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Lung cancer is the leading cause of cancer-related death worldwide, and ~85% of lung cancers are non-small cell lung cancer (NSCLC), which has a low 5-year overall survival rate and high mortality. Several therapeutic strategies have been developed, such as targeted therapy, immuno-oncotherapy and combination therapy. However, the low survival rate indicates the urgent need for new NSCLC treatments. Vasculogenic mimicry (VM) is an endothelial cell-free tumor blood supply system of aggressive and metastatic tumor cells present during tumor neovascularization. VM is clinically responsible for tumor metastasis and resistance, and is correlated with poor prognosis in NSCLC, making it a potential therapeutic target. In the present study, A549 cells formed glycoprotein-rich lined tubular structures, and transcript levels of VM-related genes were markedly upregulated in VM-forming cells. Based on a drug repurposing strategy, it was demonstrated that doxazosin (an antihypertensive drug) displayed inhibitory activity on VM formation at non-cytotoxic concentrations. Doxazosin significantly reduced the levels of vascular endothelial growth factor A (VEGF-A) and matrix metalloproteinase-2 (MMP-2) in the cell media during VM formation. Further experiments revealed that the protein expression levels of VEGF-A and vascular endothelial-cadherin (VE-cadherin), which contribute to tumor aggressiveness and VM formation, were downregulated following doxazosin treatment. Moreover, the downstream signaling Ephrin type-A receptor 2 (EphA2)/AKT/mTOR/MMP/Laminin-5γ2 network was inhibited in response to doxazosin treatment. In conclusion, the present study demonstrated that doxazosin displayed anti-VM activity in an NSCLC cell model through the downregulation of VEGF-A and VE-cadherin levels, and the suppression of signaling pathways related to the receptor tyrosine kinase, EphA2, protein kinases, AKT and mTOR, and proteases, MMP-2 and MMP-9. These results support the add-on anti-VM effect of doxazosin as a potential agent against NSCLC.
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The treatment of non-small cell lung cancer (NSCLC) is known as a significant level of unmet medical need in spite of the progress in targeted therapy and personalized therapy. Overexpression of the Na+/K+-ATPase contributes to NSCLC progression, suggesting its potentiality in antineoplastic approaches. Epi-reevesioside F, purified from Reevesia formosana, showed potent anti-NSCLC activity through inhibiting the Na+/K+-ATPase, leading to internalization of α1- and α3-subunits in Na+/K+-ATPase and suppression of Akt-independent mTOR-p70S6K-4EBP1 axis. Epi-reevesioside F caused a synergistic amplification of apoptosis induced by gefitinib but not cisplatin, docetaxel, etoposide, paclitaxel, or vinorelbine in both NCI-H460 and A549 cells. The synergism was validated by enhanced activation of the caspase cascade. Bax cleavage, tBid formation, and downregulation of Bcl-xL and Bcl-2 contributed to the synergistic apoptosis induced by the combination treatment of epi-reevesioside F and gefitinib. The increase of membrane DR4 and DR5 levels, intracellular Ca2+ concentrations, and active m-calpain expression were responsible for the caspase-8 activation and Bax cleavage. The increased α-tubulin acetylation and activation of MAPK (i.e., p38 MAPK, Erk, and JNK) depending on cell types contributed to the synergistic mechanism under combination treatment. These signaling pathways that converged on profound c-Myc downregulation led to synergistic apoptosis in NSCLC. In conclusion, the data suggest that epi-reevesioside F inhibits the Na+/K+-ATPase and displays potent anti-NSCLC activity. Epi-reevesioside F sensitizes gefitinib-induced apoptosis through multiple pathways that converge on c-Myc downregulation. The data support the inhibition of Na+/K+-ATPase as a switch-on mechanism to sensitize gefitinib-induced anti-NSCLC activity.
