-neoplastic effect, which is also seen in the presence of histidine and LA-LH. Despite the fact that histamine has a strong effect on cell viability, the formation of histamine is not responsible for the effects on the cell viability of carnosine, LA-LH, and histidine.
Assuntos
Carnosina , Glioblastoma , Alanina , Carnosina/metabolismo , Fibroblastos/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Histamina/metabolismo , Histamina/farmacologia , Histidina/metabolismo , Humanos , Imidazóis/farmacologia , beta-AlaninaRESUMO
The naturally occurring dipeptide carnosine (ß-alanyl-l-histidine) has beneficial effects in different diseases. It is also frequently used as a food supplement to improve exercise performance and because of its anti-aging effects. Nevertheless, after oral ingestion, the dipeptide is not detectable in human serum because of rapid degradation by serum carnosinase. At the same time, intact carnosine is excreted in urine up to five hours after intake. Therefore, an unknown compartment protecting the dipeptide from degradation has long been hypothesized. Considering that erythrocytes may constitute this compartment, we investigated the uptake and intracellular amounts of carnosine in human erythrocytes cultivated in the presence of the dipeptide and human serum using liquid chromatography-mass spectrometry. In addition, we studied carnosine's effect on ATP production in red blood cells and on their response to oxidative stress. Our experiments revealed uptake of carnosine into erythrocytes and protection from carnosinase degradation. In addition, no negative effect on ATP production or defense against oxidative stress was observed. In conclusion, our results for the first time demonstrate that erythrocytes can take up carnosine, and, most importantly, thereby prevent its degradation by human serum carnosinase.
Assuntos
Trifosfato de Adenosina/metabolismo , Carnosina/metabolismo , Dipeptidases/metabolismo , Eritrócitos/metabolismo , Estresse Oxidativo , Soro/enzimologia , Carnosina/química , Eritrócitos/patologia , HumanosRESUMO
Hepatoblastoma (HB), the most common pediatric primary liver neoplasm, shows nuclear localization of ß-catenin and yes-associated protein 1 (YAP1) in almost 80% of the cases. Co-expression of constitutively active S127A-YAP1 and ΔN90 deletion-mutant ß-catenin (YAP1-ΔN90-ß-catenin) causes HB in mice. Because heterogeneity in downstream signaling is being identified owing to mutational differences even in the ß-catenin gene alone, we investigated if co-expression of point mutants of ß-catenin (S33Y or S45Y) with S127A-YAP1 led to similar tumors as YAP1-ΔN90-ß-catenin. Co-expression of S33Y/S45Y-ß-catenin and S127A-YAP1 led to activation of Yap and Wnt signaling and development of HB, with 100% mortality by 13 to 14 weeks. Co-expression with YAP1-S45Y/S33Y-ß-catenin of the dominant-negative T-cell factor 4 or dominant-negative transcriptional enhanced associate domain 2, the respective surrogate transcription factors, prevented HB development. Although histologically similar, HB in YAP1-S45Y/S33Y-ß-catenin, unlike YAP1-ΔN90-ß-catenin HB, was glutamine synthetase (GS) positive. However, both ΔN90-ß-catenin and point-mutant ß-catenin comparably induced GS-luciferase reporter in vitro. Finally, using a previously reported 16-gene signature, it was shown that YAP1-ΔN90-ß-catenin HB tumors exhibited genetic similarities with more proliferative, less differentiated, GS-negative HB patient tumors, whereas YAP1-S33Y/S45Y-ß-catenin HB exhibited heterogeneity and clustered with both well-differentiated GS-positive and proliferative GS-negative patient tumors. Thus, we demonstrate that ß-catenin point mutants can also collaborate with YAP1 in HB development, albeit with a distinct molecular profile from the deletion mutant, which may have implications in both biology and therapy.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Mutação , Fatores de Transcrição/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Prognóstico , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Proteínas de Sinalização YAP , beta Catenina/genéticaRESUMO
Transdifferentiation (TD) is the direct reprogramming of adult cells into cells of alternate fate and function. We have previously shown that liver cells can be transdifferentiated into beta-like, insulin-producing cells through ectopic expression of pancreatic transcription factors (pTFs). However, the efficiency of the process was consistently limited to <15% of the human liver cells treated in culture. The data in the current study suggest that liver-to-pancreas TD is restricted to a specific population of liver cells that is predisposed to undergo reprogramming. We isolated TD-predisposed subpopulation of liver cells from >15 human donors using a lineage tracing system based on the Wnt response element, part of the pericentral-specific promoter of glutamine synthetase. The cells, that were propagated separately, consistently exhibited efficient fate switch and insulin production and secretion in >60% of the cells upon pTF expression. The rest of the cells, which originated from 85% of the culture, resisted TD. Both populations expressed the ectopic pTFs with similar efficiencies, followed by similar repression of hepatic genes. Our data suggest that the TD-predisposed cells originate from a distinct population of liver cells that are enriched for Wnt signaling, which is obligatory for efficient TD. In TD-resistant populations, Wnt induction is insufficient to induce TD. An additional step of chromatin opening enables TD of these cells. CONCLUSION: Liver-to-pancreas TD occurs in defined predisposed cells. These cells' predisposition is maintained by Wnt signaling that endows the cells with the plasticity needed to alter their transcriptional program and developmental fate when triggered by ectopic pTFs. These results may have clinical implications by drastically increasing the efficacy of TD in future clinical uses. (Hepatology 2018).
