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Although targeting the androgen signaling pathway by androgen receptor (AR) inhibitors, including enzalutamide, has shown therapeutic effectiveness, inevitable emergence of acquired resistance remains a critical challenge in the treatment of advanced prostate cancer (PCa). Recognizing targetable genomic aberrations that trigger endocrine treatment failure holds great promise for advancing therapeutic interventions. Here, we characterized PLXNA1, amplified in a subset of PCa patients, as a contributor to enzalutamide resistance (ENZR). Elevated PLXNA1 expression facilitated PCa proliferation under enzalutamide treatment due to AKT signaling activation. Mechanistically, PLXNA1 recruited NRP1 forming a PLXNA1-NRP1 complex, which in turn potentiated the phosphorylation of the AKT. Either inhibiting PLXNA1-NRP1 complex with an NRP1 inhibitor, EG01377, or targeting PLXNA1-mediated ENZR with AKT inhibitors, abolished the pro-resistance phenotype of PLXNA1. Taken together, combination of AKT inhibitor and AR inhibitors presents a promising therapeutic strategy for PCa, especially in advanced PCa patients exhibiting PLXNA1 overexpression.
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Objective: Baicalein, one of the most abundant flavonoids found in Chinese herb Scutellaria baicalensis Georgi, exhibits pharmacological activities against various cancers. However, the precise pharmacological mechanism of baicalein in treating castration-resistant prostate cancer (CRPC) remains elusive. This study aimed to elucidate the potential mechanism of baicalein against CRPC through a combination of network pharmacology and experimental approaches, thereby providing new avenues for research in CRPC treatment. Methods: The pharmacological and molecular properties of baicalein were obtained using the TCMSP database. Baicalein-related targets were collected from multiple sources including SwissTargetPrediction, PharmMapper and CTD. Targets related to CRPC were acquired from DisGeNET, GeneCards, and CTD. The protein-protein interaction (PPI) was analyzed using STRING 11.5, and Cytoscape 3.7.2 software was utilized to explore the core targets of baicalein on CRPC. GO and KEGG pathway enrichment analysis were performed using DAVID database. Cell experiments were carried out to confirm the validity of the targets. Results: A total of 131 potential targets of baicalein for the treatment of CRPC were obtained. Among them, TP53, AKT1, ALB, CASP3, and HSP90AA1, etc., were recognized as core targets by Cytoscape 3.7.2. GO function enrichment analysis yielded 926 entries, including 703 biological process (BP) terms, 84 cellular component (CC) terms and 139 molecular function (MF) terms. The KEGG pathway enrichment analysis unveiled 159 signaling pathways, mainly involved in Pathways in cancer, prostate cancer, AGE-RAGE signaling pathway in diabetic complications, TP53 signaling pathway, and PI3K-Akt signaling pathway, etc. Cell experiments confirmed that baicalein may inhibit the proliferation of CRPC cells and induce cell cycle arrest in the G1 phase. This effect could be associated with the TP53/CDK2/cyclin E1 pathway. In addition, the results of CETSA suggest that baicalein may directly bind to TP53. Conclusion: Based on network pharmacology analysis and cell experiments, we have predicted and validated the potential targets and related pathways of baicalein for CRPC treatment. This comprehensive approach provides a scientific basis for elucidating the molecular mechanism underlying the action of baicalein in CRPC treatment. Furthermore, these findings offer valuable insights and serve as a reference for the research and development of novel anti-CRPC drugs.