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BACKGROUND: Metastatic castration-resistant prostate cancer (CRPC), the most refractory prostate cancer, inevitably progresses and becomes unresponsive to hormone therapy, revealing a pressing unmet need for this disease. Novel agents targeting HDAC6 and microtubule dynamics can be a potential anti-CRPC strategy. METHODS: Cell proliferation was examined in CRPC PC-3 and DU-145 cells using sulforhodamine B assay and anchorage-dependent colony formation assay. Flow cytometric analysis of propidium iodide staining was used to determine cell-cycle progression. Cell-based tubulin polymerization assay and confocal immunofluorescence microscopic examination determine microtubule assembly/disassembly status. Protein expressions were determined using Western blot analysis. RESULTS: A total of 82 novel derivatives targeting HDAC6 were designed and synthesized, and Compound 25202 stood out, showing the highest efficacy in blocking HDAC6 (IC50, 3.5 nM in enzyme assay; IC50, 1.0 µM in antiproliferative assay in CRPC cells), superior to tubastatin A (IC50, 5.4 µM in antiproliferative assay). The selectivity and superiority of 25202 were validated by examining the acetylation of both α-tubulin and histone H3, detecting cell apoptosis and HDACs enzyme activity assessment. Notably, 25202 but not tubastatin A significantly decreased HDAC6 protein expression. 25202 prolonged mitotic arrest through the detection of cyclin B1 upregulation, Cdk1 activation, mitotic phosphoprotein levels, and Bcl-2 phosphorylation. Compound 25202 did not mimic docetaxel in inducing tubulin polymerization but disrupted microtubule organization. Compound 25202 also increased the phosphorylation of CDC20, BUB1, and BUBR1, indicating the activation of the spindle assembly checkpoint (SAC). Moreover, 25202 profoundly sensitized cisplatin-induced cell death through impairment of cisplatin-evoked DNA damage response and DNA repair in both ATR-Chk1 and ATM-Chk2 pathways. CONCLUSION: The data suggest that 25202 is a novel selective and potent HDAC6 inhibitor. Compound 25202 blocks HDAC6 activity and interferes microtubule dynamics, leading to SAC activation and mitotic arrest prolongation that eventually cause apoptosis of CRPC cells. Furthermore, 25202 sensitizes cisplatin-induced cell apoptosis through impeding DNA damage repair pathways.
Assuntos
Cisplatino , Neoplasias de Próstata Resistentes à Castração , Masculino , Humanos , Cisplatino/farmacologia , Neoplasias de Próstata Resistentes à Castração/patologia , Tubulina (Proteína)/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular , Linhagem Celular Tumoral , Apoptose , Proliferação de Células , Microtúbulos/metabolismo , Microtúbulos/patologia , Desacetilase 6 de Histona/metabolismoRESUMO
BACKGROUND: Castration-resistant prostate cancer (CRPC) is refractory to hormone treatment and the therapeutic options are continuously advancing. This study aims to discover the anti-CRPC effects and underlying mechanisms of small-molecule compounds targeting topoisomerase (TOP) II and cellular components of DNA damage repair. METHODS: Cell proliferation was determined in CRPC PC-3 and DU-145 cells using anchorage-dependent colony formation, sulforhodamine B assay and flow cytometric analysis of CFSE staining. Flow cytometric analyses of propidium iodide staining and JC-1 staining were used to examine the population of cell-cycle phases and mitochondrial membrane potential, respectively. Nuclear extraction was performed to detect the nuclear localization of cellular components in DNA repair pathways. Protein expressions were determined using Western blot analysis. RESULTS: A series of azathioxanthone-based derivatives were synthesized and examined for bioactivities in which WC-A13, WC-A14, WC-A15, and WC-A16 displayed potent anti-CRPC activities in both PC-3 and DU-145 cell models. These WC-A compounds selectively downregulated both TOP IIα and TOP IIß but not TOP I protein expression. WC-A13, WC-A14, and WC-A15 were more potent than WC-A16 on TOP II inhibition, mitochondrial dysfunction, and induction of caspase cascades indicating the key role of amine-containing side chain of the compounds in determining anti-CRPC activities. Furthermore, WC-A compounds induced an increase of γH2AX and activated ATR-Chk1 and ATM-Chk2 signaling pathways. P21 protein expression was also upregulated by WC-A compounds in which WC-A16 showed the least activity. Notably, WC-A compounds exhibited different regulation on Rad51, a major protein in homologous recombination of DNA in double-stranded break repair. WC-A13, WC-A14, and WC-A15 inhibited, whereas WC-A16 induced, the nuclear translocation of Rad51. CONCLUSION: The data suggest that WC-A compounds exhibit anti-CRPC effects through the inhibition of TOP II activities, leading to mitochondrial stress-involved caspase activation and apoptosis. Moreover, WC-A13, WC-A14, and WC-A15 but not WC-A16 display inhibitory activities of Rad51-mediated DNA repair pathway which may increase apoptotic effect of CRPC cells.
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Antineoplásicos , Neoplasias de Próstata Resistentes à Castração , Masculino , Humanos , Antineoplásicos/uso terapêutico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/metabolismo , Linhagem Celular Tumoral , Apoptose , Proliferação de Células , Caspases/metabolismo , Caspases/farmacologia , Caspases/uso terapêutico , Reparo do DNA , DNA Topoisomerases Tipo II/metabolismo , DNA Topoisomerases Tipo II/farmacologia , DNA Topoisomerases Tipo II/uso terapêuticoRESUMO
Monocots are a major taxon within flowering plants, have unique morphological traits, and show an extraordinary diversity in lifestyle. To improve our understanding of monocot origin and evolution, we generate chromosome-level reference genomes of the diploid Acorus gramineus and the tetraploid Ac. calamus, the only two accepted species from the family Acoraceae, which form a sister lineage to all other monocots. Comparing the genomes of Ac. gramineus and Ac. calamus, we suggest that Ac. gramineus is not a potential diploid progenitor of Ac. calamus, and Ac. calamus is an allotetraploid with two subgenomes A, and B, presenting asymmetric evolution and B subgenome dominance. Both the diploid genome of Ac. gramineus and the subgenomes A and B of Ac. calamus show clear evidence of whole-genome duplication (WGD), but Acoraceae does not seem to share an older WGD that is shared by most other monocots. We reconstruct an ancestral monocot karyotype and gene toolkit, and discuss scenarios that explain the complex history of the Acorus genome. Our analyses show that the ancestors of monocots exhibit mosaic genomic features, likely important for that appeared in early monocot evolution, providing fundamental insights into the origin, evolution, and diversification of monocots.