Assuntos
Linhagem da Célula , Transdiferenciação Celular/genética , Proteínas Wnt/genética , Via de Sinalização Wnt/genética , beta Catenina/genética , Animais , Causalidade , Células Cultivadas , Reprogramação Celular , Hepatócitos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pâncreas/citologia , Sensibilidade e EspecificidadeRESUMO
Carnosine (ß-alanyl-L-histidine) affects a plethora of signaling pathways and genes in different biological systems. Although known as a radical scavenger, not all of these effects can simply be ascribed to its chemical nature. As previous experiments pointed towards the possibility that carnosine affects epigenetic regulation via histone acetylation, we investigated this hypothesis using the glioblastoma cell lines U87 and T98G in which carnosine's anti-neoplastic effect is accompanied by increased expression of pyruvate dehydrogenase kinase 4. Viability and expression of PDK4 was analyzed after incubation in carnosine and different histone deacetylase inhibitors (HDACi) using cell-based assays and qRT-PCR. In addition, chromatin immunoprecipitation (ChIP) experiments were performed and the global influence of carnosine on histone H3 acetylation was analyzed by Western blot. Carnosine as well as the HDACi used increased expression of PDK4. In addition, all compounds reduced cell viability, although differences were observed with regard to magnitude and required concentrations. ChIP analysis revealed increased acetylation of histone H3 in the PDK4 promoter of U87 and T98G cells (~ 1.3- and ~ 1.7-fold, respectively) 6 h after the addition of carnosine (50 mM) followed by increased expression of PDK4 mRNA. Western blots did not detect a general increase of H3 acetylation at a genome-wide scale under the influence of carnosine. Our experiments for the first time demonstrate that carnosine influences epigenetic regulation via increased histone acetylation.
Assuntos
Carnosina/farmacologia , Epigênese Genética/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Glioblastoma/enzimologia , Histonas/metabolismo , Proteínas Quinases/genética , Transcrição Gênica/genética , Acetilação , Linhagem Celular Tumoral , Inibidores de Histona Desacetilases/farmacologia , Humanos , Regiões Promotoras Genéticas , Processamento de Proteína Pós-TraducionalRESUMO
The previous studies demonstrated that carnosine (ß-alanyl-L-histidine) inhibits the growth of tumor cells in vitro and in vivo. Considering carnosine for the treatment of glioblastoma, we investigated which proton-coupled oligopeptide transporters (POTs) are present in glioblastoma cells and how they contribute to the uptake of carnosine. Therefore, mRNA expression of the four known POTs (PEPT1, PEPT2, PHT1, and PHT2) was examined in three glioblastoma cell lines, ten primary tumor cell cultures, in freshly isolated tumor tissue and in healthy brain. Using high-performance liquid chromatography coupled to mass spectrometry, the uptake of carnosine was investigated in the presence of competitive inhibitors and after siRNA-mediated knockdown of POTs. Whereas PEPT1 mRNA was not detected in any sample, expression of the three other transporters was significantly increased in tumor tissue compared to healthy brain. In cell culture, PHT1 expression was comparable to expression in tumor tissue, PHT2 exhibited a slightly reduced expression, and PEPT2 expression was reduced to normal brain tissue levels. In the cell line LN405, the competitive inhibitors ß-alanyl-L-alanine (inhibits all transporters) and L-histidine (inhibitor of PHT1/2) both inhibited the uptake of carnosine. SiRNA-mediated knockdown of PHT1 and PHT2 revealed a significantly reduced uptake of carnosine. Interestingly, despite its low expression at the level of mRNA, knockdown of PEPT2 also resulted in decreased uptake. In conclusion, our results demonstrate that the transporters PEPT2, PHT1, and PHT2 are responsible for the uptake of carnosine into glioblastoma cells and full function of all three transporters is required for maximum uptake.
