RESUMO
Amsacrine, an anticancer drug first synthesised in 1970 by Professor Cain and colleagues, showed excellent preclinical activity and underwent clinical trial in 1978 under the auspices of the US National Cancer Institute, showing activity against acute lymphoblastic leukaemia. In 1984, the enzyme DNA topoisomerase II was identified as a molecular target for amsacrine, acting to poison this enzyme and to induce DNA double-strand breaks. One of the main challenges in the 1980s was to determine whether amsacrine analogues could be developed with activity against solid tumours. A multidisciplinary team was assembled in Auckland, and Professor Denny played a leading role in this approach. Among a large number of drugs developed in the programme, N-[2-(dimethylamino)-ethyl]-acridine-4-carboxamide (DACA), first synthesised by Professor Denny, showed excellent activity against a mouse lung adenocarcinoma. It underwent clinical trial, but dose escalation was prevented by ion channel toxicity. Subsequent work led to the DACA derivative SN 28049, which had increased potency and reduced ion channel toxicity. Mode of action studies suggested that both amsacrine and DACA target the enzyme DNA topoisomerase II but with a different balance of cellular consequences. As primarily a topoisomerase II poison, amsacrine acts to turn the enzyme into a DNA-damaging agent. As primarily topoisomerase II catalytic inhibitors, DACA and SN 28049 act to inhibit the segregation of daughter chromatids during anaphase. The balance between these two actions, one cell cycle phase specific and the other nonspecific, together with pharmacokinetic, cytokinetic and immunogenic considerations, provides links between the actions of acridine derivatives and anthracyclines such as doxorubicin. They also provide insights into the action of cytotoxic DNA-binding drugs.
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Adenocarcinoma de Pulmão/tratamento farmacológico , Antineoplásicos , DNA de Neoplasias/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Inibidores da Topoisomerase II , Adenocarcinoma de Pulmão/história , Adenocarcinoma de Pulmão/metabolismo , Amsacrina/química , Amsacrina/história , Amsacrina/farmacocinética , Amsacrina/uso terapêutico , Anáfase/efeitos dos fármacos , Animais , Antineoplásicos/química , Antineoplásicos/história , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Cromátides/metabolismo , Segregação de Cromossomos/efeitos dos fármacos , DNA Topoisomerases Tipo II/metabolismo , História do Século XX , História do Século XXI , Humanos , Neoplasias Pulmonares/história , Neoplasias Pulmonares/metabolismo , Camundongos , Naftiridinas/química , Naftiridinas/farmacocinética , Naftiridinas/uso terapêutico , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacocinética , Inibidores da Topoisomerase II/uso terapêuticoRESUMO
The long non-coding RNA ANRIL, antisense to the CDKN2B locus, is transcribed from a gene that encompasses multiple disease-associated polymorphisms. Despite the identification of multiple isoforms of ANRIL, expression of certain transcripts has been found to be tissue-specific and the characterisation of ANRIL transcripts remains incomplete. Several functions have been associated with ANRIL. In our judgement, studies on ANRIL functionality are premature pending a more complete appreciation of the profusion of isoforms. We found differential expression of ANRIL exons, which indicates that multiple isoforms exist in melanoma cells. In addition to linear isoforms, we identified circular forms of ANRIL (circANRIL). Further characterisation of circANRIL in two patient-derived metastatic melanoma cell lines (NZM7 and NZM37) revealed the existence of a rich assortment of circular isoforms. Moreover, in the two melanoma cell lines investigated, the complements of circANRIL isoforms were almost completely different. Novel exons were also discovered. We also found the family of linear ANRIL was enriched in the nucleus, whilst the circular isoforms were enriched in the cytoplasm and they differed markedly in stability. With respect to the variable processing of circANRIL species, bioinformatic analysis indicated that intronic Arthrobacter luteus (Alu) restriction endonuclease inverted repeats and exon skipping were not involved in selection of back-spliced exon junctions. Based on our findings, we hypothesise that "ANRIL" has wholly distinct dual sets of functions in melanoma. This reveals the dynamic nature of the locus and constitutes a basis for investigating the functions of ANRIL in melanoma.