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Acorus , Tetraploidia , Filogenia , Diploide , GenomaRESUMO
Breast cancer is the most prevalent cancer and the second leading cause of cancer death in women. Cisplatin is a commonly used chemotherapeutic drug for breast cancer treatment. Owing to serious side effects, the combination of cisplatin with other drugs is an effective strategy to simultaneously reduce side effects and increase the anticancer efficacy. GLUT1 is an emerging target for cancer treatment since cancer cells usually consume more glucose, a phenomenon called the Warburg effect. In this study, we found that the combination of cisplatin and a novel GLUT1 inhibitor #43 identified from our previous high-throughput screening exerted a synergistic anticancer effect in MCF-7 and MDA-MB-231 breast cancer cells. Mechanism studies in MCF-7 cells revealed that combination of cisplatin and #43 significantly induced apoptosis, intracellular reactive oxygen species, and loss of mitochondrial membrane potential. Furthermore, #43 enhanced the DNA damaging effect of cisplatin. Akt/mTOR downstream signaling and the ERK signaling pathway usually involved in cell growth and survival were inhibited by the combination treatment. On the other hand, phosphorylation of p38 and JNK, which may be associated with apoptosis, was induced by the combination treatment. Altogether, our data indicate that oxidative stress, DNA damage, the Akt/mTOR and MAPK signaling pathways, and apoptosis may be involved in the synergism of cisplatin and #43 in breast cancer cells.
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To improve our understanding of the origin and evolution of mycoheterotrophic plants, we here present the chromosome-scale genome assemblies of two sibling orchid species: partially mycoheterotrophic Platanthera zijinensis and holomycoheterotrophic Platanthera guangdongensis. Comparative analysis shows that mycoheterotrophy is associated with increased substitution rates and gene loss, and the deletion of most photoreceptor genes and auxin transporter genes might be linked to the unique phenotypes of fully mycoheterotrophic orchids. Conversely, trehalase genes that catalyse the conversion of trehalose into glucose have expanded in most sequenced orchids, in line with the fact that the germination of orchid non-endosperm seeds needs carbohydrates from fungi during the protocorm stage. We further show that the mature plant of P. guangdongensis, different from photosynthetic orchids, keeps expressing trehalase genes to hijack trehalose from fungi. Therefore, we propose that mycoheterotrophy in mature orchids is a continuation of the protocorm stage by sustaining the expression of trehalase genes. Our results shed light on the molecular mechanism underlying initial, partial and full mycoheterotrophy.
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Micorrizas , Orchidaceae , Micorrizas/genética , Orchidaceae/genética , Orchidaceae/metabolismo , Orchidaceae/microbiologia , Simbiose , Trealase/metabolismo , Trealose/metabolismoRESUMO
Non-small cell lung cancer (NSCLC), an aggressive subtype of pulmonary carcinomas with high mortality, accounts for 85% of all lung cancers. Drug resistance and high recurrence rates impede the chemotherapeutic effect, making it urgent to develop new anti-NSCLC agents. Recently, we have demonstrated that para-toluenesulfonamide is a potential anti-tumor agent in human castration-resistant prostate cancer (CRPC) through inhibition of Akt/mTOR/p70S6 kinase pathway and lipid raft disruption. In the current study, we further addressed the critical role of cholesterol-enriched membrane microdomain and autophagic activation to para-toluenesulfonamide action in killing NSCLC. Similar in CRPC, para-toluenesulfonamide inhibited the Akt/mTOR/p70S6K pathway in NSCLC cell lines NCI-H460 and A549, leading to G1 arrest of the cell cycle and apoptosis. Para-toluenesulfonamide significantly decreased the cholesterol levels of plasma membrane. External cholesterol supplement rescued para-toluenesulfonamide-mediated effects. Para-toluenesulfonamide induced a profound increase of LC3-II protein expression and a significant decrease of p62 expression. Double staining of lysosomes and cellular cholesterol showed para-toluenesulfonamide-induced lysosomal transportation of cholesterol, which was validated using flow cytometric analysis of lysosome staining. Moreover, autophagy inhibitors could blunt para-toluenesulfonamide-induced effect, indicating autophagy induction. In conclusion, the data suggest that para-toluenesulfonamide is an effective anticancer agent against NSCLC through G1 checkpoint arrest and apoptotic cell death. The disturbance of membrane cholesterol levels and autophagic activation may play a crucial role to para-toluenesulfonamide action.