Assuntos
Neoplasias Encefálicas/metabolismo , Carnosina/metabolismo , Glioblastoma/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Simportadores/metabolismo , Adulto , Idoso , Encéfalo/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Feminino , Histidina/metabolismo , Humanos , Masculino , Espectrometria de Massas , Proteínas de Membrana Transportadoras/genética , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Oligopeptídeos/metabolismo , Transportador 1 de Peptídeos/metabolismo , RNA Interferente Pequeno/metabolismo , Simportadores/genéticaRESUMO
The naturally occurring dipeptide carnosine (ß-alanyl-L-histidine) inhibits the growth of tumor cells. As its component L-histidine mimics the effect, we investigated whether cleavage of carnosine is required for its antineoplastic effect. Using ten glioblastoma cell lines and cell cultures derived from 21 patients suffering from this malignant brain tumor, we determined cell viability under the influence of carnosine and L-histidine. Moreover, we determined expression of carnosinases, the intracellular release of L-histidine from carnosine, and whether inhibition of carnosine cleavage attenuates carnosine's antineoplastic effect. We observed a significantly higher response of the cells to L-histidine than to carnosine with regard to cell viability in all cultures. In addition, we detected protein and mRNA expression of carnosinases and a low but significant release of L-histidine in cells incubated in the presence of 50 mM carnosine (p < 0.05), which did not correlate with carnosine's effect on viability. Furthermore, the carnosinase 2 inhibitor bestatin did not attenuate carnosine's effect on viability. Interestingly, we measured a ~ 40-fold higher intracellular abundance of L-histidine in the presence of 25 mM extracellular L-histidine compared to the amount of L-histidine in the presence of 50 mM carnosine, both resulting in a comparable decrease in viability. In addition, we also examined the expression of pyruvate dehydrogenase kinase 4 mRNA, which was comparably influenced by L-histidine and carnosine, but did not correlate with effects on viability. In conclusion, we demonstrate that the antineoplastic effect of carnosine is independent of its cleavage.
Assuntos
Carnosina/química , Carnosina/farmacologia , Proliferação de Células/efeitos dos fármacos , Glioblastoma/patologia , Histidina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Ciclo Celular , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Células Tumorais CultivadasRESUMO
Aim: The naturally occurring dipeptide carnosine (CAR) has been considered for glioblastoma therapy. As CAR also protects against ionizing irradiation (IR), we investigated whether it may counteract standard therapy consisting of postsurgery IR and treatment with temozolomide (TMZ). Materials & methods: Four isocitrate dehydrogenase-wildtype primary cell cultures were exposed to different doses of IR and different concentrations of TMZ and CAR. After exposure, viability under the different conditions and combinations of them was determined. Results: All cultures responded to treatment with TMZ and IR with reduced viability. CAR further decreased viability when TMZ and IR were combined. Conclusion: Treatment with CAR does not counteract glioblastoma standard therapy. As the dipeptide also protects nontumor cells from IR, it may reduce deleterious side effects of treatment.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Neoplasias Encefálicas/patologia , Carnosina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Glioblastoma/patologia , Isocitrato Desidrogenase/genética , Radiação Ionizante , Temozolomida/farmacologia , Idoso , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Sobrevivência Celular/efeitos da radiação , Feminino , Glioblastoma/genética , Glioblastoma/terapia , Humanos , Isocitrato Desidrogenase/metabolismo , Masculino , Pessoa de Meia-Idade , Células Tumorais CultivadasRESUMO
Mass spectrometric analysis of glioblastoma cyst fluids has disclosed a protein peak with m/z 6424-6433. Among the proteins, potentially generating this peak are ApoC1 and LuzP6. To further elucidate protein expression of glioblastoma cells, we analyzed MALDI-TOF results of cyst fluid, performed immunohistochemistry and mRNA analysis. MALDI-TOF protein extraction from 24 glioblastoma cyst fluids was performed with a weak cation exchange. 50 glioblastoma samples were stained with two custom-made antibodies against LuzP6 and commercial antibodies against ApoC1, C12orf75 and OCC-1 and analyzed. For mRNA detection, 16 tissue samples were stored in RNAlater, extracted using the miRNeasy kit and reversely transcribed. For 12 patients, synopsis of results from all three examinations was possible. MALDI-TOF confirmed the peak at 6433 Da in 75% of samples. Immunohistochemically, LuzP6 was detected in 92% (LuzP61-29) and 96% (LuzP630-58) of samples and ApoC1 in 66%. Mean mRNA levels were highest for ApoC1, followed by LuzP6. No correlation between mRNA expression, immunohistochemical staining and intensity of the MALDI-TOF peaks was found. An unequivocal identification of one protein as the source for the 6433 peak is not possible, but our results point to ApoC1 and LuzP6 as the underlying proteins.
