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1.
Int J Mol Sci ; 22(3)2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33498210

RESUMO

Protein inhibitors of proteases are an important tool of nature to regulate and control proteolysis in living organisms under physiological and pathological conditions. In this review, we analyzed the mechanisms of inhibition of cysteine proteases on the basis of structural information and compiled kinetic data. The gathered structural data indicate that the protein fold is not a major obstacle for the evolution of a protease inhibitor. It appears that nature can convert almost any starting fold into an inhibitor of a protease. In addition, there appears to be no general rule governing the inhibitory mechanism. The structural data make it clear that the "lock and key" mechanism is a historical concept with limited validity. However, the analysis suggests that the shape of the active site cleft of proteases imposes some restraints. When the S1 binding site is shaped as a pocket buried in the structure of protease, inhibitors can apply substrate-like binding mechanisms. In contrast, when the S1 binding site is in part exposed to solvent, the substrate-like inhibition cannot be employed. It appears that all proteases, with the exception of papain-like proteases, belong to the first group of proteases. Finally, we show a number of examples and provide hints on how to engineer protein inhibitors.


Assuntos
Cisteína Proteases/química , Inibidores de Cisteína Proteinase/química , Animais , Cistatinas/química , Cistatinas/metabolismo , Cistatinas/farmacologia , Cisteína Proteases/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Humanos , Ligação Proteica , Securina/química , Securina/metabolismo , Securina/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/química , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/farmacologia
2.
Medicine (Baltimore) ; 99(9): e18497, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32118702

RESUMO

BACKGROUND: The aim of this meta-analysis was to systematically evaluate the prognostic significance of X-linked inhibitor of apoptosis protein (XIAP) in patients with gastrointestinal tract (GIT) cancers. METHODS: PubMed, Web of Science, EMBASE, Cochrane Library and China National Knowledge Infrastructure were searched for potentially eligible literature. The baseline characteristics and relevant data were extracted. Hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated to assess the prognostic role of XIAP in patients with GIT cancers. RESULTS: Twelve studies with 2,477 patients were included. The pooled HRs of higher expression of XIAP for overall survival (OS) and recurrence free survival (RFS) in patients with GIT cancers were 1.64 (95% CI, 1.27-2.13) and 1.06 (95% CI, 0.96-1.16), respectively. Subgroup analysis and sensitivity analysis were also performed. No significant publication bias was found. CONCLUSION: Our results suggested that XIAP could be a prognostic marker for OS but not RFS in patients with GIT cancers. Higher expression of XIAP was related to poorer OS. These findings may help evaluate the prognosis of patients and assist future research on novel therapeutic strategies of GIT cancers by targeting XIAP. However, more well-designed studies are warranted to verify the results.


Assuntos
Neoplasias Gastrointestinais/diagnóstico , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Neoplasias Gastrointestinais/metabolismo , Humanos , Prognóstico
3.
Mol Cells ; 43(3): 236-250, 2020 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-32050753

RESUMO

Currently, many available anti-cancer therapies are targeting apoptosis. However, many cancer cells have acquired resistance to apoptosis. To overcome this problem, simultaneous induction of other types of programmed cell death in addition to apoptosis of cancer cells might be an attractive strategy. For this purpose, we initially investigated the inhibitory role of TRIP-Br1/XIAP in necroptosis, a regulated form of necrosis, under nutrient/serum starvation. Our data showed that necroptosis was significantly induced in all tested 9 different types of cancer cell lines in response to prolonged serum starvation. Among them, necroptosis was induced at a relatively lower level in MCF-7 breast cancer line that was highly resistant to apoptosis than that in other cancer cell lines. Interestingly, TRIP-Br1 oncogenic protein level was found to be very high in this cell line. Upregulated TRIP-Br1 suppressed necroptosis by repressing reactive oxygen species generation. Such suppression of necroptosis was greatly enhanced by XIAP, a potent inhibitor of apoptosis. Our data also showed that TRIP-Br1 increased XIAP phosphorylation at serine87, an active form of XIAP. Our mitochondrial fractionation data revealed that TRIPBr1 protein level was greatly increased in the mitochondria upon serum starvation. It suppressed the export of CypD, a vital regulator in mitochondria-mediated necroptosis, from mitochondria to cytosol. TRIP-Br1 also suppressed shikoninmediated necroptosis, but not TNF-α-mediated necroptosis, implying possible presence of another signaling pathway in necroptosis. Taken together, our results suggest that TRIPBr1/XIAP can function as onco-proteins by suppressing necroptosis of cancer cells under nutrient/serum starvation.