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Melanoma/genética , Isoformas de RNA/genética , RNA Longo não Codificante/genética , Neoplasias Cutâneas/genética , Linhagem Celular Tumoral , Éxons , Regulação Neoplásica da Expressão Gênica , Humanos , Conformação de Ácido Nucleico , Isoformas de RNA/análise , Splicing de RNA , RNA Longo não Codificante/análiseRESUMO
Melanocytes are melanin-producing cells found in skin, hair follicles, eyes, inner ear, bones, heart and brain of humans. They arise from pluripotent neural crest cells and differentiate in response to a complex network of interacting regulatory pathways. Melanins are pigment molecules that are endogenously synthesized by melanocytes. The light absorption of melanin in skin and hair leads to photoreceptor shielding, thermoregulation, photoprotection, camouflage and display coloring. Melanins are also powerful cation chelators and may act as free radical sinks. Melanin formation is a product of complex biochemical events that starts from amino acid tyrosine and its metabolite, dopa. The types and amounts of melanin produced by melanocytes are determined genetically and are influenced by a variety of extrinsic and intrinsic factors such as hormonal changes, inflammation, age and exposure to UV light. These stimuli affect the different pathways in melanogenesis. In this review we will discuss the regulatory mechanisms involved in melanogenesis and explain how intrinsic and extrinsic factors regulate melanin production. We will also explain the regulatory roles of different proteins involved in melanogenesis.
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Melaninas/metabolismo , Melanócitos/citologia , Melanócitos/metabolismo , Animais , Humanos , Transdução de SinaisRESUMO
BACKGROUND: The phosphatidylinositol-3-kinase (PI3K-PKB), mitogen activated protein kinase (MEK-ERK) and the mammalian target of rapamycin (mTOR- p70S6K), are thought to regulate many aspects of tumour cell proliferation and survival. We have examined the utilisation of these three signalling pathways in a number of cell lines derived from patients with metastatic malignant melanoma of known PIK3CA, PTEN, NRAS and BRAF mutational status. METHODS: Western blotting was used to compare the phosphorylation status of components of the PI3K-PKB, MEK-ERK and mTOR-p70S6K signalling pathways, as indices of pathway utilisation. RESULTS: Normal melanocytes could not be distinguished from melanoma cells on the basis of pathway utilisation when grown in the presence of serum, but could be distinguished upon serum starvation, where signalling protein phosphorylation was generally abrogated. Surprisingly, the differential utilisation of individual pathways was not consistently associated with the presence of an oncogenic or tumour suppressor mutation of genes in these pathways. CONCLUSION: Utilisation of the PI3K-PKB, MEK-ERK and mTOR-p70S6K signalling pathways in melanoma, as determined by phosphorylation of signalling components, varies widely across a series of cell lines, and does not directly reflect mutation of genes coding these components. The main difference between cultured normal melanocytes and melanoma cells is not the pathway utilisation itself, but rather in the serum dependence of pathway utilisation.
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Melanócitos/metabolismo , Melanoma/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Western Blotting , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases , Genes ras/genética , Humanos , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Proteínas Proto-Oncogênicas B-raf/genéticaRESUMO
AIM: SN 28049 (N-[2-(dimethylamino)ethyl]-2,6-dimethyl-1-oxo-1,2-dihydrobenzo[b]-1,6-naphthyridine-4-carboxamide) is a new DNA binding drug that targets topoisomerase II. SN 28049 is curative against the murine Colon 38 adenocarcinoma (CT38) while etoposide, another topoisomerase II-directed drug, shows minimal activity; we investigated the basis for this difference in vivo and in vitro. METHODS: Colon 38 tumours were grown in C57Bl mice and in immunodeficient mice. Tumour sections were examined by staining and TUNEL assays. A new cell line (Co-38P) derived from the in vivo tumour was developed and responses were analysed using flow cytometry. RESULTS: Both SN 28049 and etoposide induced similar tumour histological changes, reducing mitotic index and increasing apoptotic index 8 h after administration. At later times however, SN 28049-treated tumours showed further progressive morphological changes while etoposide-treated tumours reverted to their original growth characteristics. The effects of SN 28049 on tumour growth were delayed and attenuated when Colon 38 tumours were grown in immunodeficient mice. SN 28049 and etoposide both induced dose-dependent increases of γ-phosphorylation of histone H2AX and cell cycle perturbation of the Co-38P cell line, indicative of DNA damage, although SN 28049 had 30-fold higher activity. Following 1-hour drug exposure of Co-38P cells, SN 28049 was more effective that etoposide in inducing persistent cycle arrest for the same degree of DNA damage. CONCLUSION: The superior antitumour activity of SN 28049 may result from its ability to induce long term cycle arrest. Host immune responses contribute to the curative activity of SN 28049 and this could result from the induction of cycle arrest.