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BACKGROUND: Endocrine resistance driven by sustained activation of androgen receptor (AR) signaling pathway in advanced prostate cancer (PCa) is fatal. Characterization of mechanisms underlying aberrant AR pathway activation to search for potential therapeutic strategy is particularly important. Rac GTPase-activating protein 1 (RACGAP1) is one of the specific GTPase-activating proteins. As a novel tumor proto-oncogene, overexpression of RACGAP1 was related to the occurrence of various tumors. METHODS: Bioinformatics methods were used to analyze the relationship of expression level between RACGAP1 and AR as well as AR pathway activation. qRT-PCR and western blotting assays were performed to assess the expression of AR/AR-V7 and RACGAP1 in PCa cells. Immunoprecipitation and immunofluorescence experiments were conducted to detect the interaction and co-localization between RACGAP1 and AR/AR-V7. Gain- and loss-of-function analyses were conducted to investigate the biological roles of RACGAP1 in PCa cells, using MTS and colony formation assays. In vivo experiments were conducted to evaluate the effect of RACGAP1 inhibition on the tumor growth. RESULTS: RACGAP1 was a gene activated by AR, which was markedly upregulated in PCa patients with CRPC and enzalutamide resistance. AR transcriptionally activated RACGAP1 expression by binding to its promoter region. Reciprocally, nuclear RACGAP1 bound to the N-terminal domain (NTD) of both AR and AR-V7, blocking their interaction with the E3 ubiquitin ligase MDM2. Consequently, this prevented the degradation of AR/AR-V7 in a ubiquitin-proteasome-dependent pathway. Notably, the positive feedback loop between RACGAP1 and AR/AR-V7 contributed to endocrine therapy resistance of CRPC. Combination of enzalutamide and in vivo cholesterol-conjugated RIG-I siRNA drugs targeting RACGAP1 induced potent inhibition of xenograft tumor growth of PCa. CONCLUSION: In summary, our results reveal that reciprocal regulation between RACGAP1 and AR/AR-V7 contributes to the endocrine resistance in PCa. These findings highlight the therapeutic potential of combined RACGAP1 inhibition and enzalutamide in treatment of advanced PCa.
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Resistencia a Medicamentos Antineoplásicos , Proteínas Ativadoras de GTPase , Neoplasias da Próstata , Receptores Androgênicos , Masculino , Humanos , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores Androgênicos/metabolismo , Receptores Androgênicos/genética , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Linhagem Celular Tumoral , Animais , Proto-Oncogene Mas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Feniltioidantoína/farmacologia , Camundongos Nus , Nitrilas/farmacologia , Camundongos , Benzamidas/farmacologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Taking a previously discovered indazole derivative 1 as a lead, systematic structural modifications were performed with an indazole core at the 1- and 6-positions to improve its aqueous solubility. Among the designed indazole derivatives, 6-methylpyridin-3-yl indazole derivative 8l and 1H-indol-4-yl indazole derivative 8m exhibited high potency in the low nanomolar range against A549, Huh-7, and T24 cancer cells, including Taxol-resistant variant cells (A549/Tax). As a hydrochloride salt, 8l exhibited much improved aqueous solubility, and its log P value fell into a favorable range. In mechanistic studies, 8l impeded tubulin polymerization through interacting with the colchicine site, resulting in cell cycle arrest and cellular apoptosis. In addition, compared to lead compound 1, 8l reduced cell migration and led to more potent inhibition of tumor growth in vivo without apparent toxicity. In summary, indazole derivative 8l could work as a potential anticancer agent and deserves further investigation for cancer therapy.
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Antineoplásicos , Indazóis , Indazóis/farmacologia , Polimerização , Proliferação de Células , Antineoplásicos/farmacologia , Antineoplásicos/química , Paclitaxel/farmacologia , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/metabolismo , Colchicina/farmacologia , Microtúbulos/metabolismo , Linhagem Celular Tumoral , Relação Estrutura-AtividadeRESUMO
Neoadjuvant hormonal therapy (NHT) prior to radical prostatectomy (RP) is an approach that can potentially maximize survival outcomes in prostate cancer (PCa) patients with high-risk disease. Unfortunately, subsets of patients do not respond well to such hormonal therapy. We previously identified several pathological parameters in predicting differences in response to NHT of PCa. However, little is known about the potential role and mechanism of miRNAs mediated NHT resistance (NHT-R) in PCa. Here we demonstrate that miR-l42-3p, miR-150-5p and miR-342-3p are the top downregulated miRNAs in PCa tissues with NHT-R. Functional analysis reveals that the three miRNAs inhibit cell proliferation in vitro. Transfection of miRNAs mimics strengthens the inhibitory effects of bicalutamide and enzalutamide to PCa cells. Luciferase reporter assay reveals that CREB5 is the common target of these three miRNAs. Clinically, high expression level of CREB5 correlates with high Gleason score, advanced tumor stage and NHT-R in PCa tissues. CREB5 expression promotes antiandrogen therapy resistance in LNCaP cells and IL6 signaling pathway may be involved in this process. In all, our findings highlight an important role of miR-142-3p, miR-150-5p, and miR-342-3p in contributing NHT-R by targeting CREB5 in PCa.