Assuntos
Neoplasias/metabolismo , Nutrientes/deficiência , Fatores de Transcrição/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Células A549 , Apoptose/fisiologia , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , Células MCF-7 , Necroptose/fisiologia , Neoplasias/patologia , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética
4.
Int J Oncol ; 56(1): 368-378, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31789392

RESUMO

Meridianin C is a marine natural product with anticancer activity. Several meridianin C derivatives (compounds 7a­j) were recently synthesized, and their inhibitory effects on pro­viral integration site for Moloney murine leukemia virus (PIM) kinases, as well as their antiproliferative effects on human leukemia cells, were reported. However, the anti­leukemic effects and mechanisms of action of meridianin C and its derivatives remain largely unknown. The aim of the present study was to investigate the effects of meridianin C and its derivatives on MV4­11 human acute myeloid leukemia cell growth. The parent compound meridianin C did not markedly affect the viability and survival of MV4­11 cells. By contrast, MV4­11 cell viability and survival were reduced by meridianin C derivatives, with compound 7a achieving the most prominent reduction. Compound 7a notably inhibited the expression and activity of PIM kinases, as evidenced by reduced B­cell lymphoma­2 (Bcl­2)­associated death promoter phosphorylation at Ser112. However, meridianin C also suppressed PIM kinase expression and activity, and the pan­PIM kinase inhibitor AZD1208 only slightly suppressed the survival of MV4­11 cells. Thus, the anti­survival effect of compound 7a on MV4­11 cells was unrelated to PIM kinase inhibition. Moreover, compound 7a induced apoptosis, caspase­9 and ­3 activation and poly(ADP­ribose) polymerase (PARP) cleavage, but did not affect death receptor (DR)­4 or DR­5 expression in MV4­11 cells. Compound 7a also induced the generation of cleaved Bcl­2, and the downregulation of myeloid cell leukemia (Mcl)­1 and X­linked inhibitor of apoptosis (XIAP) in MV4­11 cells. Furthermore, compound 7a increased eukaryotic initiation factor (eIF)­2α phosphorylation and decreased S6 phosphorylation, whereas GRP­78 expression was unaffected. Importantly, treatment with a pan­caspase inhibitor (z­VAD­fmk) significantly attenuated compound 7a­induced apoptosis, caspase­9 and ­3 activation, PARP cleavage, generation of cleaved Bcl­2 and downregulation of Mcl­1 and XIAP in MV4­11 cells. Collectively, these findings demonstrated the strong anti­survival and pro­apoptotic effects of compound 7a on MV4­11 cells through regulation of caspase­9 and ­3, Bcl­2, Mcl­1, XIAP, eIF­2α and S6 molecules.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Proliferação de Células , Indóis/química , Indóis/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Pirimidinas/química , Pirimidinas/farmacologia , Proteínas Reguladoras de Apoptose/genética , Caspase 9/genética , Caspase 9/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Fosforilação , Inibidores de Proteínas Quinases/química , Células Tumorais Cultivadas , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
5.
Cell Oncol (Dordr) ; 43(1): 81-93, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31512195

RESUMO

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is the most common and lethal subtype of pancreatic cancer, with a 5-year survival rate of < 3%. Early tumor dissemination, late diagnosis and insensitivity to conventional treatment are the major reasons for its high mortality rate. Members of the vascular endothelial growth factor (VEGF) family are overexpressed in PDAC and play important roles in its malignant progression, suggesting that VEGF-targeted therapies may interrupt the proliferation and motility of PDAC cells. Here, we evaluated the anti-tumor activity of cediranib, a pan-VEGF receptor inhibitor, on PDAC cells. METHODS: Anti-proliferative effects of cediranib were determined using cell proliferation and crystal violet staining assays. Annexin V/PI staining, radiation therapy, and cell migration and invasion assays were carried out to examine the effects of cediranib on apoptosis, radio-sensitivity and cell motility, respectively. Quantitative reverse transcription-PCR (qRT-PCR) and Western blot analyses were applied to elucidate the molecular mechanisms underlying the anti-tumor activity of cediranib. RESULTS: We found that cediranib decreased PDAC cell proliferation and clonogenic survival and induced apoptotic cell death through inhibition of the anti-apoptotic proteins cIAP1, XIAP, MCL-1 and survivin. Combination with cediranib synergistically increased the sensitivity of PDAC cells to chemotherapeutic agents such as gemcitabine and paclitaxel, and potentiated the effects of radiation therapy on PDAC cell growth inhibition and apoptosis induction. Furthermore, we found that treatment with cediranib impaired PDAC cell migration and invasion via expression reduction of the epithelial-to-mesenchymal transition (EMT) markers ZEB1, N-cadherin and Snail. CONCLUSIONS: Our data indicate that cediranib may exhibit anti-tumor activity in PDAC cells and provide a rationale for further investigation of the potential of VEGF receptor-targeted therapies for the treatment of PDAC.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Ductal Pancreático/metabolismo , Proliferação de Células/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias Pancreáticas/metabolismo , Quinazolinas/farmacologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Apoptose/efeitos da radiação , Proteína 3 com Repetições IAP de Baculovírus/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Sinergismo Farmacológico , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Paclitaxel/farmacologia , Tolerância a Radiação , Fatores de Transcrição da Família Snail/metabolismo , Survivina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo
6.
Nat Prod Res ; 34(18): 2616-2620, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30587055