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Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Membrana Celular/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Autofagia/fisiologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/metabolismo , Colesterol/metabolismo , Gefitinibe/administração & dosagem , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Sulfonamidas/administração & dosagem , Sulfonamidas/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Tolueno/administração & dosagem , Tolueno/análogos & derivados , Tolueno/farmacologiaRESUMO
MutT Homolog 1 (MTH1) has been proven to hydrolyze oxidized nucleotide triphosphates during DNA repair. It can prevent the incorporation of wrong nucleotides during DNA replication and mitigate cell apoptosis. In a cancer cell, abundant reactive oxygen species can lead to substantial DNA damage and DNA mutations by base-pairing mismatch. MTH1 could eliminate oxidized dNTP and prevent cancer cells from entering cell death. Therefore, inhibition of MTH1 activity is considered to be an anti-cancer therapeutic target. In this study, high-throughput screening techniques were combined with a fragment-based library containing 2,313 compounds, which were used to screen for lead compounds with MTH1 inhibitor activity. Four compounds with MTH1 inhibitor ability were selected, and compound MI0639 was found to have the highest effective inhibition. To discover the selectivity and specificity of this action, several derivatives based on the MTH1 and MI0639 complex structure were synthesized. We compared 14 complex structures of MTH1 and the various compounds in combination with enzymatic inhibition and thermodynamic analysis. Nanomolar-range IC50 inhibition abilities by enzyme kinetics and Kd values by thermodynamic analysis were obtained for two compounds, named MI1020 and MI1024. Based on structural information and compound optimization, we aim to provide a strategy for the development of MTH1 inhibitors with high selectivity and specificity.
Assuntos
Antineoplásicos/farmacologia , Enzimas Reparadoras do DNA/antagonistas & inibidores , Diaminas/farmacologia , Desenvolvimento de Medicamentos , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Enzimas Reparadoras do DNA/metabolismo , Diaminas/síntese química , Diaminas/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Monoéster Fosfórico Hidrolases/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , TermodinâmicaRESUMO
Hepatocellular carcinoma (HCC) is the most common primary liver malignancy in adults and accounts for 85-90% of all primary liver cancer. Based on the estimation by the International Agency for Research on Cancer in 2018, liver cancer is the fourth leading cause of cancer death globally. Dihydroartemisinin (DHA), the main active metabolite of artemisinin derivatives, is a well-known drug for the treatment of malaria. Previous studies have demonstrated that DHA exhibits antitumor effects toward a variety of human cancers and has a potential for repurposing as an anticancer drug. However, its short half-life is a concern and may limit the application in cancer therapy. We have reported that UDC-DHA, a hybrid of bile acid ursodeoxycholic acid (UDCA) and DHA, is â¼12 times more potent than DHA against a HCC cell line HepG2. In this study, we found that UDC-DHA was also effective against another HCC cell line Huh-7 with an IC50 of 2.16 µM, which was 18.5-fold better than DHA with an IC50 of 39.96 µM. UDC-DHA was much more potent than the combination of DHA and UDCA at 1:1 molar ratio, suggesting that the covalent linkage rather than a synergism between UDCA and DHA is critical for enhancing DHA potency in HepG2 cells. Importantly, UDC-DHA was much less toxic to normal cells than DHA. UDC-DHA induced G0/G1 arrest and apoptosis. Both DHA and UDC-DHA significantly elevated cellular reactive oxygen species generation but with different magnitude and timing in HepG2 cells; whereas only DHA but not UDC-DHA induced reactive oxygen species in Huh-7 cells. Depolarization of mitochondrial membrane potential was detected in both HepG2 and Huh-7 cells and may contribute to the anticancer effect of DHA and UDC-DHA. Furthermore, UDC-DHA was much more stable than DHA based on activity assays and high performance liquid chromatography-MS/MS analysis. In conclusion, UDC-DHA and DHA may exert anticancer actions via similar mechanisms but a much lower concentration of UDC-DHA was required, which could be attributed to a better stability of UDC-DHA. Thus, UDC-DHA could be a better drug candidate than DHA against HCC and further investigation is warranted.