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MicroRNAs , Neoplasias da Próstata , Masculino , Humanos , MicroRNAs/genética , Terapia Neoadjuvante , Genes Supressores de Tumor , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Próstata/patologia , Proteína A de Ligação a Elemento de Resposta do AMP Cíclico/genéticaRESUMO
Although enzalutamide improves the overall survival of patients with metastatic prostate cancers, enzalutamide resistance (ENZR) will be inevitably developed. Emerging evidence support that alternative oncogenic pathways may bypass the androgen receptor (AR) signaling to promote ENZR progression, however, the underpinning mechanisms remain poorly defined. Here, we report that the expression of RuvB like AAA ATPase 1 (RUVBL1) is upregulated in ENZR cells and xenograft models and prostate tumors in patients. Enzalutamide increases RUVBL1 accumulation in the cytoplasm, which in turn enhances the recruitment of CRAF proto-oncogene serine/threonine kinase protein to plexin A1 (PLXNA1) and the subsequent activation of the downstream MAPK pathway. Co-overexpression of RUVBL1 and PLXNA1 defines a subgroup of prostate cancer (PCa) patients with a poor prognosis. Furthermore, pharmacological inhibition of RUVBL1 by CB-6644 suppresses ENZR cell proliferation and xenograft growth and allows re-sensitization of ENZR cells and xenografts to enzalutamide, indicating that RUVBL1 may act to substitute the AR signaling to promote cancer cell survival and ENZR development. Together, these findings may lead to the identification of RUVBL1 as a potential therapeutic target for ENZR tumors.
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Neoplasias de Próstata Resistentes à Castração , ATPases Associadas a Diversas Atividades Celulares/genética , Benzamidas , Proteínas de Transporte , Linhagem Celular Tumoral , Proliferação de Células , DNA Helicases/genética , DNA Helicases/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Masculino , Proteínas do Tecido Nervoso/metabolismo , Nitrilas/uso terapêutico , Feniltioidantoína , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
Farnesoid X receptor (FXR) agonist obeticholic acid (OCA) has emerged as a potential therapy for nonalcoholic fatty liver disease (NAFLD). However, the side effects of OCA may limit its application in clinics. We identified previously that isotschimgine (ITG) is a non-steroidal FXR selective agonist and has potent therapeutic effects on NAFLD in mice. Here, we aimed to evaluate the therapeutic effects of ITG on nonalcoholic steatohepatitis (NASH) and fibrosis in mice. We used methionine and choline deficient (MCD) diet-induced NASH mice, bile duct ligation (BDL), and carbon tetrachloride (CCl4 )-treated hepatic fibrosis mice to investigate the effects of ITG on NASH, fibrosis, and cholestatic liver injury. Our results showed that ITG improved steatosis and inflammation in the liver of MCD diet-fed mice, as well as alleviated fibrosis and inflammation in the liver of CCl4 -treated mice. Furthermore, ITG attenuated serum bile acid levels, and reduced vacuolization, inflammatory infiltration, hepatic parenchymal necrosis, and collagen accumulation in the liver of BDL mice. Mechanistically, ITG increased the expression of FXR target genes. These data suggest that ITG is an FXR agonist and may be developed as a novel therapy for NASH, hepatic fibrosis, or primary biliary cholangitis.