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Antineoplásicos/farmacologia , Neoplasias do Colo/tratamento farmacológico , DNA Topoisomerases Tipo II/metabolismo , Naftiridinas/farmacologia , Animais , Antineoplásicos/administração & dosagem , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Dano ao DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Etoposídeo/administração & dosagem , Etoposídeo/farmacologia , Feminino , Citometria de Fluxo , Marcação In Situ das Extremidades Cortadas , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Naftiridinas/administração & dosagem , Fosforilação/efeitos dos fármacosRESUMO
AIM: We have examined the cellular action of SN 28049 (N-[2-(dimethylamino)ethyl]-2,6-dimethyl-1-oxo-1,2-dihydrobenzo[b]-1,6-naphthyridine-4-carboxamide), a DNA binding drug with curative activity against the Colon 38 transplantable murine carcinoma, on human tumour cells. Its action has been compared with that of two topoisomerase II-targetted drugs, etoposide and doxorubicin. METHODS: The NZM3 melanoma and HCT116 colon carcinoma cell lines, each expressing wild-type p53, were cultured and responses were compared by flow cytometry, electrophoresis, microscopy, and growth of tumour xenografts. RESULTS: Responses of NZM3 cells to all three drugs, as measured by histone H2AX γ-phosphorylation, induction of the p53 pathway and cell cycle arrest, were comparable and typical of those of topoisomerase II poisons. Xenografts of NZM3 cells responded to SN 28049 with a tumour growth delay of 16 days. In contrast, HCT116 cells had an attenuated DNA damage response to the drugs and SN 28049 had no in vivo activity, consistent with low topoisomerase II activity. However, SN 28049 inhibited HCT116 cell growth in vitro and activated the p53 pathway to induce a state with G(2)/M-phase DNA content, low mitotic index and a high proportion of binucleate cells. Treated cells expressed cyclin E and the senescence marker ß-galactosidase but showed low expression of cyclin B and survivin. In comparison, etoposide caused little p53 expression or cycle arrest, and doxorubicin had an intermediate effect. CONCLUSION: The action of SN 28049 in NZM3 cells is typical of a topoisomerase II poison, but the low topoisomerase IIα activity of HCT116 cells allowed the detection of a second antiproliferative action of SN 28049 in which cells undergo post-mitotic cycle arrest and induction of p53.
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Antineoplásicos/farmacologia , Proteínas de Ligação a DNA/antagonistas & inibidores , DNA/metabolismo , Doxorrubicina/farmacologia , Etoposídeo/farmacologia , Naftiridinas/farmacologia , Animais , Antígenos de Neoplasias/metabolismo , Antineoplásicos/química , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Citometria de Fluxo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Células HCT116 , Humanos , Concentração Inibidora 50 , Camundongos , Naftiridinas/química , Transdução de Sinais/efeitos dos fármacos , Tetraploidia , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Receptor fas/metabolismoRESUMO
Electric cell-substrate impedance sensing (ECIS) is an impedance-based method for monitoring changes in cell behaviour in real-time. In this paper, we highlight the importance of ECIS in measuring the kinetics of human melanoma cell invasion across human brain endothelium. ECIS data can be mathematically modelled to assess which component of the endothelial paracellular and basolateral barriers is being affected and when. Our results reveal that a range of human melanoma cells can mediate disruption of human brain endothelium, primarily involving the paracellular route, as demonstrated by ECIS. The sensitivity of ECIS also reveals that the paracellular barrier weakens within 30-60 min of the melanoma cells being added to the apical face of the endothelial cells. Imaging reveals pronounced localisation of the melanoma cells at the paracellular junctions consistent with paracellular migration. Time-lapse imaging further reveals junctional opening and disruption of the endothelial monolayer by the invasive melanoma cells all within several hours. We suggest that the ability of ECIS to resolve changes to barrier integrity in real time, and to determine the route of migration, provides a powerful tool for future studies investigating the key molecules involved in the invasive process of cancer cells.