RESUMO

Xanthatin is a natural plant bicyclic sesquiterpene lactone extracted from Xanthium plants (Asteraceae). In the present study, we demonstrated for the first time that Xanthatin inhibited cell proliferation and mediated G2/M phase arrest in human colon cancer cells. Xanthatin also activated caspase and mediated apoptosis in these cells. Concomitantly, Xanthatin triggered cell autophagic response. We found down-regulation of X-linked inhibitor of apoptosis protein (XIAP) contribute to the induction of apoptosis and autophagy. Moreover, reactive oxygen species (ROS) production was triggered upon exposure to Xanthatin in colon cancer cells. ROS inhibitor N-acetylcysteine (NAC) significantly reversed Xanthatin-mediated XIAP down-regulation, G2/M phase arrest, apoptosis and autophagosome accumulation. In summary, our findings demonstrated that Xanthatin caused G2/M phase arrest and mediated apoptosis and autophagy through ROS/XIAP in human colon cancer cells. We provided molecular bases for developing Xanthatin as a promising antitumor candidate for colon cancer therapy. AbbreviationsROSreactive oxygen speciesDMSOdimethyl sulfoxide5-FU5-Fluorouracil3-MA3-MethyladenineDCFH-DA2'7'-dichlorfluorescein-diacetateNACN-acetylcysteineXIAPX-linked inhibitor of apoptosis protein.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Furanos/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Asteraceae/química , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Furanos/isolamento & purificação , Furanos/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular , Humanos , Espécies Reativas de Oxigênio/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Xanthium/química
7.
Gastroenterology ; 158(4): 1000-1015, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31743734

RESUMO

BACKGROUND & AIMS: Mutations in the tetratricopeptide repeat domain 7A gene (TTC7A) cause intestinal epithelial and immune defects. Patients can become immune deficient and develop apoptotic enterocolitis, multiple intestinal atresia, and recurrent intestinal stenosis. The intestinal disease in patients with TTC7A deficiency is severe and untreatable, and it recurs despite resection or allogeneic hematopoietic stem cell transplant. We screened drugs for those that prevent apoptosis of in cells with TTC7A deficiency and tested their effects in an animal model of the disease. METHODS: We developed a high-throughput screen to identify compounds approved by the US Food and Drug Administration that reduce activity of caspases 3 and 7 in TTC7A-knockout (TTC7A-KO) HAP1 (human haploid) cells and reduce the susceptibility to apoptosis. We validated the effects of identified agents in HeLa cells that stably express TTC7A with point mutations found in patients. Signaling pathways in cells were analyzed by immunoblots. We tested the effects of identified agents in zebrafish with disruption of ttc7a, which develop intestinal defects, and colonoids derived from biopsy samples of patients with and without mutations in TTC7A. We performed real-time imaging of intestinal peristalsis in zebrafish and histologic analyses of intestinal tissues from patients and zebrafish. Colonoids were analyzed by immunofluorescence and for ion transport. RESULTS: TTC7A-KO HAP1 cells have abnormal morphology and undergo apoptosis, due to increased levels of active caspases 3 and 7. We identified drugs that increased cell viability; leflunomide (used to treat patients with inflammatory conditions) reduced caspase 3 and 7 activity in cells by 96%. TTC7A-KO cells contained cleaved caspase 3 and had reduced levels of phosphorylated AKT and X-linked inhibitor of apoptosis (XIAP); incubation of these cells with leflunomide increased levels of phosphorylated AKT and XIAP and reduced levels of cleaved caspase 3. Administration of leflunomide to ttc7a-/- zebrafish increased gut motility, reduced intestinal tract narrowing, increased intestinal cell survival, increased sizes of intestinal luminal spaces, and restored villi and goblet cell morphology. Exposure of patient-derived colonoids to leflunomide increased cell survival, polarity, and transport function. CONCLUSIONS: In a drug screen, we identified leflunomide as an agent that reduces apoptosis and activates AKT signaling in TTC7A-KO cells. In zebrafish with disruption of ttc7a, leflunomide restores gut motility, reduces intestinal tract narrowing, and increases intestinal cell survival. This drug might be repurposed for treatment of TTC7A deficiency.