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Cirrose Hepática/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Éteres Fenílicos/farmacologia , Animais , Tetracloreto de Carbono , Ácido Quenodesoxicólico/análogos & derivados , Ácido Quenodesoxicólico/farmacologia , Dieta , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Natural killer (NK) cells, a key member of innate lymphocytes, are a promising immunotherapeutic target for ischemic stroke. Astragaloside IV (ASIV) is isolated from Astragalus mongholicus Bunge (Fabaceae), a herbal medicine possessing immunomodulatory ability. This study investigated the effect of ASIV on NK cells during the acute stage of brain ischemic injury in a mouse model of middle cerebral artery occlusion (MCAO). MCAO mice treated with ASIV had better functional outcomes, smaller brain infarction and less NK cell brain infiltration. NK cell depletion echoed the protective effect of ASIV. Notably, ASIV did not enhance the protective effect of NK cell depletion against brain ischemic injury. ASIV inhibited glial cell-derived CCL2-mediated chemotaxis to prevent post-ischemic NK cell brain recruitment. Meanwhile, ASIV also abrogated NK cell-mediated cytolytic killing of neurons subjected to oxygen-glucose deprivation and suppressed NK cell-derived IFN-γ and NKG2D expression in the ischemic brain. The inhibitory effect of ASIV on NK cell brain infiltration and activation was mimicked by cryptotanshinone, a STAT3 inhibitor. There was no additive effect when ASIV and cryptotanshinone were used together. In conclusion, ASIV inhibits post-ischemic brain infiltration and activation of NK cells through STAT3 suppression, and this inhibitory effect of ASIV on NK cells plays a key role in its protection against acute ischemic brain injury. Our findings suggest that ASIV is a promising therapeutic candidate in NK cell-based immunotherapy for the treatment of acute ischemic stroke and pave the way for potential clinical trials.
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Natural killer (NK) cells, a type of cytotoxic lymphocytes, can infiltrate into ischemic brain and exacerbate neuronal cell death. Astragaloside IV (ASIV) is the major bioactive ingredient of Astragalus membranaceus, a Chinese herbal medicine, and possesses potent immunomodulatory and neuroprotective properties. This study investigated the effects of ASIV on post-ischemic brain infiltration and activation of NK cells. ASIV reduced brain infarction and alleviated functional deficits in MCAO rats, and these beneficial effects persisted for at least 7 days. Abundant NK cells infiltrated into the ischemic hemisphere on day 1 after brain ischemia, and this infiltration was suppressed by ASIV. Strikingly, ASIV reversed NK cell deficiency in the spleen and blood after brain ischemia. ASIV inhibited astrocyte-derived CCL2 upregulation and reduced CCR2+ NK cell levels in the ischemic brain. Meanwhile, ASIV attenuated NK cell activating receptor NKG2D levels and reduced interferon-γ production. ASIV restored acetylation of histone H3 and the p65 subunit of nuclear factor-κB in the ischemic brain, suggesting inhibition of histone deacetylase (HDAC). Simultaneously, ASIV prevented p65 nuclear translocation. The effects of ASIV on reducing CCL2 production, restoring acetylated p65 levels and preventing p65 nuclear translocation were mimicked by valproate, an HDAC inhibitor, in astrocytes subjected to oxygen-glucose deprivation. Our findings suggest that ASIV inhibits post-ischemic NK cell brain infiltration and activation and reverses NK cell deficiency in the periphery, which together contribute to the beneficial effects of ASIV against brain ischemia. Furthermore, ASIV's effects on suppressing NK cell brain infiltration and activation may involve HDAC inhibition.
