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1.
FEBS Open Bio ; 13(6): 1056-1066, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37079001

RESUMO

Combination chemotherapy with gemcitabine and cisplatin (GC) is recommended as the primary treatment for advanced bladder cancer (BC). However, the benefits of this approach are limited owing to the acquisition of drug resistance. Here, we found that gemcitabine-resistant and cisplatin-resistant BCs do not exhibit cross-resistance, and that these BCs exhibit different mRNA patterns, as revealed using RNA sequence analysis. To overcome drug resistance, we used the newly developed pan-RAS inhibitor Compound 3144. Compound 3144 inhibited cell viability through suppression of RAS-dependent signaling in gemcitabine- and cisplatin-resistant BCs. RNA sequencing revealed that several genes and pathways, particularly those related to the cell cycle, were significantly downregulated in Compound 3144-treated BCs. These findings provide insights into potential therapeutic strategies for treating BC.


Assuntos
Antineoplásicos , Neoplasias da Bexiga Urinária , Humanos , Gencitabina , Cisplatino , Resistencia a Medicamentos Antineoplásicos/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/genética , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico
2.
Biochem Biophys Res Commun ; 611: 99-106, 2022 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-35487063

RESUMO

In recent years, cancer metabolism has attracted attention as a therapeutic target, and glutamine metabolism is considered one of the most important metabolic processes in cancer. Solute carrier family 1 member 5 (SLC1A5) is a sodium channel that functions as a glutamine transporter. In various cancer types, SLC1A5 gene expression is enhanced, and cancer cell growth is suppressed by inhibition of SLC1A5. However, the involvement of SLC1A5 in clear cell renal cell carcinoma (ccRCC) is unclear. Therefore, in this study, we evaluated the clinical importance of SLC1A5 in ccRCC using The Cancer Genome Atlas database. Our findings confirmed that SLC1A5 was a prognosis factor for poor survival in ccRCC. Furthermore, loss-of-function assays using small interfering RNAs or an SLC1A5 inhibitor (V9302) in human ccRCC cell lines (A498 and Caki1) showed that inhibition of SLC1A5 significantly suppressed tumor growth, invasion, and migration. Additionally, inhibition of SLC1A5 by V9302 in vivo significantly suppressed tumor growth, and the antitumor effects of SLC1A5 inhibition were related to cellular senescence. Our findings may improve our understanding of ccRCC and the development of new treatment strategies for ccRCC.


Assuntos
Sistema ASC de Transporte de Aminoácidos , Carcinoma de Células Renais , Senescência Celular , Neoplasias Renais , Antígenos de Histocompatibilidade Menor , Sistema ASC de Transporte de Aminoácidos/genética , Sistema ASC de Transporte de Aminoácidos/metabolismo , Carcinoma de Células Renais/genética , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Glutamina/metabolismo , Humanos , Neoplasias Renais/genética , Antígenos de Histocompatibilidade Menor/genética , RNA Interferente Pequeno/genética
3.
Mol Oncol ; 16(6): 1329-1346, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35148461

RESUMO

Patients with advanced bladder cancer are generally treated with a combination of chemotherapeutics, including gemcitabine, but the effect is limited due to acquisition of drug resistance. Thus, in this study, we investigated the mechanism of gemcitabine resistance. First, gemcitabine-resistant cells were established and resistance confirmed in vitro and in vivo. Small RNA sequencing analyses were performed to search for miRNAs involved in gemcitabine resistance. miR-99a-5p, selected as a candidate miRNA, was downregulated compared to its parental cells. In gain-of-function studies, miR-99a-5p inhibited cell viabilities and restored sensitivity to gemcitabine. RNA sequencing analysis was performed to find the target gene of miR-99a-5p. SMARCD1 was selected as a candidate gene. Dual-luciferase reporter assays showed that miR-99a-5p directly regulated SMARCD1. Loss-of-function studies conducted with si-RNAs revealed suppression of cell functions and restoration of gemcitabine sensitivity. miR-99a-5p overexpression and SMARCD1 knockdown also suppressed gemcitabine-resistant cells in vivo. Furthermore, ß-galactosidase staining showed that miR-99a-5p induction and SMARCD1 suppression contributed to cellular senescence. In summary, tumor-suppressive miR-99a-5p induced cellular senescence in gemcitabine-resistant bladder cancer cells by targeting SMARCD1.