Assuntos
Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Doenças Inflamatórias Intestinais/tratamento farmacológico , Leflunomida/farmacologia , Proteínas/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Colo/citologia , Técnicas de Inativação de Genes , Haploidia , Humanos , Doenças Inflamatórias Intestinais/genética , Fosforilação/efeitos dos fármacos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
8.
Acta Haematol ; 143(1): 51-59, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31434075

RESUMO

BACKGROUND: SAM domain- and HD domain-containing protein 1 (SAMHD1) is a cellular enzyme which is responsible for blocking replication in viruses and participates in the progression of many cancers. OBJECTIVE: The aim of this study was to correlate the expression level of SAMHD1 with other apoptotic and autophagic genes in acute myeloid leukemia (AML) patients. METHODS: In the present study, mRNA levels of SAMHD1 with other apoptotic and autophagic-related genes were evaluated in patients who were newly diagnosed with AML. RESULTS: SAMHD1, Bcl-xl, Bax, Bak, XIAP, and cIAP1 were downregulated in the AML group compared to the non-AML group (p < 0.05). SAMHD1 expression did not correlate with the other genes, while most apoptotic genes were positively correlated with each other. SAMHD1 expression was not associated with the blood routine or blast percentage of the AML patients, while Bax, Bak, cIAP2, and LC3 were significantly correlated with white blood cells. No statistically significant differences were found between the studied genes and prognosis stratifications, but Bcl-xl, Bak, cIAP1, and Mcl-1, LC3 were expressed at lower levels in the unfavorable AML group compared to the controls. CONCLUSION: SAMHD1 and Bcl-xl, Bax, Bak, XIAP, and cIAP1 were downregulated in AML patients, while there were no significant differences in the clinical characteristics and prognosis with reference to SAMHD1 expression.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Leucemia Mieloide Aguda/diagnóstico , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteínas Reguladoras de Apoptose/genética , Regulação para Baixo , Feminino , Expressão Gênica , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucócitos/citologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteína 1 com Domínio SAM e Domínio HD/genética , Taxa de Sobrevida , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismo
9.
Cell Commun Signal ; 17(1): 168, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31842909

RESUMO

BACKGROUND: Our previous study has demonstrated that NAD(P)H: quinone oxidoreductase 1 (NQO1) is significantly upregulated in human liver cancer where it potentiates the apoptosis evasion of liver cancer cell. However, the underlying mechanisms of the oncogenic function of NQO1 in HCC have not been fully elucidated. METHODS: Expression of NQO1, SIRT6, AKT and X-linked inhibitor of apoptosis protein (XIAP) protein were measured by western blotting and immunohistochemistry. Additionally, the interaction between NQO1 and potential proteins were determined by immunoprecipitation assays. Furthermore, the effect of NQO1 and SIRT6 on tumor growth was determined in cell model and orthotopic tumor implantation model. RESULTS: We found that NQO1 overexpression in HCC enhanced SIRT6 protein stability via inhibiting ubiquitin-mediated 26S proteasome degradation. High level of SIRT6 reduced acetylation of AKT which resulted in increased phosphorylation and activity of AKT. Activated AKT subsequently phosphorylated anti-apoptotic protein XIAP at Ser87 which determined its protein stability. Reintroduction of SIRT6 or AKT efficiently rescued NQO1 knock-out-mediated inhibition of growth and induction of apoptosis. In orthotopic mouse model, NQO1 knock-out inhibited tumor growth and induced apoptosis while this effect was effectively rescued by SIRT6 overexpression or MG132 treatment partially. CONCLUSIONS: Collectively, these results reveal an oncogenic function of NQO1 in sustaining HCC cell proliferation through SIRT6/AKT/XIAP signaling pathway.


Assuntos
Apoptose , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Sirtuínas/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Neoplasias Hepáticas/patologia , NAD(P)H Desidrogenase (Quinona)/deficiência , Fosforilação , Estabilidade Proteica , Transdução de Sinais , Regulação para Cima
10.
Anticancer Res ; 39(12): 6635-6643, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31810928

RESUMO

BACKGROUND/AIM: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis of cancer cells and, when used in combination with other anticancer drugs, is regarded as an effective strategy for anticancer treatment. In this study, we investigated the efficacy of combination treatment with TRAIL-secreting human mesenchymal stem cells (MSC-TRAIL) and compound C, an AMP-activated protein kinase (AMPK inhibitor), on glioblastoma. MATERIALS AND METHODS: The anticancer effect using MSC-TRAIL and compound C on glioma was evaluated in vitro and on in vivo models. RESULTS: Combination treatment of MSC-TRAIL and compound C increased apoptosis by enhancing expression of B-cell lymphoma 2 (BCL2)-associated X protein (BAX) and reducing that of anti-apoptotic proteins cellular FLICE-inhibitory protein (FLIP), X-linked inhibitor of apoptosis (XIAP), and BCL2 in glioma. In addition, MSC-TRAIL and compound C combination increased caspase-3 cleavage and apoptotic cells in a mouse glioma model compared with the group treated with the agents alone. CONCLUSION: Our results suggest that MSC-TRAIL and compound C are a novel combination for treatment of glioma.