Assuntos
Apolipoproteína C-I/genética , Apolipoproteína C-I/metabolismo , Glioblastoma/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Feminino , Humanos , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
BACKGROUND: Glioblastoma (GBM) is a tumor of the central nervous system. After surgical removal and standard therapy, recurrence of tumors is observed within 6-9 months because of the high migratory behavior and the infiltrative growth of cells. Here, we investigated whether carnosine (ß-alanine-l-histidine), which has an inhibitory effect on glioblastoma proliferation, may on the opposite promote invasion as proposed by the so-called "go-or-grow concept". METHODS: Cell viability of nine patient derived primary (isocitrate dehydrogenase wildtype; IDH1R132H non mutant) glioblastoma cell cultures and of eleven patient derived fibroblast cultures was determined by measuring ATP in cell lysates and dehydrogenase activity after incubation with 0, 50 or 75 mM carnosine for 48 h. Using the glioblastoma cell line T98G, patient derived glioblastoma cells and fibroblasts, a co-culture model was developed using 12 well plates and cloning rings, placing glioblastoma cells inside and fibroblasts outside the ring. After cultivation in the presence of carnosine, the number of colonies and the size of the tumor cell occupied area were determined. RESULTS: In 48 h single cultures of fibroblasts and tumor cells, 50 and 75 mM carnosine reduced ATP in cell lysates and dehydrogenase activity when compared to the corresponding untreated control cells. Co-culture experiments revealed that after 4 week exposure to carnosine the number of T98G tumor cell colonies within the fibroblast layer and the area occupied by tumor cells was reduced with increasing concentrations of carnosine. Although primary cultured tumor cells did not form colonies in the absence of carnosine, they were eliminated from the co-culture by cell death and did not build colonies under the influence of carnosine, whereas fibroblasts survived and were healthy. CONCLUSIONS: Our results demonstrate that the anti-proliferative effect of carnosine is not accompanied by an induction of cell migration. Instead, the dipeptide is able to prevent colony formation and selectively eliminates tumor cells in a co-culture with fibroblasts.
RESUMO
BACKGROUND: The Special AT-rich Sequence Binding Protein 1 (SATB1) regulates the expression of many genes by acting as a global chromatin organizer. While in many tumor entities SATB1 overexpression has been observed and connected to pro-tumorigenic processes, somewhat contradictory evidence exists in brain tumors with regard to SATB1 overexpression in glioblastoma and its association with poorer prognosis and tumor progression. On the functional side, initial data indicate that SATB1 may be involved in several tumor cell-relevant processes. METHODS: For the detailed analysis of the functional relevance and possible therapeutic potential of SATB1 inhibition, we employ transient siRNA-mediated knockdown and comprehensively analyze the cellular and molecular role of SATB1 in glioblastoma. RESULTS: In various cell lines with different SATB1 expression levels, a SATB1 gene dose-dependent inhibition of anchorage-dependent and -independent proliferation is observed. This is due to cell cycle-inhibitory and pro-apoptotic effects of SATB1 knockdown. Molecular analyses reveal SATB1 knockdown effects on multiple important (proto-) oncogenes, including Myc, Bcl-2, Pim-1, EGFR, ß-catenin and Survivin. Molecules involved in cell cycle, EMT and cell adhesion are affected as well. The putative therapeutic relevance of SATB1 inhibition is further supported in an in vivo tumor xenograft mouse model, where the treatment with polymeric nanoparticles containing SATB1-specific siRNAs exerts antitumor effects. CONCLUSION: Our results demonstrate that SATB1 may represent a promising target molecule in glioblastoma therapy whose inhibition or knockdown affects multiple crucial pathways.