Assuntos
MicroRNAs , Neoplasias da Bexiga Urinária , Linhagem Celular Tumoral , Proliferação de Células , Senescência Celular/genética , Proteínas Cromossômicas não Histona/genética , Desoxicitidina/análogos & derivados , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/genética , Gencitabina
4.
BMC Cancer ; 21(1): 48, 2021 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-33430801

RESUMO

BACKGROUND: Cisplatin-based chemotherapy is recommended as the primary treatment for advanced bladder cancer (BC) with unresectable or metastatic disease. However, the benefits are limited due to the acquisition of drug resistance. The mechanisms of resistance remain unclear. Although there are some reports that some molecules are associated with cisplatin resistance in advanced BC, those reports have not been fully investigated. Therefore, we undertook a new search for cisplatin resistance-related genes targeted by tumor suppressive microRNAs as well as genes that were downregulated in cisplatin-resistant BC cells and clinical BC tissues. METHODS: First, we established cisplatin-resistant BOY and T24 BC cell lines (CDDP-R-BOY, CDDP-R-T24). Then, Next Generation Sequence analysis was performed with parental and cisplatin-resistant cell lines to search for the microRNAs responsible for cisplatin resistance. We conducted gain-of-function analysis of microRNAs and their effects on cisplatin resistance, and we searched target genes comprehensively using Next Generation mRNA sequences. RESULTS: A total of 28 microRNAs were significantly downregulated in both CDDP-R-BOY and CDDP-R-T24. Among them, miR-486-5p, a tumor suppressor miRNA, was negatively correlated with the TNM classification of clinical BC samples in The Cancer Genome Atlas (TCGA) database. Transfection of miRNA-486-5p significantly inhibited cancer cell proliferation, migration, and invasion, and also improved the cells' resistance to cisplatin. Among the genes targeted by miRNA-486-5p, we focused on enoyl-CoA, hydratase/3-hydroxyacyl CoA dehydrogenase (EHHADH), which is involved in the degradation of fatty acids. EHHADH was directly regulated by miRNA-486-5p as determined by a dual-luciferase reporter assay. Loss-of-function study using EHHADH si-RNA showed significant inhibitions of cell proliferation, migration, invasion and the recovery of cisplatin sensitivity. CONCLUSION: Identification of EHHADH as a target of miRNA-486-5p provides novel insights into the potential mechanisms of cisplatin resistance in BC.


Assuntos
Biomarcadores Tumorais/metabolismo , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Enzima Bifuncional do Peroxissomo/metabolismo , Neoplasias da Bexiga Urinária/patologia , Animais , Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Movimento Celular , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Enzima Bifuncional do Peroxissomo/genética , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
5.
PLoS One ; 15(5): e0232545, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32379831

RESUMO

Exosomes are 40-100 nm nano-sized extracellular vesicles. They are released from many cell types and move into the extracellular space, thereby transferring their components to recipient cells. Exosomes are receiving increasing attention as novel structures participating in intracellular communication. RAB27B is one of the leading proteins involved in exosome secretion, and oncogenic effects have been reported in several cancers. In recent years, molecularly targeted agents typified by sunitinib are widely used for the treatment of metastatic or recurrent renal cell carcinoma (RCC). However, intrinsic or acquired resistance to sunitinib has become a major issue. The present study aimed to elucidate the role of RAB27B in RCC including sunitinib-resistant and its role in exosomes. Bioinformatic analyses revealed that high expression of RAB27B correlates with progression of RCC. The expression of RAB27B protein in RCC cell lines was significantly enhanced compared with that in normal kidney cell lines. Furthermore, RAB27B protein expression was enhanced in all of the tested sunitinib-resistant RCC cell lines compared to parental cells. Although no specific effect of RAB27B on exosomes was identified in RCC cells, loss-of-function studies demonstrated that knockdown of RAB27B suppressed cell proliferation, migration and invasive activities. Moreover, anti-tumor effects of RAB27B downregulation were also observed in sunitinib-resistant RCC cells. RNA sequence and pathway analysis suggested that the oncogenic effects of RAB27B might be associated with MAPK and VEGF signaling pathways. These results showed that RAB27B is a prognostic marker and a novel therapeutic target in sunitinib-sensitive and -resistant RCCs. Further analyses should improve our understanding of sunitinib resistance in RCC.