Assuntos
Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Transplante de Células-Tronco Mesenquimais , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Animais , Apoptose , Neoplasias Encefálicas/metabolismo , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 3/metabolismo , Terapia Combinada , Glioblastoma/metabolismo , Xenoenxertos , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Proteína X Associada a bcl-2/metabolismo
11.
Cell Death Dis ; 10(11): 846, 2019 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-31699976

RESUMO

According to recent studies, Cannabidiol (CBD), one of the main components of Cannabis sativa, has anticancer effects in several cancers. However, the exact mechanism of CBD action is not currently understood. Here, CBD promoted cell death in gastric cancer. We suggest that CBD induced apoptosis by suppressing X-linked inhibitor apoptosis (XIAP), a member of the IAP protein family. CBD reduced XIAP protein levels while increasing ubiquitination of XIAP. The expression of Smac, a known inhibitor of XIAP, was found to be elevated during CBD treatment. Moreover, CBD treatment increased the interaction between XIAP and Smac by increasing Smac release from mitochondria to the cytosol. CBD has also been shown to affect mitochondrial dysfunction. Taken together, these results suggest that CBD may have potential as a new therapeutic target in gastric cancer.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Biomarcadores Tumorais/metabolismo , Canabidiol/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Neoplasias Gástricas/patologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Biomarcadores Tumorais/genética , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Mitocondriais/genética , Invasividade Neoplásica , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Células Tumorais Cultivadas , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Cell Death Dis ; 10(11): 837, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31685811

RESUMO

TRAIL specifically induces apoptosis in cancer cells without affecting healthy cells. However, TRAIL's cancer cytotoxicity was insufficient in clinical trials. Circulatory-shear stress is known to sensitize cancer cells to TRAIL. In this study, we examine the mechanism of this TRAIL sensitization with the goal of translating it to static conditions. GsMTx-4, a Piezo1 inhibitor, was found to reduce shear stress-related TRAIL sensitization, implicating Piezo1 activation as a potential TRAIL-sensitizer. The Piezo1 agonist Yoda1 recreated shear stress-induced TRAIL sensitization under static conditions. A significant increase in apoptosis occurred when PC3, COLO 205, or MDA-MB-231 cells were treated with Yoda1 and TRAIL in combination, but not in Bax-deficient DU145 cells. Calpastatin inhibited apoptosis in Yoda1-TRAIL treated cells, indicating that calpain activation is necessary for apoptosis by Yoda1 and TRAIL. Yoda1 and TRAIL treated PC3 cells showed increased mitochondrial outer membrane permeability (MOMP), mitochondrial depolarization, and activated Bax. This implies that Piezo1 activation sensitizes cancer cells to TRAIL through a calcium influx that activates calpains. The Calpains then induce MOMP by enhancing Bax activation. From these experiments a computational model was developed to simulate apoptosis for cells treated with TRAIL and increased calcium. The computational model elucidated the proapoptotic or antiapoptotic roles of Bax, Bcl-2, XIAP, and other proteins important in the mitochondrial-apoptotic signaling pathway.


Assuntos
Apoptose , Canais Iônicos/metabolismo , Membranas Mitocondriais/metabolismo , Modelos Biológicos , Neoplasias/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/genética , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Células PC-3 , Permeabilidade , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pirazinas/farmacologia , Venenos de Aranha/farmacologia , Tiadiazóis/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Proteína X Associada a bcl-2/metabolismo
13.
Cell Commun Signal ; 17(1): 150, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31744505
14.
Int J Mol Sci ; 20(20)2019 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-31614718