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Técnicas Biossensoriais , Barreira Hematoencefálica/patologia , Encéfalo/patologia , Células Endoteliais/patologia , Melanoma/patologia , Neoplasias Cutâneas/patologia , Impedância Elétrica , Humanos , Fatores de Tempo , Melanoma Maligno CutâneoRESUMO
PURPOSE: SN 28049 (N-[2-(dimethylamino)ethyl]-2,6-dimethyl-1-oxo-1,2-dihydrobenzo[b]-1,6-naphthyridine-4-carboxamide) is a DNA intercalating drug that binds selectively to GC-rich DNA and shows curative activity against the Colon 38 adenocarcinoma in mice. We wished to investigate the roles of topoisomerase (topo) I, topo II and RNA transcription in the action of SN 28049. METHODS: We used clonogenic assays to study the cytotoxicity of SN 28049; RNA interference and enzyme assays to examine the role of topo I in SN 28049 action; 3H uridine incorporation and reporter assays to study its effects on transcription; and RT-PCR to examine its ability to reduce endogenous h-TERT expression. RESULTS: In clonogenic assays, SN 28049 showed a biphasic cytotoxic dose response curve in H460 cells typical of acridine derivatives such as N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA) although it was approximately 16-fold more potent. Down-regulation of topo IIalpha in HTETOP cells reduced the cytotoxicity of SN 28049, establishing its action as a topo IIalpha poison. Surprisingly, down-regulation of topo I in H460 cells by RNA interference sensitised them to the actions of SN 28049 and other topo II poisons. SN 28049 also inhibited topo I-mediated relaxation of supercoiled plasmid DNA. SN 28049 was also an inhibitor of transcription in HEK293 cells and was more potent at reducing luciferase expression from a GC-rich SP-1 binding promoter than from a non-GC-rich AP-1 binding promoter. The drug also reduced luciferase reporter gene expression driven by the SP-1-binding survivin promoter as well as reducing endogenous h-TERT expression in HEK293 cells whose promoter also contains SP-1 binding sites. CONCLUSION: We conclude that SN 28049 has a complex action that may involve poisoning of topo IIalpha, suppression of topo I and inhibition of gene transcription from promoters with SP-1 sites. These actions may contribute to the promising experimental solid tumour anticancer activity of SN 28049.
Assuntos
Antineoplásicos/farmacologia , DNA Topoisomerases Tipo I/fisiologia , Naftiridinas/farmacologia , RNA Neoplásico/biossíntese , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Expressão Gênica/efeitos dos fármacos , Genes Reporter/genética , Humanos , Luciferases/genética , Inibidores da Síntese de Proteínas/farmacologia , Interferência de RNA , RNA Neoplásico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tetraciclina/farmacologia , Transcrição Gênica/fisiologia , Uridina/metabolismoRESUMO
Background: Most human breast cancer cell lines currently in use were developed and are cultured under ambient (21%) oxygen conditions. While this is convenient in practical terms, higher ambient oxygen could increase oxygen radical production, potentially modulating signaling pathways. We have derived and grown a series of four human breast cancer cell lines under 5% oxygen, and have compared their properties to those of established breast cancer lines growing under ambient oxygen. Methods: Cell lines were characterized in terms of appearance, cellular DNA content, mutation spectrum, hormone receptor status, pathway utilization and drug sensitivity. Results: Three of the four lines (NZBR1, NZBR2, and NZBR4) were triple negative (ER-, PR-, HER2-), with NZBR1 also over-expressing EGFR. NZBR3 was HER2+ and ER+ and also over-expressed EGFR. Cell lines grown in 5% oxygen showed increased expression of the hypoxia-inducible factor 1 (HIF-1) target gene carbonic anhydrase 9 (CA9) and decreased levels of ROS. As determined by protein phosphorylation, NZBR1 showed low AKT pathway utilization while NZBR2 and NZBR4 showed low p70S6K and rpS6 pathway utilization. The lines were characterized for sensitivity to 7-hydroxytamoxifen, doxorubicin, paclitaxel, the PI3K inhibitor BEZ235 and the HER inhibitors lapatinib, afatinib, dacomitinib, and ARRY-380. In some cases they were compared to established breast cancer lines. Of particular note was the high sensitivity of NZBR3 to HER inhibitors. The spectrum of mutations in the NZBR lines was generally similar to that found in commonly used breast cancer cell lines but TP53 mutations were absent and mutations in EVI2B, LRP1B, and PMS2, which have not been reported in other breast cancer lines, were detected. The results suggest that the properties of cell lines developed under low oxygen conditions (5% O2) are similar to those of commonly used breast cancer cell lines. Although reduced ROS production and increased HIF-1 activity under 5% oxygen can potentially influence experimental outcomes, no difference in sensitivity to estrogen or doxorubicin was observed between cell lines cultured in 5 vs. 21% oxygen.