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Chalcones are responsible for biological activity throughout fruits, vegetables, and medicinal plants in preventing and treating a variety of inflammation-related diseases. However, their structure-activity relationship (SAR) in inhibiting inflammasome activation has not been explored. We synthesized numerous chalcones and determined their SAR on lipopolysaccharide (LPS)-primed ATP-induced NLRP3 inflammasome activation. 11Cha1 displayed good inhibitory activity on release reaction of caspase-1, IL-1ß, and IL-18. It significantly inhibited LPS-induced phosphorylation and proteolytic degradation of IĸB-α and nuclear translocation of NF-ĸB, but had little effect on mitogen-activated protein kinases (MAPKs) activities. Furthermore, 11Cha1 blocked LPS-induced up-regulation of NLRP3, pro-caspase-1, ASC, IL-18, and IL-1ß, indicating the suppression on priming step of inflammasome activation. ASC dimerization and oligomerization are considered to be direct evidence for inflammasome activation. 11Cha1 profoundly inhibited ATP-induced formation of ASC dimers, trimers, and oligomers, and the assembly of ASC, pro-caspase-1, and NLRP3 in inflammasome formation. Decrease of intracellular K+ levels is the common cellular activity elicited by all NLRP3 inflammasome activators. 11Cha1 substantially diminished ATP-mediated K+ efflux, confirming the anti-NLRP3 inflammasome activity of 11Cha1. In summary, the SAR of chalcone derivatives in anti-inflammasome activities was examined. Besides, 11Cha1 inhibited both priming and activation steps of NLRP3 inflammasome activation. It inhibited NF-ĸB activation and subsequently suppressed the up-regulation of NLRP3 inflammasome components including NLRP3, ASC, pro-caspase-1, pro-IL-18, and pro-IL-1ß. Next, 11Cha1 blocked ATP-mediated K+ efflux and suppressed the assembly and activation of NLRP3 inflammasome, leading to the inhibition of caspase-1 activation and proteolytic cleavage, maturation, and secretion of IL-1ß and IL-18.
Assuntos
Chalconas/farmacologia , Inflamassomos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Trifosfato de Adenosina/farmacologia , Caspase 1/metabolismo , Linhagem Celular , Dimerização , Humanos , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Piroptose/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Combination therapies that display cancer-killing activities through either coexistent targeting of several cellular factors or more efficient suppression of a specific pathway are generally used in cancer treatment. Sildenafil, a specific phosphodiesterase type 5 (PDE5) inhibitor, has been suggested to display both cardioprotective and neuroprotective activities that provide a rationale for the combination with vincristine on the treatment against castration-resistant prostate cancer (CRPC). In the present work, vincristine arrested cells in the metaphase stage of mitosis. Vincristine-induced mitotic arrest was identified by Cdk1 activation (i.e., increased Cdk1Thr161 phosphorylation and decreased Cdk1Tyr15 phosphorylation), cyclin B1 upregulation, and increased phosphorylation of multiple mitotic proteins and stathmin. Sildenafil synergistically potentiated vincristine-induced mitotic arrest and a dramatic increase of mitotic index. Furthermore, sildenafil potentiated vincristine-induced mitochondrial damage, including Mcl-1 downregulation, Bcl-2 phosphorylation and downregulation, Bak upregulation and loss of mitochondrial membrane potential, and sensitized caspase-dependent apoptotic cell death. Sildenafil-mediated synergistic effects were mimicked by other PDE5 inhibitors including vardenafil and tadalafil, and also by PDE5A knockdown in cells, suggesting PDE5-involved mechanism. Notably, sildenafil amplified vincristine-induced phosphorylation and cleavage of BUBR1, a protein kinase in spindle assembly checkpoint (SAC) function and chromosome segregation. Sildenafil also significantly decreased kinetochore tension during SAC activation. Moreover, sildenafil synergized with vincristine on suppressing tumor growth in an in vivo model. In conclusion, the data suggest that sildenafil, in a PDE5-dependent manner, potentiates vincristine-induced mitotic arrest signaling, and sensitizes mitochondria damage-involved apoptosis in CRPC. Both in vitro and in vivo data suggest the combination potential of PDE5 inhibitors and vincristine on CRPC treatment.
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Non-small cell lung cancer (NSCLC) accounts about 80% of all lung cancers. More than two-thirds of NSCLC patients have inoperable, locally advanced or metastatic tumors. Non-toxic agents that synergistically potentiate cancer-killing activities of chemotherapeutic drugs are in high demand. YL-9 was a novel and non-cytotoxic compound with the structure related to sildenafil but showing much less activity against phosphodiesterase type 5 (PDE5). NCI-H460, an NSCLC cell line with low PDE5 expression, was used as the cell model. YL-9 synergistically potentiated vinorelbine-induced anti-proliferative and apoptotic effects in NCI-H460 cells. Vinorelbine induced tubulin acetylation and Bub1-related kinase (BUBR1) phosphorylation, a necessary component in spindle assembly checkpoint. These effects, as well as BUBR1 cleavage, were substantially enhanced in co-treatment with YL-9. Several mitotic arrest signals were enhanced under combinatory treatment of vinorelbine and YL-9, including an increase of mitotic spindle abnormalities, increased cyclin B1 expression, B-cell lymphoma 2 (Bcl-2) phosphorylation and increased phosphoproteins. Moreover, YL-9 also displayed synergistic activity in combining with vinorelbine to induce apoptosis in A549 cells which express PDE5. In conclusion. the data suggest that YL-9 is a novel agent that synergistically amplifies vinorelbine-induced NSCLC apoptosis through activation of spindle assembly checkpoint and increased mitotic arrest of the cell cycle. YL-9 shows the potential for further development in combinatory treatment against NSCLC.