RESUMO

Drug resistance represents a major issue in treating breast cancer, despite the identification of novel therapeutic strategies, biomarkers, and subgroups. We have previously identified the LQB-223, 11a-N-Tosyl-5-deoxi-pterocarpan, as a promising compound in sensitizing doxorubicin-resistant breast cancer cells, with little toxicity to non-neoplastic cells. Here, we investigated the mechanisms underlying LQB-223 antitumor effects in 2D and 3D models of breast cancer. MCF-7 and MDA-MB-231 cells had migration and motility profile assessed by wound-healing and phagokinetic track motility assays, respectively. Cytotoxicity in 3D conformation was evaluated by measuring spheroid size and performing acid phosphatase and gelatin migration assays. Protein expression was analyzed by immunoblotting. Our results show that LQB-223, but not doxorubicin treatment, suppressed the migratory and motility capacity of breast cancer cells. In 3D conformation, LQB-223 remarkably decreased cell viability, as well as reduced 3D culture size and migration. Mechanistically, LQB-223-mediated anticancer effects involved decreased proteins levels of XIAP, c-IAP1, and Mcl-1 chemoresistance-related proteins, but not survivin. Survivin knockdown partially potentiated LQB-223-induced cytotoxicity. Additionally, cell treatment with LQB-223 resulted in changes in the mRNA levels of epithelial-mesenchymal transition markers, suggesting that it might modulate cell plasticity. Our data demonstrate that LQB-223 impairs 3D culture growth and migration in 2D and 3D models of breast cancer exhibiting different phenotypes.


Assuntos
Antineoplásicos/farmacologia , Apoptose , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos , Pterocarpanos/farmacologia , Antineoplásicos/toxicidade , Movimento Celular , Proliferação de Células , Feminino , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Células MCF-7 , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Pterocarpanos/toxicidade , Esferoides Celulares/efeitos dos fármacos , Survivina/genética , Survivina/metabolismo , Células Tumorais Cultivadas , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
15.
Reprod Biol Endocrinol ; 17(1): 81, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31647034

RESUMO

BACKGROUND: Gestational diabetes mellitus (GDM) is associated with important factors that influence fetal development. Sphingolipids are known to be associated with the development of diabetes. Our objective was to examine ceramide, a key sphingolipid, hyperosmolarity, and apoptosis in placentas from GDM patients treated with insulin or diet. METHODS: Ceramide levels were assessed in placental tissues using immunohistochemistry. Immunoblot was performed to quantify serine palmitoyltransferase (SPT), the rate-limiting enzyme in ceramide biosynthesis, NFAT5, SMIT, AR, caspase 3 and the X-linked inhibitor of apoptosis. Trophoblast cells were treated with insulin or ceramide and assessments for mitochondrial respiration, caspase 3 and XIAP were also performed. RESULTS: Immunohistochemistry showed increased ceramides in the placental villous trophoblasts of the insulin-treated GDM patients. Nuclear SPT was upregulated only in the insulin-treated GDM placenta when compared to controls. Nuclear NFAT5 was also increased in the GDM placenta. Active caspase 3 was elevated in placentas from both insulin- and diet-treated GDM patients. Mitochondrial respiration was decreased in trophoblasts treated with ceramide. Active caspase was not changed while XIAP protein was increased in trophoblasts treated with ceramide. CONCLUSIONS: Our findings confirm the presence of ceramide in the human placenta of control and GDM patients. Furthermore, we conclude that ceramide is increased in the placental trophoblast during insulin treatment and that its upregulation correlates with elevated NFAT5, SMIT, increased apoptosis and decreased trophoblast mitochondrial respiration.


Assuntos
Ceramidas/metabolismo , Diabetes Gestacional/metabolismo , Mitocôndrias/metabolismo , Placenta/metabolismo , Trofoblastos/metabolismo , Adulto , Apoptose/efeitos dos fármacos , Ceramidas/farmacologia , Diabetes Gestacional/tratamento farmacológico , Dieta , Feminino , Humanos , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêutico , Mitocôndrias/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Gravidez , Serina C-Palmitoiltransferase/metabolismo , Trofoblastos/efeitos dos fármacos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
16.
Cells ; 8(10)2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31635307