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INTRODUCTION: Endocrine therapy of breast cancer, which either deprives cancer tissue of estrogen or prevents estrogen pathway signaling, is the most common treatment after surgery and radiotherapy. We have previously shown for the estrogen-responsive MCF-7 cell line that exposure to tamoxifen, or deprivation of estrogen, leads initially to inhibition of cell proliferation, followed after several months by the emergence of resistant sub-lines that are phenotypically different from the parental line. We examined the early responses of MCF-7 cells following either exposure to 4-hydroxytamoxifen or deprivation of estrogen for periods of 2 days-4 weeks. METHODS: Endocrine-sensitive or -resistant breast cancer cell lines were used to examine the expression of the stem cell gene SOX2, and the Wnt effector genes AXIN2 and DKK1 using quantitative PCR analysis. Breast cancer cell lines were used to assess the anti-proliferative effects (as determined by IC50 values) of Wnt pathway inhibitors LGK974 and IWP-2. RESULTS: Hormone therapy led to time-dependent increases of up to 10-fold in SOX2 expression, up to threefold in expression of the Wnt target genes AXIN2 and DKK1, and variable changes in NANOG and OCT4 expression. The cells also showed increased mammosphere formation and increased CD24 surface protein expression. Some but not all hormone-resistant MCF-7 sub-lines, emerging after long-term hormonal stress, showed up to 50-fold increases in SOX2 expression and smaller increases in AXIN2 and DKK1 expression. However, the increase in Wnt target gene expression was not accompanied by an increase in sensitivity to Wnt pathway inhibitors LGK974 and IWP-2. A general trend of lower IC50 values was observed in 3-dimensional spheroid culture conditions (which allowed enrichment of cells with cancer stem cell phenotype) relative to monolayer cultures. The endocrine-resistant cell lines showed no significant increase in sensitivity to Wnt inhibitors. CONCLUSION: Hormone treatment of cultured MCF-7 cells leads within 2 days to increased expression of components of the SOX2 and Wnt pathways and to increased potential for mammosphere formation. We suggest that these responses are indicative of early adaptation to endocrine stress with features of stem cell character and that this facilitates the survival of emerging hormone-resistant cell populations.
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BACKGROUND: Most of the eukaryotic genome is transcribed, yielding a complex network of transcripts including thousands of lncRNAs that generally lack protein coding potential. However, only a small percentage of these molecules has been functionally characterised, and discoveries of specific functions demonstrate layers of complexity. A large percentage of lncRNAs is located in the cytoplasm, associated with ribosomes but the function of the majority of these transcripts is unclear. The current study analyses putative mechanisms of action of the lncRNA species member ZFAS1 that was initially discovered by microarray analysis of murine tissues undergoing mammary gland development. As developmental genes are often deregulated in cancer, here we have studied its function in breast cancer cell lines. RESULTS: Using human breast cancer cell lines, ZFAS1 was found to be expressed in all cell lines tested, albeit at different levels of abundance. Following subcellular fractionation, human ZFAS1 was found in both nucleus and cytoplasm (as is the mouse orthologue) in an isoform-independent manner. Sucrose gradients based on velocity sedimentation were utilised to separate the different components of total cell lysate, and surprisingly ZFAS1 was primarily co-localised with light polysomes. Further investigation into ribosome association through subunit dissociation studies showed that ZFAS1 was predominantly associated with the 40S small ribosomal subunit. The expression levels of ZFAS1 and of mRNAs encoding several ribosomal proteins that have roles in ribosome assembly, production and maturation were tightly correlated. ZFAS1 knockdown significantly reduced RPS6 phosphorylation. CONCLUSION: A large number of lncRNAs associate with ribosomes but the function of the majority of these lncRNAs has not been elucidated. The association of the lncRNA ZFAS1 with a subpopulation of ribosomes and the correlation with expression of mRNAs for ribosomal proteins suggest a ribosome-interacting mechanism pertaining to their assembly or biosynthetic activity. ZFAS1 may represent a new class of lncRNAs which associates with ribosomes to regulate their function. REVIEWERS: This article was reviewed by Christine Vande Velde, Nicola Aceto and Haruhiko Siomi.