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Antineoplásicos Fitogênicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/genética , Proteínas Serina-Treonina Quinases/genética , Células A549 , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Microtúbulos/genética , Inibidores da Fosfodiesterase 5/farmacologia , Fuso Acromático/efeitos dos fármacos , Vinorelbina/farmacologiaRESUMO
Because conventional chemotherapy is not sufficiently effective against prostate cancer, various examinations have been performed to identify anticancer activity of naturally occurring components and their mechanisms of action. The (+)-brevipolide H, an α-pyrone-based natural compound, induced potent and long-term anticancer effects in human castration-resistant prostate cancer (CRPC) PC-3 cells. Flow cytofluorometric analysis with propidium iodide staining showed (+)-brevipolide H-induced G1 arrest of cell cycle and subsequent apoptosis through induction of caspase cascades. Since Akt/mTOR pathway has been well substantiated in participating in cell cycle progression in G1 phase, its signaling and downstream regulators were examined. Consequently, (+)-brevipolide H inhibited the signaling pathway of Akt/mTOR/p70S6K. The c-Myc inhibition and downregulation of G1 phase cyclins were also attributed to (+)-brevipolide H action. Overexpression of myristoylated Akt significantly rescued mTOR/p70S6K and downstream signaling under (+)-brevipolide H treatment. ROS and Ca2+, two key mediators in regulating intracellular signaling, were determined, showing that (+)-brevipolide H interactively induced ROS production and an increase of intracellular Ca2+ levels. The (+)-Brevipolide H also induced the downregulation of anti-apoptotic Bcl-2 family proteins (Bcl-2 and Bcl-xL) and loss of mitochondrial membrane potential, indicating the contribution of mitochondrial dysfunction to apoptosis. In conclusion, the data suggest that (+)-brevipolide H displays anticancer activity through crosstalk between ROS production and intracellular Ca2+ mobilization. In addition, suppression of Akt/mTOR/p70S6K pathway associated with downregulation of G1 phase cyclins contributes to (+)-brevipolide H-mediated anticancer activity, which ultimately causes mitochondrial dysfunction and cell apoptosis. The data also support the biological significance and, possibly, clinically important development of natural product-based anticancer approaches.
Assuntos
Apoptose , Ciclopropanos/farmacologia , Pontos de Checagem da Fase G1 do Ciclo Celular , Estresse Oxidativo/efeitos dos fármacos , Neoplasias de Próstata Resistentes à Castração/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pironas/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Células Tumorais CultivadasRESUMO
Various derivatives that mimic ceramide structures by introducing a triazole to connect the aminodiol moiety and long alkyl chain have been synthesized and screened for their anti-leukemia activity. SPS8 stood out among the derivatives, showing cytotoxic selectivity between leukemic cell lines and human peripheral blood mononuclear cells (about ten times). DAPI nuclear staining and H&E staining revealed DNA fragmentation under the action of SPS8. SPS8 induced an increase in intracellular Ca2+ levels and mitochondrial stress in HL-60 cells identified by the loss of mitochondrial membrane potential, transmission electron microscopy (TEM) examination, and altered expressions of Bcl-2 family proteins. SPS8 also induced autophagy through the detection of Atg5, beclin-1, and LC3 II protein expression, as well as TEM examination. Chloroquine, an autophagy inhibitor, promoted SPS8-induced apoptosis, suggesting the cytoprotective role of autophagy in hindering SPS8 from apoptosis. Furthermore, SPS8 was shown to alter the expressions of a variety of genes using a microarray analysis and volcano plot filtering. A further cellular signaling pathways analysis suggested that SPS8 induced several cellular processes in HL-60, including the sterol biosynthesis process and cholesterol biosynthesis process, and inhibited some cellular pathways, in which STAT3 was the most critical nuclear factor. Further identification revealed that SPS8 inhibited the phosphorylation of STAT3, representing the loss of cytoprotective activity. In conclusion, the data suggest that SPS8 induces both apoptosis and autophagy in leukemic cells, in which autophagy plays a cytoprotective role in impeding apoptosis. Moreover, the inhibition of STAT3 phosphorylation may support SPS8-induced anti-leukemic activity.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Triazóis/química , Triazóis/farmacologia , Cálcio/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Redes Reguladoras de Genes , Células HL-60 , Humanos , Leucemia , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Modelos Biológicos , Estrutura Molecular , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico , Transcrição Gênica , TranscriptomaRESUMO