RESUMO

Ataxia-telegiectasia mutated (ATM), phosphatase and tensin homolog (PTEN), and p85α are key tumour suppressors. Whether ATM regulates PTEN expression and influence platinum sensitivity is unknown. We generated ATM knockdowns (KD) and CRISPR knock outs (KO) in glioblastoma (LN18, LN229) and ovarian cancer cells (OVCAR3, OVCAR4). Doxycycline inducible PTEN expression was generated in LN18 and LN229 cells. Transient KD of p85α, CK2, and XIAP was accomplished using siRNAs. Stable p85α knock-in was isolated in LN18 cells. Molecular biology assays included proteasome activity assays, PCR, flow cytometry analysis (cell cycle, double strand break accumulation, apoptosis), immunofluorescence, co-immunoprecipitation, clonogenic, invasion, migration, and 3D neurosphere assays. The clinicopathological significance of ATM, PTEN, p85α, and XIAP (X-linked inhibitor of apoptosis protein) was evaluated in 525 human ovarian cancers using immunohistochemistry. ATM regulated PTEN is p85α dependant. ATM also controls CK2α level which in turn phosphorylates and stabilizes PTEN. In addition, p85α physically interacts with CK2α and protects CK2α from ATM regulated degradation. ATM deficiency resulted in accumulation of XIAP/p-XIAP levels which ubiquitinated PTEN and CK2α thereby directing them to degradation. ATM depletion in the context of p85α deficiency impaired cancer cell migration and invasion reduced 3D-neurosphere formation and increased toxicity to cisplatin chemotherapy. Increased sensitivity to platinum was associated with DNA double strand breaks accumulation, cell cycle arrest, and induction of autophagy. In ovarian cancer patients, ATM, PTEN, p85α, and XIAP protein levels predicted better progression free survival after platinum therapy. We unravel a previously unknown function of ATM in the regulation of PTEN throµgh XIAP mediated proteasome degradation.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Platina/uso terapêutico , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Western Blotting , Caspase 3/genética , Caspase 3/metabolismo , Caspase 7/genética , Caspase 7/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Cisplatino/uso terapêutico , Doxiciclina/uso terapêutico , Feminino , Citometria de Fluxo , Células HeLa , Humanos , Neoplasias Ovarianas/genética , PTEN Fosfo-Hidrolase/genética , Plasmídeos/genética , Estabilidade Proteica/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Análise Serial de Tecidos , Ubiquitina-Proteína Ligases/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética
17.
Cell Death Dis ; 10(11): 790, 2019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31624230

RESUMO

Mammalian female fertility is limited by the number and quality of oocytes in the ovarian reserve. The number of oocytes is finite since all germ cells cease proliferation to become oocytes in fetal life. Moreover, 70-80% of the initial oocyte population is eliminated during fetal and neonatal development, restricting the ovarian reserve. Why so many oocytes are lost during normal development remains an enigma. In Meiotic Prophase I (MPI), oocytes go through homologous chromosome synapsis and recombination, dependent on formation and subsequent repair of DNA double strand breaks (DSBs). The oocytes that have failed in DSB repair or synapsis get eliminated mainly in neonatal ovaries. However, a large oocyte population is eliminated before birth, and the cause or mechanism of this early oocyte loss is not well understood. In the current paper, we show that the oocyte loss in fetal ovaries was prevented by a deficiency of Caspase 9 (CASP9), which is the hub of the mitochondrial apoptotic pathway. Furthermore, CASP9 and its downstream effector Caspase 3 were counteracted by endogenous X-linked Inhibitor of Apoptosis (XIAP) to regulate the oocyte population; while XIAP overexpression mimicked CASP9 deficiency, XIAP deficiency accelerated oocyte loss. In the CASP9 deficiency, more oocytes were accumulated at the pachytene stage with multiple γH2AFX foci and high LINE1 expression levels, but with normal levels of synapsis and overall DSB repair. We conclude that the oocytes with LINE1 overexpression were preferentially eliminated by CASP9-dependent apoptosis in balance with XIAP during fetal ovarian development. When such oocytes were retained, however, they get eliminated by a CASP9-independent mechanism during neonatal development. Thus, the oocyte is equipped with multiple surveillance mechanisms during MPI progression to safe-guard the quality of oocytes in the ovarian reserve.


Assuntos
Caspase 9/metabolismo , Desenvolvimento Fetal/fisiologia , Proteínas Inibidoras de Apoptose/metabolismo , Oócitos/enzimologia , Animais , Apoptose/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Oócitos/citologia , Ovário/citologia , Ovário/enzimologia , Ovário/crescimento & desenvolvimento , Gravidez , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
18.
Cell Death Dis ; 10(11): 792, 2019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31624242

RESUMO

Prostate cancer (PCa) is one of the major men's malignancies with high mortality worldwide. Circular RNAs (circRNAs) have been shown to serve as essential regulators in human cancers. CircRNA can exert their functions by cooperating with their host genes. In the present study, microarray analysis revealed an upregulated mRNA in PCa samples. X-linked inhibitor of apoptosis protein (XIAP), a key regulator in the progression of human cancers. Through bioinformatics analysis, we determined that XIAP is a host gene for circRNA0005276. Therefore, this study focused on the interaction between circ0005276 and XIAP as well as their functions in PCa progression. The upregulation of XIAP and circ0005276 was determined in PCa tissues and cell lines. Moreover, we confirmed the positive regulation of circ0005276 on XIAP expression. Functionally, we validated that circ0005276 and XIAP promoted cell proliferation, migration and epithelial-mesenchymal transition. Mechanistically, we verified that circ0005276 interacted with FUS binding protein (FUS) so as to activate the transcription of XIAP. Rescue assays were conducted to determine the crucial role of XIAP in circ0005276 and FUS-mediated PCa cellular processes. Collectively, our study revealed the mechanism and function of circ0005276 and its host gene XIAP in PCa progression.