Assuntos
Neoplasias da Mama/genética , RNA Longo não Codificante/genética , Ribossomos/genética , Linhagem Celular Tumoral , Núcleo Celular/genética , Citoplasma/genética , Humanos , Isoformas de Proteínas/química , RNA Longo não Codificante/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismoRESUMO
GRIN2A mutations are frequent in melanoma tumours but their role in disease is not well understood. GRIN2A encodes a modulatory subunit of the N-methyl-d-aspartate receptor (NMDAR). We hypothesized that certain GRIN2A mutations increase NMDAR function and support melanoma growth through oncogenic effects. This hypothesis was tested using 19 low-passage melanoma cell lines, four of which carried novel missense mutations in GRIN2A that we previously reported. We examined NMDAR expression, function of a calcium ion (Ca2+) channel and its contribution to cell growth using pharmacological modulators; findings were correlated with the presence or absence of GRIN2A mutations. We found that NMDAR expression was low in all melanoma cell lines, independent of GRIN2A mutations. In keeping with this, NMDAR-mediated Ca2+ influx and its contribution to cell proliferation were weak in most cell lines. However, certain GRIN2A mutations and culture media with lower glutamate levels enhanced NMDAR effects on cell growth and invasion. The main finding was that G762E was associated with higher glutamate-mediated Ca2+ influx and stronger NMDAR contribution to cell proliferation, compared with wild-type GRIN2A and other GRIN2A mutations. The pro-invasive phenotype of mutated cell lines was increased in culture medium containing less glutamate, implying environmental modulation of mutation effects. In conclusion, NMDAR ion channel function is low in cultured melanoma cells but supports cell proliferation and invasion. Selected GRIN2A mutations, such as G762E, are associated with oncogenic consequences that can be modulated by extracellular glutamate. Primary cultures may be better suited to determine the role of the NMDAR in melanoma in vivo.
Assuntos
Ácido Glutâmico/farmacologia , Melanoma/genética , Receptores de N-Metil-D-Aspartato/genética , Cálcio/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Melanoma/patologia , Mutação , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Relação Estrutura-AtividadeRESUMO
The mammalian target of rapamycin (mTOR), a vital component of signaling pathways involving PI3K/AKT, is an attractive therapeutic target in breast cancer. Everolimus, an allosteric mTOR inhibitor that inhibits the mTOR functional complex mTORC1, is approved for treatment of estrogen receptor positive (ER+) breast cancer. Other mTOR inhibitors show interesting differences in target specificities: BEZ235 and GSK2126458 are ATP competitive mTOR inhibitors targeting both PI3K and mTORC1/2; AZD8055, AZD2014 and KU-0063794 are ATP competitive mTOR inhibitors targeting both mTORC1 and mTORC2; and GDC-0941 is a pan-PI3K inhibitor. We have addressed the question of whether mTOR inhibitors may be more effective in combination than singly in inhibiting the proliferation of breast cancer cells. We selected a panel of 30 human breast cancer cell lines that included ER and PR positive, HER2 over-expressing, and "triple negative" variants, and determined whether signaling pathway utilization was related to drug-induced inhibition of proliferation. A significant correlation (p = 0.005) was found between everolimus IC50 values and p70S6K phosphorylation, but not with AKT or ERK phosphorylation, consistent with the mTOR pathway being a principal target. We then carried out combination studies with four everolimus resistant triple-negative breast cancer cell lines, and found an unexpectedly high degree of synergy between everolimus and the other inhibitors tested. The level of potentiation of everolimus inhibitory activity (measured by IC50 values) was found to be cell line-specific for all the kinase inhibitors tested. The results suggest that judicious combination of mTOR inhibitors with different modes of action could have beneficial effects in the treatment of breast cancer.
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Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Complexos Multiproteicos/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Everolimo/farmacologia , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células MCF-7 , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The development and progression of melanoma have been attributed to independent or combined genetic and epigenetic events. There has been remarkable progress in understanding melanoma pathogenesis in terms of genetic alterations. However, recent studies have revealed a complex involvement of epigenetic mechanisms in the regulation of gene expression, including methylation, chromatin modification and remodeling, and the diverse activities of non-coding RNAs. The roles of gene methylation and miRNAs have been relatively well studied in melanoma, but other studies have shown that changes in chromatin status and in the differential expression of long non-coding RNAs can lead to altered regulation of key genes. Taken together, they affect the functioning of signaling pathways that influence each other, intersect, and form networks in which local perturbations disturb the activity of the whole system. Here, we focus on how epigenetic events intertwine with these pathways and contribute to the molecular pathogenesis of melanoma.