Assuntos
Antineoplásicos/uso terapêutico , Carcinoma de Células Renais/metabolismo , Exossomos/metabolismo , Neoplasias Renais/metabolismo , Sunitinibe/uso terapêutico , Proteínas rab de Ligação ao GTP/metabolismo , Western Blotting , Carcinoma de Células Renais/tratamento farmacológico , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Humanos , Rim/metabolismo , Neoplasias Renais/tratamento farmacológico
6.
Cancer Sci ; 111(5): 1607-1618, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32232883

RESUMO

Sunitinib, a multitargeted receptor tyrosine kinase inhibitor including vascular endothelial growth factor, has been widely used as a first-line treatment against metastatic renal cell carcinoma (mRCC). However, mRCC often acquires resistance to sunitinib, rendering it difficult to treat with this agent. Recently, Rapalink-1, a drug that links rapamycin and the mTOR kinase inhibitor MLN0128, has been developed with excellent therapeutic effects against breast cancer cells carrying mTOR resistance mutations. The aim of the present study was to evaluate the in vitro and in vivo therapeutic efficacy of Rapalink-1 against renal cell carcinoma (RCC) compared to temsirolimus, which is commonly used as a small molecule inhibitor of mTOR and is a derivative of rapamycin. In comparison with temsirolimus, Rapalink-1 showed significantly greater effects against proliferation, migration, invasion and cFolony formation in sunitinib-naïve RCC cells. Inhibition was achieved through suppression of the phosphorylation of substrates in the mTOR signal pathway, such as p70S6K, eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) and AKT. In addition, Rapalink-1 had greater tumor suppressive effects than temsirolimus against the sunitinib-resistant 786-o cell line (SU-R 786-o), which we had previously established, as well as 3 additional SU-R cell lines established here. RNA sequencing showed that Rapalink-1 suppressed not only the mTOR signaling pathway but also a part of the MAPK signaling pathway, the ErbB signaling pathway and ABC transporters that were associated with resistance to several drugs. Our study suggests the possibility of a new treatment option for patients with RCC that is either sunitinib-sensitive or sunitinib-resistant.


Assuntos
Carcinoma de Células Renais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Renais/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Sirolimo/análogos & derivados , Sunitinibe/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Camundongos , Camundongos Nus , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Sunitinibe/uso terapêutico , Serina-Treonina Quinases TOR/metabolismo
7.
Sci Rep ; 9(1): 13977, 2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31562379

RESUMO

Protein kinase N1 (PKN1) knockout (KO) mice spontaneously form germinal centers (GCs) and develop an autoimmune-like disease with age. Here, we investigated the function of PKN1 kinase activity in vivo using aged mice deficient in kinase activity resulting from the introduction of a point mutation (T778A) in the activation loop of the enzyme. PKN1[T778A] mice reached adulthood without external abnormalities; however, the average spleen size and weight of aged PKN1[T778A] mice increased significantly compared to aged wild type (WT) mice. Histologic examination and Southern blot analyses of spleens showed extramedullary hematopoiesis and/or lymphomagenesis in some cases, although without significantly different incidences between PKN1[T778A] and WT mice. Additionally, flow cytometry revealed increased numbers in B220+, CD3+, Gr1+ and CD193+ leukocytes in the spleen of aged PKN1[T778A] mice, whereas the number of lymphocytes, neutrophils, eosinophils, and monocytes was reduced in the peripheral blood, suggesting an advanced impairment of leukocyte trafficking with age. Moreover, aged PKN1[T778A] mice showed no obvious GC formation nor autoimmune-like phenotypes, such as glomerulonephritis or increased anti-dsDNA antibody titer, in peripheral blood. Our results showing phenotypic differences between aged Pkn1-KO and PKN1[T778A] mice may provide insight into the importance of PKN1-specific kinase-independent functions in vivo.


Assuntos
Leucopenia/genética , Proteína Quinase C/genética , Esplenomegalia/genética , Fatores Etários , Animais , Técnicas de Introdução de Genes , Centro Germinativo/metabolismo , Leucopenia/metabolismo , Camundongos , Camundongos Knockout , Fosforilação , Proteína Quinase C/metabolismo , Transdução de Sinais/fisiologia , Esplenomegalia/metabolismo
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