Assuntos
Neoplasias da Próstata/genética , RNA Circular/metabolismo , Proteína FUS de Ligação a RNA/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Animais , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Progressão da Doença , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Nus , Células PC-3 , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Circular/biossíntese , RNA Circular/genética , Proteína FUS de Ligação a RNA/metabolismo , Ativação Transcricional , Regulação para Cima , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/biossíntese , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
19.
Hypertension ; 74(4): 957-966, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31476900

RESUMO

Persistent pulmonary hypertension of the newborn (PPHN) is a failure of pulmonary vascular resistance to decline at birth rapidly. One principal mechanism implicated in PPHN development is mitochondrial oxidative stress. Expression and activity of mitochondrial SOD2 (superoxide dismutase) are decreased in PPHN; however, the mechanism remains unknown. Recently, OLA1 (Obg-like ATPase-1) was shown to act as a critical regulator of proteins controlling cell response to stress including Hsp70, an obligate chaperone for SOD2. Here, we investigated whether OLA1 is causally linked to PPHN. Compared with controls, SOD2 expression is reduced in distal-pulmonary arteries (PAs) from patients with PPHN and fetal-lamb models. Disruptions of the SOD2 gene reproduced PPHN phenotypes, manifested by elevated right ventricular systolic pressure, PA-endothelial cells apoptosis, and PA-smooth muscle cells proliferation. Analyses of SOD2 protein dynamics revealed higher ubiquitinated-SOD2 protein levels in PPHN-lambs, suggesting dysregulated protein ubiquitination. OLA1 controls multiple proteostatic mechanisms and is overexpressed in response to stress. We demonstrated that OLA1 acts as a molecular chaperone, and its activity is induced by stress. Strikingly, OLA1 expression is decreased in distal-PAs from PPHN-patients and fetal-lambs. OLA1 deficiency enhanced CHIP affinity for Hsp70-SOD2 complexes, facilitating SOD2 degradation. Consequently, mitochondrial H2O2 formation is impaired, leading to XIAP (X-linked inhibitor of apoptosis) overexpression that suppresses caspase activity in PA-smooth muscle cells, allowing them to survive and proliferate, contributing to PA remodeling. In-vivo, ola1-/- downregulated SOD2 expression, induced distal-PA remodeling, and right ventricular hypertrophy. We conclude that decreased OLA1 expression accounts for SOD2 downregulation and, therefore, a therapeutic target in PPHN treatments.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Pulmão/metabolismo , Síndrome da Persistência do Padrão de Circulação Fetal/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Superóxido Dismutase/metabolismo , Ubiquitina/metabolismo , Animais , Apoptose , Regulação para Baixo , Feminino , Hemodinâmica/fisiologia , Humanos , Peróxido de Hidrogênio/metabolismo , Recém-Nascido , Masculino , Mitocôndrias/metabolismo , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Ovinos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
20.
Sci Adv ; 5(9): eaaw9162, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31517048

RESUMO

Extrinsic pathway agonists have failed repeatedly in the clinic for three core reasons: Inefficient ligand-induced receptor multimerization, poor pharmacokinetic properties, and tumor intrinsic resistance. Here, we address these factors by (i) using a highly potent death receptor agonist (DRA), (ii) developing an injectable depot for sustained DRA delivery, and (iii) leveraging a CRISPR-Cas9 knockout screen in DRA-resistant colorectal cancer (CRC) cells to identify functional drivers of resistance. Pharmacological blockade of XIAP and BCL-XL by targeted small-molecule drugs strongly enhanced the antitumor activity of DRA in CRC cell lines. Recombinant fusion of the DRA to a thermally responsive elastin-like polypeptide (ELP) creates a gel-like depot upon subcutaneous injection that abolishes tumors in DRA-sensitive Colo205 mouse xenografts. Combination of ELPdepot-DRA with BCL-XL and/or XIAP inhibitors led to tumor growth inhibition and extended survival in DRA-resistant patient-derived xenografts. This strategy provides a precision medicine approach to overcome similar challenges with other protein-based cancer therapies.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Proteína bcl-X/antagonistas & inibidores , Animais , Antineoplásicos/química , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Células HCT116 , Células HT29 , Humanos , Camundongos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína bcl-X/genética , Proteína bcl-X/metabolismo
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