Assuntos
Neoplasias Encefálicas/patologia , Encéfalo/patologia , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Animais , Apoptose , Encéfalo/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Ciclo Celular , Proliferação de Células , Transição Epitelial-Mesenquimal , Técnicas de Silenciamento de Genes , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Proteínas de Ligação à Região de Interação com a Matriz/antagonistas & inibidores , Proteínas de Ligação à Região de Interação com a Matriz/genética , Camundongos , Camundongos Nus , RNA Interferente Pequeno/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The energy metabolism of most tumor cells relies on aerobic glycolysis (Warburg effect) characterized by an increased glycolytic flux that is accompanied by the increased formation of the cytotoxic metabolite methylglyoxal (MGO). Consequently, the rate of detoxification of this reactive glycolytic byproduct needs to be increased in order to prevent deleterious effects to the cells. This is brought about by an increased expression of glyoxalase 1 (GLO1) that is the rate-limiting enzyme of the MGO-detoxifying glyoxalase system. Here, we overexpressed GLO1 in HEK 293 cells and silenced it in MCF-7 cells using shRNA. Tumor-related properties of wild type and transformed cells were compared and key glycolytic enzyme activities assessed. Furthermore, the cells were subjected to hypoxic conditions to analyze the impact on cell proliferation and enzyme activities. Our results demonstrate that knockdown of GLO1 in the cancer cells significantly reduced tumor-associated properties such as migration and proliferation, whereas no functional alterations where found by overexpression of GLO1 in HEK 293 cells. In contrast, hypoxia caused inhibition of cell growth of all cells except of those overexpressing GLO1. Altogether, we conclude that GLO1 on one hand is crucial to maintaining tumor characteristics of malignant cells, and, on the other hand, supports malignant transformation of cells in a hypoxic environment when overexpressed.
Assuntos
Movimento Celular/genética , Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Glicólise/genética , Lactoilglutationa Liase/genética , Aldeído Pirúvico/metabolismo , Neoplasias da Mama/patologia , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/patologia , Feminino , Glutationa/metabolismo , Células HEK293 , Humanos , Lactoilglutationa Liase/biossíntese , Lactoilglutationa Liase/metabolismo , Células MCF-7 , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
The naturally occurring dipeptide carnosine (ß-alanyl-L-histidine) has been shown to inhibit, selectively, growth of transformed cells mediated, at least in part, by depleting glycolytic ATP levels. The mechanism(s) responsible has/have yet to be determined. Here, we discuss a number of probable and/or possible processes which could, theoretically, suppress glycolytic activity which would decrease ATP supply and generation of metabolic intermediates required for continued cell reproduction. Possibilities include effects on (i) glycolytic enzymes, (ii) metabolic regulatory activities, (iii) redox biology, (iv) protein glycation, (v) glyoxalase activity, (vi) apoptosis, (vii) gene expression and (viii) metastasis. It is possible, by acting at various sites that this pluripotent dipeptide may be an example of an endogenous "smart drug".
Assuntos
Antineoplásicos/farmacologia , Carnosina/farmacologia , Proliferação de Células/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Glicólise/efeitos dos fármacos , Humanos , Oxirredução , Transdução de Sinais/efeitos dos fármacosRESUMO
Carnosine (ß-alanyl-L-histidine) is a naturally occurring dipeptide that shows antineoplastic effects in cell culture as well as in animal experiments. Since its mode of action and the targets at the molecular level have not yet been elucidated, we performed qRT-PCR experiments with RNA isolated from glioblastoma cell lines treated with carnosine, ß-alanine, L-alanine, L-histidine and the dipeptide L-alanine-L-histidine. The experiments identified a strong induction of expression of the gene encoding pyruvate dehydrogenase 4 (PDK4) under the influence of carnosine and L-histidine, but not by the other substances employed. In addition, inhibition of cell viability was only detected in cells treated with carnosine and L-histidine, with the latter showing a significantly stronger effect than carnosine. Since the tumor cells expressed the tissue form of carnosinase (CN2) but almost no serum carnosinase (CN1), we conclude that cleavage by CN2 is a prerequisite for the antineoplastic effect of carnosine. In addition, enhanced expression of PDK4 under the influence of carnosine/L-histidine opens a new perspective for the interpretation of the ergogenic potential of dietary ß-alanine supplementation and adds a new contribution to a growing body of evidence that single amino acids can regulate key metabolic pathways important in health and disease.