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Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Carcinogênese/genética , Montagem e Desmontagem da Cromatina , Metilação de DNA , Histonas/metabolismo , Humanos , Melanoma/patologia , Processamento de Proteína Pós-Traducional , Proto-Oncogene Mas , RNA Longo não Codificante/metabolismo , Transdução de SinaisRESUMO
The majority of the human genome is transcribed, even though only 2% of transcripts encode proteins. Non-coding transcripts were originally dismissed as evolutionary junk or transcriptional noise, but with the development of whole genome technologies, these non-coding RNAs (ncRNAs) are emerging as molecules with vital roles in regulating gene expression. While shorter ncRNAs have been extensively studied, the functional roles of long ncRNAs (lncRNAs) are still being elucidated. Studies over the last decade show that lncRNAs are emerging as new players in a number of diseases including cancer. Potential roles in both oncogenic and tumor suppressive pathways in cancer have been elucidated, but the biological functions of the majority of lncRNAs remain to be identified. Accumulated data are identifying the molecular mechanisms by which lncRNA mediates both structural and functional roles. LncRNA can regulate gene expression at both transcriptional and post-transcriptional levels, including splicing and regulating mRNA processing, transport, and translation. Much current research is aimed at elucidating the function of lncRNAs in breast cancer and mammary gland development, and at identifying the cellular processes influenced by lncRNAs. In this paper we review current knowledge of lncRNAs contributing to these processes and present lncRNA as a new paradigm in breast cancer development.
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The MCF-7 line, derived in 1973 from a malignant pleural effusion, is one of the most commonly used culture models for human breast cancer. Despite its long history, MCF-7 is a surprisingly heterogeneous line. We previously showed that if MCF-7 cells were cultured for a prolonged period either in the absence of estrogen or in the presence of the antiestrogen tamoxifen, sub-lines were selected that differed from the parental line in ploidy, mean cell volume, signaling pathway usage, and drug sensitivity. This suggests a process of selection of preexisting variants rather than of adaptation of the parental line. All the sublines were estrogen receptor (ER) positive, raising the question of whether MCF-7 also contains ER negative variants. Here, we have looked for such variants by culturing for a prolonged period in the presence of fulvestrant, an estrogen antagonist that has no estrogen agonist activity. Three sublines were developed, each of which was ER negative, progesterone receptor (PR) negative and expressed only a low level of HER2. Each of the variants differed from the original MCF-7 line in ploidy, modal cell volume, and signaling pathway usage. Control experiments in which cells were cultured for a prolonged period in the absence of estrogen selected for variants that were ER and PR positive. The properties of the triple-negative MCF-7 were compared with those of an existing triple-negative cell line, MDA-MB-231, and human epidermal growth factor receptor 2 (HER2)+ SKBr3, as well as from those of the "immortalized" breast epithelial line MCF10A. The results suggest that new variants or phenotypes of MCF-7 might be generated continuously in culture, and by implication this might apply to breast cancer development and even normal breast epithelial development in vivo.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Linhagem Celular Tumoral , Feminino , HumanosRESUMO
PURPOSE: SN 28049 is a new DNA-binding topoisomerase II poison identified by its curative activity against the murine colon 38 carcinoma. Previous studies showed activity to be associated with selective drug accumulation and retention in tumour tissue. Retention varied widely among different tumours and was related to antitumour activity. We determined whether differences in the uptake and retention of SN 28049 could be observed in vitro. METHODS: The Co38P and LLTC lines were derived from the murine colon 38 carcinoma and Lewis lung carcinoma (3LL), respectively. The NZM4, NZM10 and NZM52 human melanoma lines, as well as the CCRF/CEM, CEM/VLB100 and CEM/E1000 human leukaemia lines were also utilised. Cell-associated drug was measured by liquid chromatography-mass spectrometry, laser-scanning confocal microscopy and fluorescence microscopy. Data for SN 28049 were compared for four SN 28049 analogues, for the structurally related drug N-[2-(dimethylamino)-ethyl]acridine-4-carboxamide (DACA) and for doxorubicin. RESULTS: Cellular uptake of SN 28049 was rapid and associated with increased fluorescence in cytoplasmic vesicles or bodies. SN 28049 uptake after an incubation time of 1 h varied widely with different cell lines (2-98 pmol/106 cells) and did not correlate with growth inhibitory concentrations (IC50 values), which also varied widely (1.2-19 nM). Changes in the length of the N-linked side chain of SN 28049 had large effects on drug uptake by Co38P cells. SN 28049 uptake by CCRF/CEM cells was only slightly affected by the expression of P-glycoprotein (CEM/VLB100) or MRP1 protein (CEM/E1000). As measured by cytoplasmic fluorescence, SN 28049 was taken up rapidly and retained strongly by Co38P cells, DACA was taken up rapidly and retained poorly, and doxorubicin was taken up slowly and retained moderately. CONCLUSIONS: The results suggest that SN 28049 is actively transported into cytoplasmic vesicles. While vesicle-associated drug is not important for intrinsic cytotoxicity, it may play a key role as a "slow release" form that modifies pharmacokinetics in multicellular structures such as tumours.