BACKGROUND: Cardiac glycosides, which inhibit Na+ /K+ -ATPase, display inotropic effects for the treatment of congestive heart failure and cardiac arrhythmia. Recent studies have suggested signaling downstream of Na+ /K+ -ATPase action in the regulation of cell proliferation and apoptosis and have revealed the anticancer activity of cardiac glycosides. The study aims to characterize the anticancer potential of ascleposide, a natural cardenolide, and to uncover its primary target and underlying mechanism against human castration-resistant prostate cancer (CRPC). METHODS: Cell proliferation was examined in CRPC PC-3 and DU-145 cells using sulforhodamine B assay, carboxyfluorescein succinimidyl ester staining assay and clonogenic examination. Flow cytometric analysis was used to detect the distribution of cell cycle phase, mitochondrial membrane potential, intracellular Na+ and Ca2+ levels, and reactive oxygen species production. Protein expression was examined using Western blot analysis. Endocytosis of Na+ /K+ -ATPase was determined using confocal immunofluorescence microscopic examination. RESULTS: Ascleposide induced an increase of intracellular Na+ and a potent antiproliferative effect. It also induced a decrease of G1 phase distribution while an increase in both G2/M and apoptotic sub-G1 phases, and downregulated several cell cycle regulator proteins, including cyclins, Cdk, p21, and p27 Cip/Kip proteins, Rb and c-Myc. Ascleposide decreased the expression of antiapoptotic Bcl-2 members (eg, Bcl-2 and Mcl-1) but upregulated proapoptotic member (eg, Bak), leading to a significant loss of mitochondrial membrane potential and activation of both caspase-9 and caspase-3. Ascleposide also dramatically induced tubulin acetylation, leading to inhibition of the catalytic activity of Na+ /K+ -ATPase. Notably, extracellular high K+ (16 mM) significantly blunted ascleposide-mediated effects. Furthermore, ascleposide induced a p38 MAPK-dependent endocytosis of Na+ /K+ -ATPase and downregulated the protein expression of Na+ /K+ -ATPase α1 subunit. CONCLUSION: Ascleposide displays antiproliferative and apoptotic activities dependent on the inhibition of Na+ /K+ -ATPase pumping activity through p38 MAPK-mediated endocytosis of Na+ /K+ -ATPase and downregulation of α1 subunit, which in turn cause tubulin acetylation and cell cycle arrest. Cell apoptosis is ultimately triggered by the activation of caspase cascade attributed to mitochondrial damage through the downregulation of Bcl-2 and Mcl-1 protein expressions while upregulation of Bak protein levels. The data also suggest the potential of ascleposide in anti-CRPC development.
Assuntos
Cardenolídeos/farmacologia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Tubulina (Proteína)/metabolismo , Acetilação/efeitos dos fármacos , Antineoplásicos Fitogênicos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Humanos , Masculino , Malvaceae/química , Células PC-3 , Extratos Vegetais/farmacologia , Neoplasias de Próstata Resistentes à Castração/patologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Breast cancer is the most commonly diagnosed cancer and the second leading cause of cancer death in women. Hormone receptor-positive breast cancer is usually subjected to hormone therapy, while triple-negative breast cancer is more formidable and poses a therapeutic challenge. Glucose transporters are potential targets for the development of anticancer drugs. In search of anticancer agents whose effect could be enhanced by a GLUT1 inhibitor WZB117, we found that MK-2206, a potent allosteric Akt inhibitor, when combined with WZB117, showed a synergistic effect on growth inhibition and apoptosis induction in breast cancer cells, including ER(+) MCF-7 cells and triple-negative MDA-MB-231 cells. The combination index values at 50% growth inhibition were 0.45 and 0.21, respectively. Mechanism studies revealed that MK-2206 and WZB117 exert a synergistic cytotoxic effect in both MCF-7 and MDA-MB-231 breast cancer cells by inhibiting Akt phosphorylation and inducing DNA damage. The combination may also compromise DNA damage repair and ultimately lead to apoptosis. Our findings suggest that the combination of Akt inhibitors and GLUT1 inhibitors could be a novel strategy to combat breast cancer.