Assuntos
Antineoplásicos/metabolismo , Carnosina/metabolismo , Histidina/metabolismo , Proteínas Quinases/genética , Alanina/metabolismo , Linhagem Celular Tumoral , Humanos , Proteínas Quinases/metabolismo , Ácido Pirúvico/metabolismo , Regulação para CimaRESUMO
Homozygous CDKN2A/B deletion has been associated with an increased risk of recurrence in meningiomas. However, the evidence is confined to a limited number of studies, and the importance of heterozygous CDKN2A/B deletions remains insufficiently investigated. Hence, the present meta-analysis reconstructs individual patient data (IPD) and reconstructs the probabilities of progression-free survival (PFS) stratified by CDKN2A/B status. IPD of PFS rates were extracted from published Kaplan-Meier plots using the R package IPDfromKM in R studio (RStudio, Boston, MA, USA). Reconstructed Kaplan-Meier Plots of the pooled IPD data were created. One-stage and two-stage meta-analyses were performed. Hazard ratios (HR) were used as effective measures. Of 181 records screened, four articles with 2521 participants were included. The prevalence of homozygous CDKN2A/B deletions in the included studies was 0.049 (95% CI 0.040-0.057), with higher tumor grades associated with a significantly greater proportion of CDKN2A/B deletions. The reconstructed PFS curves for the pooled cohort showed that the median PFS time of patients with a CDKN2A/B wild-type status, heterozygous or homozygous CDKN2A/B deletion was 180.0 (95% CI 145.7-214.3), 26.1 (95% CI 23.3-29.0), and 11.00 (95% CI 8.6-13.3) months, respectively (p < 0.0001). Both hetero- or homozygous CDKN2A/B deletions were significantly associated with shortened time to meningioma progression. One-stage meta-analysis showed that hetero- (HR: 5.5, 95% CI 4.0-7.6, p < 0.00001) and homozygous CDKN2A/B deletions (HR: 8.4, 95% CI 6.4-11.0, p < 0.00001) are significantly associated with shortened time to meningioma progression. Multivariable Cox regression analysis of progression in a subgroup with available covariates (age, sex, WHO grade, and TERT status) and also two-stage meta-analysis confirmed and validated the results of the one-stage analysis that both heterozygous and homozygous CDKN2A/B deletions are of prognostic importance. Further large-scale studies of WHO grade 2 and 3 meningiomas are needed to validate the importance of heterozygous CDKN2A/B deletions with consideration of established factors.
Assuntos
Neoplasias Meníngeas , Meningioma , Humanos , Meningioma/genética , Meningioma/patologia , Prognóstico , Intervalo Livre de Progressão , Neoplasias Meníngeas/genética , Neoplasias Meníngeas/patologia , Inibidor p16 de Quinase Dependente de Ciclina/genéticaRESUMO
The application of carnosine in medicine has been discussed since several years, but many claims of therapeutic effects have not been substantiated by rigorous experimental examination. In the present perspective, a possible use of carnosine as an anti-neoplastic therapeutic, especially for the treatment of malignant brain tumours such as glioblastoma is discussed. Possible mechanisms by which carnosine may perform its anti-tumourigenic effects are outlined and its expected bioavailability and possible negative and positive side effects are considered. Finally, alternative strategies are examined such as treatment with other dipeptides or ß-alanine.