Assuntos
Antineoplásicos/metabolismo , Carcinoma/metabolismo , Drogas em Investigação/metabolismo , Leucemia/metabolismo , Melanoma/metabolismo , Naftiridinas/metabolismo , Inibidores da Topoisomerase II/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Transporte Biológico Ativo , Carcinoma/tratamento farmacológico , Carcinoma/ultraestrutura , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/metabolismo , Vesículas Citoplasmáticas/ultraestrutura , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Drogas em Investigação/química , Drogas em Investigação/farmacologia , Humanos , Cinética , Leucemia/tratamento farmacológico , Leucemia/patologia , Melanoma/tratamento farmacológico , Melanoma/ultraestrutura , Camundongos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Naftiridinas/química , Naftiridinas/farmacologia , Proteínas de Neoplasias/metabolismo , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacologiaRESUMO
The transcription factor SOX2 is essential for maintaining pluripotency in a variety of stem cells. It has important functions during embryonic development, is involved in cancer stem cell maintenance, and is often deregulated in cancer. The mechanism of SOX2 regulation has yet to be clarified, but the SOX2 gene lies in an intron of a long multi-exon non-coding RNA called SOX2 overlapping transcript (SOX2OT). Here, we show that the expression of SOX2 and SOX2OT is concordant in breast cancer, differentially expressed in estrogen receptor positive and negative breast cancer samples and that both are up-regulated in suspension culture conditions that favor growth of stem cell phenotypes. Importantly, ectopic expression of SOX2OT led to an almost 20-fold increase in SOX2 expression, together with a reduced proliferation and increased breast cancer cell anchorage-independent growth. We propose that SOX2OT plays a key role in the induction and/or maintenance of SOX2 expression in breast cancer.
Assuntos
Neoplasias da Mama/genética , RNA Longo não Codificante/genética , Fatores de Transcrição SOXB1/genética , Animais , Neoplasias da Mama/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Mamárias Experimentais , Camundongos , Células-Tronco Neoplásicas/metabolismo , Regulação para CimaRESUMO
Cellular signaling pathways involving mTOR, PI3K and ERK have dominated recent studies of breast cancer biology, and inhibitors of these pathways have formed a focus of numerous clinical trials. We have chosen trametinib, a drug targeting MEK in the ERK pathway, to address two questions. Firstly, does inhibition of a signaling pathway, as measured by protein phosphorylation, predict the antiproliferative activity of trametinib? Secondly, do inhibitors of the mTOR and PI3K pathways synergize with trametinib in their effects on cell proliferation? A panel of 30 human breast cancer cell lines was chosen to include lines that could be classified according to whether they were ER and PR positive, HER2 over-expressing, and "triple negative". Everolimus (targeting mTOR), NVP-BEZ235 and GSK2126458 (both targeting PI3K/mTOR) were chosen for combination experiments. Inhibition of cell proliferation was measured by IC50 values and pathway utilization was measured by phosphorylation of signaling kinases. Overall, no correlation was found between trametinib IC50 values and inhibition of ERK signaling. Inhibition of ERK phosphorylation was observed at trametinib concentrations not affecting proliferation, and sensitivity of cell proliferation to trametinib was found in cell lines with low ERK phosphorylation. Evidence was found for synergy between trametinib and either everolimus, NVP-BEZ235 or GSK2126458, but this was cell line specific. The results have implications for the clinical application of PI3K/mTOR and MEK inhibitors.