RESUMO
Castration-resistant prostate cancer (CRPC) cells can resist many cellular stresses to ensure survival. There is an unmet medical need to fight against the multiple adaptive mechanisms in cells to achieve optimal treatment in patients. Para-toluenesulfonamide (PTS) is a small molecule that inhibited cell proliferation of PC-3 and DU-145, two CRPC cell lines, through p21- and p27-independent G1 arrest of cell cycle in which cyclin D1 was down-regulated and Rb phosphorylation was inhibited. PTS also induced a significant loss of mitochondrial membrane potential that was attributed to up-regulation of both Bak and PUMA, two pro-apoptotic Bcl-2 family members, leading to apoptosis. PTS inhibited the phosphorylation of m-TOR, 4E-BP1, and p70S6K in both cell lines. Overexpression of constitutively active Akt rescued the inhibition of mTOR/p70S6K signaling in PC-3 cells indicating an Akt-dependent pathway. In contrast, Akt-independent effect was observed in DU-145 cells. Lipid rafts serve as functional platforms for multiple cellular signaling and trafficking processes. Both cell lines expressed raft-associated Akt, mTOR, and p70S6K. PTS induced decreases of expressions in both raft-associated total and phosphorylated forms of these kinases. PTS-induced inhibitory effects were rescued by supplement of cholesterol, an essential constituent in lipid raft, indicating a key role of cholesterol contents. Moreover, the tumor xenograft model showed that PTS inhibited tumor growth with a T/C (treatment/control) of 0.44 and a 56% inhibition of growth rate indicating the in vivo efficacy. In conclusion, the data suggest that PTS is an effective anti-tumor agent with in vitro and in vivo efficacies through inhibition of both Akt-dependent and -independent mTOR/p70S6K pathways. Moreover, disturbance of lipid raft and cholesterol contents may at least partly explain PTS-mediated anti-tumor mechanism.
RESUMO
Human castration-resistant prostate cancer (CRPC) is a significant target of clinical research. The use of DNA-damaging agents has a long history in cancer chemotherapy but is limited by their toxicities. The combination with a safer drug can be a strategy in reducing dosage and toxicity while increasing anticancer activity in CRPC treatment. Phosphodiesterase type 5 (PDE5) inhibitors are used to treat erectile dysfunction through the selective inhibition of PDE5 that is responsible for cGMP degradation in the corpus cavernosum. Several studies have reported that PDE5 inhibitors display protective effect against doxorubicin-induced cardiotoxicity. The combinatory treatment of CRPC with doxorubicin and PDE5 inhibitors has been studied accordingly. The data demonstrated that sildenafil or vardenafil (two structure-related PDE5 inhibitors) but not tadalafil (structure-unrelated to sildenafil) sensitized doxorubicin-induced apoptosis in CRPC cells with deteriorating the down-regulation of anti-apoptotic Bcl-2 family members, including Bcl-xL and Mcl-1, and amplifying caspase activation. Homologous recombination (HR) and non-homologous end joining (NHEJ) DNA repair systems were inhibited in the apoptotic sensitization through detection of nuclear foci formation of Rad51 and DNA end-binding of Ku80. PDE5 knockdown to mimic the exposure to PDE5 inhibitors did not reproduce apoptotic sensitization, suggesting a PDE5-independent mechanism. Not only doxorubicin, sildenafil combined with other inhibitors of topoisomerase II but not topoisomerase I also triggered apoptotic sensitization. In conclusion, the data suggest that sildenafil and vardenafil induce PDE5-independent apoptotic sensitization to doxorubicin (or other topoisomerase II inhibitors) through impairment of both HR and NHEJ repair systems that are evident by a decrease of nuclear Rad51 levels and their foci formation in the nucleus, and an inhibition of Ku80 DNA end-binding capability. The combinatory treatment may enable an important strategy for anti-CRPC development.
RESUMO
The use of peptides that target cancer cells and induce anticancer activities through various mechanisms is developing as a potential anticancer strategy. KUD983, an enantiomerically pure ß-dipeptide derivative, displays potent activity against hormone-refractory prostate cancer (HRPC) PC-3 and DU145 cells with submicromolar IC50. KUD983 induced G1 arrest of the cell cycle and subsequent apoptosis associated with down-regulation of several related proteins including cyclin D1, cyclin E and Cdk4, and the de-phosphorylation of RB. The levels of nuclear and total c-Myc protein, which could increase the expression of both cyclin D1 and cyclin E, were profoundly inhibited by KUD983. Furthermore, it inhibited PI3K/Akt and mTOR/p70S6K/4E-BP1 pathways, the key signaling in multiple cellular functions. The transient transfection of constitutively active myristylated Akt (myr-Akt) cDNA significantly rescued KUD983-induced caspase activation but did not blunt the inhibition of mTOR/p70S6K/4E-BP1 signaling cascade suggesting the presence of both Akt-dependent and -independent pathways. Moreover, KUD983-induced effect was enhanced with the down-regulation of anti-apoptotic Bcl-2 members (e.g., Bcl-2, and Mcl-1) and IAP family members (e.g., survivin). Notably, KUD983 induced autophagic cell death using confocal microscopic examination, tracking the level of conversion of LC3-I to LC3-II and flow cytometric detection of acidic vesicular organelles-positive cells. In conclusion, the data suggest that KUD983 is an anticancer ß-dipeptide against HRPCs through the inhibition of cell proliferation and induction of apoptotic and autophagic cell death. The suppression of signaling pathways regulated by c-Myc, PI3K/Akt and mTOR/p70S6K/4E-BP1 and the collaboration with down-regulation of Mcl-1 and survivin may explain KUD983-induced anti-HRPC mechanism.