Assuntos
Antineoplásicos/uso terapêutico , Carnosina/uso terapêutico , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/sangue , Antineoplásicos/farmacologia , Carnosina/efeitos adversos , Carnosina/sangue , Carnosina/farmacologia , Humanos , Camundongos , beta-Alanina/administração & dosagemRESUMO
One of the major challenges in cancer research is finding models that closely resemble tumors within patients. Human tissue slice cultures are a promising approach to provide a model of the patient's tumor biology ex vivo. Recently, it was shown that these slices can be successfully analyzed by whole transcriptome sequencing as well as automated histochemistry, increasing their usability as preclinical model. Glioblastoma multiforme (GBM) is a highly malignant brain tumor with poor prognosis and little is known about its genetic background and heterogeneity regarding therapy success. In this study, tissue from the tumors of 25 patients with primary GBM was processed into slice cultures and treated with standard therapy (irradiation and temozolomide). Total RNA sequencing and automated histochemistry were performed to enable analysis of treatment effects at a transcriptional and histological level. Slice cultures from long-term survivors (overall survival [OS] > 24 months) exhibited more apoptosis than cultures from patients with shorter OS. Proliferation within these slices was slightly increased in contrast to other groups, but not significantly. Among all samples, 58 protein-coding genes were upregulated and 32 downregulated in treated vs. untreated slice cultures. In general, an upregulation of DNA damage-related and cell cycle checkpoint genes as well as enrichment of genotoxicity pathways and p53-dependent signaling was found after treatment. Overall, the current study reproduces knowledge from former studies regarding the feasibility of transcriptomic analyses and automated histology in tissue slice cultures. We further demonstrate that the experimental data merge with the clinical follow-up of the patients, which improves the applicability of our model system.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Neoplasias Encefálicas/genética , Glioblastoma/metabolismo , Humanos , Análise de Sequência de RNA , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Sequenciamento do ExomaRESUMO
Human glioblastoma multiforme is the most malignant brain tumor in adults and is difficult to treat. Recently, it was demonstrated that the dipeptide carnosine inhibits tumor growth, but the main molecular targets are not known. Therefore, a proteomics study with glioblastoma cells treated with carnosine was performed. Two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight detected 31 proteins expressed differentially under the influence of carnosine. Finally, peptide mass fingerprinting identified the "BCL2-associated athanogene 2" and the "von Hippel-Lindau binding protein 1" among other proteins, linking the action of carnosine to protein folding and HIF-1α signalling.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/metabolismo , Carnosina/farmacologia , Proliferação de Células/efeitos dos fármacos , Glioblastoma/metabolismo , Proteínas de Neoplasias/metabolismo , Proteômica , Idoso , Idoso de 80 Anos ou mais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Proteínas do Citoesqueleto , Eletroforese em Gel Bidimensional , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Pessoa de Meia-Idade , Chaperonas Moleculares/metabolismo , Proteínas de Neoplasias/genética , Mapeamento de Peptídeos , Dobramento de Proteína , Proteômica/métodos , RNA Mensageiro/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Células Tumorais CultivadasRESUMO
We tested two CO (2) extracts of Petasites hybridus L. rhizomes, A (rich in furanoeremophilanes) and B (rich in petasins), for IN VITRO cytotoxicity in rat hepatocytes by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (EC (50) values of 0.64 mg/mL for A and 0.32 mg/mL for B). Eight eremophilane sesquiterpene lactones (SL) (1- 8) and one petasin (9) isolated from A were nontoxic or showed moderate cytotoxicity. The presence and type of the ester side chain most probably control the extent of cytotoxicity of the eremophilanolides. (8 R)-2-[(angeloyl)oxy]eremophil-7(11)-en-12,8-olide (1) damaged the hepatocytes most. The 8 α-stereoisomers of both 8-H epimeric couples of the 2-angeloyloxy- and 2-methacroyloxy-esters seem to be more cytotoxic (up to approx. 10-fold) than the corresponding 8 ß-H stereoisomers. Moreover, the results of the MTT assay depended on the cell density being more pronounced with both 8 α-stereoisomers. Further investigations were conducted to study the influence of the stereochemistry on cell respiration, energy metabolism, and membrane integrity [release of lactate dehydrogenase (LDH)] with both couples of the 2-angeloyloxy- and 2-methacroyloxy-esters. In the LDH-leakage assay, (8 R)-2-[(methacroyl)oxy]eremophil-7(11)-en-12,8-olide (2) was the most toxic eremophilane. The stereoselectivity of cell damage of some SL points to a specific, yet unidentified molecular cytotoxicity target.