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Saponinas , Triterpenos , Animais , Encéfalo , Histona Desacetilases , Células Matadoras Naturais , Ratos , Saponinas/farmacologia , Triterpenos/farmacologiaRESUMO
Cycloastragenol (CAG) is the active form of astragaloside IV isolated from Astragalus Radix, which displays multiple pharmacological effects. Silent information regulator 1 (SIRT1), a class III histone deacetylase, has been shown to play an important role in neuroprotection against cerebral ischemia. In this study, we investigated whether CAG protected against ischemic brain injury and, if so, whether the beneficial effects were associated with the regulation of SIRT1 in the ischemic brain. Mice were subjected to 45 min of middle cerebral artery occlusion (MCAO) followed by reperfusion. CAG (5, 10, 20 mg/kg) was injected intraperitoneally at the onset of reperfusion, 12 h later and then twice daily for up to three days. CAG dose-dependently reduced brain infarct volume, significantly ameliorated functional deficits, and prevented neuronal cell loss in MCAO mice. Meanwhile, CAG significantly reduced matrix metalloproteinase-9 activity, prevented tight junction degradation and subsequently ameliorated blood-brain barrier disruption. Moreover, CAG significantly upregulated SIRT1 expression in the ischemic brain but did not directly activate its enzymatic activity. Concomitant with SIRT1 upregulation, CAG reduced p53 acetylation and the ratio of Bax to Bcl-2 in the ischemic brain. CAG also inhibited NF-κB p65 nuclear translocation. As a result, CAG suppressed the mRNA expression of pro-inflammatory cytokines, including TNF-α and IL-1ß, and inhibited the activation of microglia and astrocytes in the ischemic brain. Our findings suggest that CAG is neuroprotective against ischemic brain injury in mice and that its beneficial effect may involve SIRT1 upregulation and the inhibition of apoptosis and neuroinflammation in the ischemic brain.
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Apoptose/efeitos dos fármacos , Infarto da Artéria Cerebral Média/tratamento farmacológico , Inflamação/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Sapogeninas/uso terapêutico , Sirtuína 1/metabolismo , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Subunidade p50 de NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Junções Íntimas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND/AIMS: Natural killer (NK) cells are among the first immune cells that respond to an ischemic insult in human brains. The infiltrated NK cells damage blood-brain barrier (BBB) and exacerbate brain infarction. Buyang Huanwu Decoction (BHD), a classic Chinese traditional herbal prescription, has long been used for the treatment of ischemic stroke. The present study investigated whether BHD can prevent brain infiltration of NK cells, attenuate BBB disruption and improve ischemic outcomes. METHODS: Transient focal cerebral ischemia was induced in rats by a 60-minute middle cerebral artery occlusion, and BHD was orally administrated at the onset of reperfusion, 12 hours later, then twice daily. Assessed parameters on Day 3 after ischemia were: neurological and motor functional deficits through neurological deficit score and rotarod test, respectively; brain infarction through TTC staining; BBB integrity through Evans blue extravasation; matrix metalloproteinase-2/9 activities through gelatin zymography; tight junction protein, nuclear factor-kB (NF-kB) p65 and phospho-p65 levels through Western blotting; NK cell brain infiltration and CXCR3 levels on NK cells through flow cytometry; interferon-γ production through ELISA; CXCL10 mRNA levels through real-time PCR; CXCL10 expression and p65 nuclear translocation through immunofluorescence staining. RESULTS: BHD markedly reduced brain infarction, improved rotarod performance, and attenuated BBB breakdown. Concurrently, BHD attenuated the upregulation of matrix metalloproteinase-2/9 activities and the degradation of tight junction proteins in the ischemic brain. Infiltration of NK cells was observed in the ischemic hemisphere, and this infiltration was blunted by treatment with BHD. BHD suppressed brain ischemia-induced interferon-γ and chemokine CXCL10 production. Furthermore, BHD significantly reduced the expression of CXCR3 on brain-infiltrated NK cells. Strikingly, BHD did not affect NK cell levels or its CXCR3 expression in the spleen or peripheral blood after brain ischemia. The nuclear translocation of NF-kB p65 and phospho-p65 in the ischemic brain was inhibited by BHD. CONCLUSION: Our findings suggest that BHD prevents brain infiltration of NK cells, preserves BBB integrity and eventually improves ischemic outcomes. The inhibitory effects of BHD on NK cell brain invasion may involve its ability of suppressing NF-kB-associated CXCL10-CXCR3-mediated chemotaxis. Notably, BHD only suppresses NK cells and their CXCR3 expression in the ischemic brain, but not those in periphery.